1. health and fitness
2. disease

Finalists, Outstanding Early Career Investigator Award
1Opposing Roles for Endoglin and Soluble Endoglin in
Cardiac Remodeling and Heart Failure
Navin K Kapur, Szuhuei Wilson, Adil A Yunis, Corey Baker, Mark
J Aronovitz, Qing Lu, Tufts Medical Ctr MCRI, Boston, MA; Ananth
Karumanchi, Beth Israel Deaconess Medical Ctr, Boston, MA; Michelle
Letarte, The Hosp for Sick Children, Toronto, ON; David A Kass, Johns
Hopkins Hosp, Baltimore, MD; Michael E Mendelsohn, Richard H
Karas, Tufts Medical Ctr MCRI, Boston, MA
Transforming growth factor beta-1 (TGFb1) promotes cardiac
fibrosis. The transmembrane co-receptor Endoglin (Eng;
CD105) facilitates TGFb1 signaling via SMAD effector proteins.
In contrast, a circulating form of soluble endoglin (sEng)
inhibits TGFb1 signaling in vascular endothelium. We recently
reported that increased sEng levels in human serum correlate
with clinical indices of heart failure severity. Therefore, we
tested the hypothesis that Eng and sEng mediate opposing
effects on cardiac fibrosis in heart failure. In male, wild-type
mice (WT), Eng expression increased in the left ventricle (LV)
after 2, 4, and 10 weeks of thoracic aortic constriction (TAC)
accompanied by progressive LV fibrosis and hypertrophy. In
contrast to WT mice, Eng haploinsufficient (Eng+/-) mice had
preserved LV function (FS%: 78±4 vs 22±16, Eng+/- vs WT,
p<0.01) and improved survival [88%(7/8) vs 50%(4/8), Eng+/vs WT, p<0.001) after 10 weeks of TAC. Reduced LV fibrosis
was observed in Eng+/- mice, while LV mass, cardiomyocyte
hypertrophy, and calcineurin, SerCA, and bMHC expression
were comparable to WT after TAC. Capillary density was
significantly higher in Eng+/- mice after TAC compared to WT.
LV SMAD phosphorylation (pSMAD) after TAC was studied
and a preferential increase was observed in pSMAD1/5/8
expression in Eng+/- mice as compared to WT mice, in
which cardiac pSmad2/3 expression was increased. The
dependence of TGFb1 induced collagen synthesis on Eng
expression was tested in vitro using human cardiac fibroblasts
(hCF). Neutralizing antibodies and siRNA against Eng each
attenuated TGFb1 induced collagen synthesis. In contrast,
conditioned media from cells transfected with an adenovirus
over-expressing sEng attenuated pSMAD2/3 expression
and TGFb1 induced collagen synthesis in hCF. Treatment
of hCF with recombinant sEng blocked TGFb1 induced
collagen synthesis in a dose-dependent manner, confirming
an inhibitory role of sEng. These results indicate that Eng and
sEng mediate opposite effects on TGFb1 induced collagen
synthesis. Reduced Eng expression uncouples cardiac fibrosis
from cardiomyocyte hypertrophy, promotes SMAD1/5/8signaling, and enhances angiogenesis. Eng may represent a
novel therapeutic target to improve survival in heart failure.
This research has received full or partial funding support from the
American Heart Association, Founders Affiliate (Connecticut,
Maine, Massachusetts, New Hampshire, New Jersey, New York,
Rhode Island, Vermont).
2 IL-10 Modulates Mobilization of Bone Marrow Endothelial
Progenitor Cells and Enhances Their Survival and
Angiogenic Properties in Ischemic Myocardium
Prasanna Krishnamurthy, Melissa Thal, Suresh Verma, Eneda Hoxha,
Erin Lambers, Veronica Ramirez, Gangjian Qin, Douglas Losordo, Raj
Kishore, Northwestern Univ, Chicago, IL
Endothelial progenitor cells (EPC) transplantation has been
shown to enhance neovascularization and improve myocardial
infarction (MI)-induced ventricular dysfunctions. However,
persistent inflammation in the ischemic myocardium, adversely
affect EPC survival and function, thereby compromising full
benefits of EPC-mediated vascular repair. We hypothesized
P. Krishnamurthy: None. M. Thal: None. S. Verma: None. E. Hoxha:
None. E. Lambers: None. V. Ramirez: None. G. Qin: None. D.
Losordo: None. R. Kishore: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
3Interaction Between NFκB and NFAT Coordinates Cardiac
Hypertrophy and Pathological Remodeling
Qinghang Liu, Univ of Washington, Seattle, WA; Jeffery Molkentin,
HHMI, Cincinnati Children’s Hosp Medical Ctr, Cincinnati, OH
Both NFAT and NFkB are Rel homology domaincontaining family members of transcription factors whose
independent activities are critically involved in regulating
cardiac hypertrophy and failure. Here we identified a novel
transcriptional regulatory mechanism whereby NFkB
and NFAT directly interact and synergistically promote
transcriptional activation of one another in cardiomyoctes. We
showed that NFkB-p65 co-immunoprecipitates with NFAT
isoforms in cardiomyocytes, and this interaction is mapped
to the Rel homology domain (RHD) within p65. Intriguingly,
overexpression of p65-RHD disrupts the association between
endogenous p65 and NFATc1, leading to reduced NFAT
luciferase activity. Overexpression of p65-RHD or IKKβ
leads to significant nuclear translocation of NFATc1, and
expression of a constitutively nuclear NFATc1-SA similarly
facilitated p65 nuclear translocation, further suggesting a
physical interaction between p65 and NFATc1. Indeed, we
observed that combined overexpression of p65 and NFAT
leads to synergistic activation of NFAT transcriptional activity
in cardiomyocytes. Conversely, NFAT transcriptional activity
in cardiomyocytes is significantly reduced by inhibition of
NFkB with IkBαM or dominant negative IKKβ. Importantly,
hypertrophic agonist-induced NFAT activity is also significantly
reduced in NFkB-p65 null MEFs compared to wild-type
MEFs, while adenoviral-mediated expression of p65 restored
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
N.K. Kapur: None. S. Wilson: None. A.A. Yunis: None. C. Baker:
None. M.J. Aronovitz: None. Q. Lu: None. A. Karumanchi: None.
M. Letarte: None. D.A. Kass: None. M.E. Mendelsohn: None.
R.H. Karas: None.
that modulation of IL-10 signaling in EPCs enhances their
mobilization, survival and function in ischemic myocardium
after MI. GFP-labeled EPC were transplanted intramyocardially
after induction of MI, and the mice were treated with either
saline or recombinant IL-10. EPC survival and EPC-mediated
neovascularization and myocardial repair were evaluated.
IL-10-treated mice showed increased number of GFP+EPCs
retention that was associated with reduced EPC apoptosis
in the myocardium (P;0.05). The engraftment of EPC into the
vascular structures and the associated capillaries density
was significantly higher in IL-10-treated mice (P;0.05). The
above findings were corroborated with reduced infarct size,
fibrosis and enhanced LV function (echocardiography) in
IL-10+EPC group as compared to EPC+saline group. Invitro,
IL-10-deficient EPCs showed higher LPS-induced apoptosis
compared to WT-EPCs (P;0.05). IL-10 treatment induced
VEGF expression in WT-EPCs which was abrogated by STAT3
inhibition (using curcurbitacin I). Furthermore, microRNA
(miR) profile experiments identified significant increases in a
number of pro-apoptotic and anti-angiogenic-related miRs in
EPCs from IL-10 deficient mice. Interestingly, IL-10-deficient
mice showed impaired MI-induced mobilization of bone
marrow EPCs (Sca1+Flk1+ cells) into the circulation and the
associated SDF-1 mRNA expression in the myocardium.
Bone marrow transplantation studies involving replacement
of IL-10-deficient marrow with WT marrow attenuated these
effects. Invitro, LPS-induced CXCR4 expression was lower
in IL-10-deficient EPCs as compared to WT-EPC. Taken
together, our studies suggest that IL-10 enhances EPC
mobilization, possibly in an SDF1-CXCR4 dependent manner
and increased their survival and neovascularization and the
associated myocardial repair, in part via activation of STAT3
signaling cascades.
19
Poster Presentations
NFAT luciferase activity in p65 null MEFs. In vivo, cardiacspecific deletion of NFkB-p65 using a Cre-LoxP system
caused a ~ 50% reduction in NFAT activity in NFAT-luciferase
reporter mice. Moreover, ablation of p65 in the mouse heart
decreased the hypertrophic response following pressure
overload stimulation, reduced the degree of pathological
remodeling, and preserved contractile function. Taken
together, our results suggest a direct interaction between
NFAT and NFkB pathways that may serve as a novel signaling
mechanism in cardiac hypertrophy and failure.
Q. Liu: None. J. Molkentin: None.
Poster Presentations
P1HDAC1 Plays an Important Role in the Differentiation of
Embryonic Stem Cells and Induced Pluripotent Stem Cells
into Cardiovascular Lineages
ABSTRACTS
Eneda Hoxha, Erin Lambers, Veronica Ramirez, Prasanna Krishnamurthy,
Suresh Verma, Melissa Thal, Raj Kishore, Northwestern Univ, Chicago, IL
20
Despite advancements in the treatment of myocardial
infarction (MI), the majority of patients are at increased risk for
developing heart failure due to the loss of cardiomyocytes and
microvasculature. Some of the main obstacles in the realization
of the full potential of iPS/ES cells arise from incomplete and
poorly understood molecular mechanisms and epigenetic
modifications that govern their pluripotency and directed
differentiation. Real-time array experiments revealed that
HDAC1 is highly expressed in pluripotent cells. Additionally the
lack of this molecule is embryonic lethal, suggesting it plays a
key role in development. Thus, we hypothesized that HDAC1
plays a critical role in directing cardiovascular differentiation
of mES and iPS cells in vitro. HDAC1 was knocked down
in mES cells (C57BL/6) and iPS cells using a shRNA vector.
Differentiation through embryoid body (EB) was induced in
wild type mES cells and iPS cells and in their HDAC1-null
counterparts and the ability of these cells to differentiate
into three early embryonic lineages and more specifically
cardiovascular lineage was monitored. EBs lacking HDAC1
differentiated slower and showed delayed suppression of
pluripotent genes such as Oct4 and Sox2. ChiP experiments
revealed high histone acetylation levels at the promoter
regions of these genes during early differentiation. In
addition cells lacking HDAC1 showed reduced expression
of early markers for all three germ layers. HDAC1-null EBs
also showed delayed and reduced spontaneous beating.
Expression of cardiomyocite markers as well as markers of
other cardiovascular lineages was repressed in HDAC1-null
cells. However, supplementation with BMP2 during early
differentiation recovered the ability in the HDAC1-null cells to
differentiate into endodermal and mesodermal lineages, but
not ectodermal. We propose that HDAC1 plays a critical role
in early development and cardiovascular differentiation of mES
and iPS cells by repressing pluripotent genes and allowing
for expression of early developmental genes such as SOX17
and BMP2. Further research in the molecular mechanisms
involved in this process will greatly aid our understanding of
the epigenetic circuitry of pluripotency and differentiation in ES
and iPS cells.
ationale: The efficiency of adoptively transferred human
R
cardiac stem cells to repair damaged myocardium is extremely
limited. There is an urgent need to improve the potency of
human stem cells to engraft, proliferate and differentiate
into cardiac phenotypes before launching them as viable
therapeutic tool in clinics. Objective: Demonstrate that Pim-1
engineering of human cardiac stem cells (hCPCs) can improve
their long term persistence in the damaged myocardium
associated with significant augmentation of cardiac function.
Methods and Results: hCPCs positive for the putative stem
cell marker c-kit were isolated from heart biopsy samples
from patients undergoing Left Ventricular Assist Device
(LVAD) implantation. hCPCs were engineered to express
Pim-1-GFP, a fused GFP version of the kinase by using a
lentivirus expression system. Pim-1 engineering of hCPCs
significantly increased their viability, proliferation and metabolic
activity. Conversely, treatment with a Pim-1 specific inhibitor
quercetagetin completely abrogated the observed proliferative
response. Pim-1 engineered hCPCs were transplanted in
immunocompromised mice with myocardial infarction to
evaluate functional competency of the cells. Animals receiving
Pim-1 CPCs showed increased hemodynamic performance
as evidenced by dp/dt, LVEDP and LVDP measurements 10
weeks after transplantation. Cardiac function demonstrated
sustained improvement even after 20 weeks of transplantation
in Pim-1 CPC transplanted animals compared to control
cell transplanted group. Concurrent with enhanced cardiac
function, animals having Pim-1 CPCs demonstrated increased
cellular engraftment, persistence and proliferation of human
CPCs. In particular, a greater number of c-kit+ cells, improved
vasculature and reduced infarct size were observed.
Conclusion: Genetic engineering of human CPCs with Pim-1
enhances their ability to repair damaged myocardium. Ex vivo
gene delivery to enhance survival, proliferation and regeneration
in damaged myocardium has emerged as a viable option
addressing current limitations associated with stem cell therapy.
S. Mohsin: None. M. Khan: None. H. Toko: None. B. Bailey: None. S.
Siddiqi: None. K. Fischer: None. N. Gude: None. S. Truffa: None. M.A.
Sussman: None.
P3
Withdrawn
P4 Cardiac Regeneration in Neonatal Mice Following
Ischemic Injury
Enzo R Porrello, Ahmed I Mahmoud, Emma Simpson, Joseph A Hill,
James A Richardson, Eric N Olson, Hesham A Sadek, UT Southwestern
Medical Ctr, Dallas, TX
As a consequence of the adult mammalian heart’s limited
regenerative capacity, ischemic heart disease is the leading
cause of morbidity and mortality in the developed world.
We have recently identified a brief window during post-natal
development when the mammalian heart retains significant
cardiac regenerative potential. Similar to adult zebrafish, the 1
day-old neonatal mouse heart is capable of complete cardiac
regeneration following amputation of the entire ventricular
apex. One major unresolved question is whether the neonatal
mouse heart can also regenerate in response to myocardial
ischemia. Here, we demonstrate that the neonatal mouse heart
is also capable of undergoing a cardiac regenerative response
to replace lost cardiomyocytes following myocardial ischemia.
Following permanent ligation of the left anterior descending
(LAD) coronary artery of 1 day-old neonatal mice, we
E. Hoxha: None. E. Lambers: None. V. Ramirez: None. P. Krishnamurthy:
documented evidence of significant cardiac injury and myocyte
None. S. Verma: None. M. Thal: None. R. Kishore: None.
death by histology and echocardiography at 1 and 4 days after
P2Pim-1 Engineering of Human CPCs Increases Their Ability
LAD ligation (Ejection Fraction (MI vs. Sham) = 70.9±10.1%
to Repair the Heart After Myocardial Infarction
vs. 98.6±0.3%, P<0.05). Remarkably, the 1 day-old neonatal
Sadia Mohsin, Mohsin Khan, Haruhiro Toko, Brandi Bailey, Sailay
mouse heart completely regenerated the necrotic myocardium
Siddiqi, Kimberlee Fischer, Natalie Gude, Silvia Truffa, Mark A Sussman,
within 21 days, with minimal fibrosis and full recovery of cardiac
San Diego State Univ, San Diego, CA
Become an AHA/ASA Professional Member and save up to $300 off next year’s registration. (Promo code MBJ050ZZ)
Poster Presentations (continued)
BM-derived cells devoid of either βArr1 or βArr2 were found
to proliferate and colonize in a significantly deficient manner
compared to BM cells isolated from wild-type (WT) mice.
In addition, the number of c-kit positive cardiac stem cells
(as a % of total BM) were significantly lower in the βArr KO
mice compared to WT. We carried out BM transplant studies
to begin to determine whether the βArrs may play a role in
cardiac repair. In our study, WT mice were irradiated and then
received BM transplants from either WT donors as a control
or BM from βArr1 or βArr2 KO mice. Subsequent to BM
reconstitution, mice underwent a myocardial infarction (MI) and
their condition was followed. Interestingly, chimeric mice with
βArr1 and βArr2 KO BM had significantly poorer outcomes
than mice receiving WT BM. This included significantly
decreased post-MI survival with βArr2 KO BM and both βArr
chimeras had significantly lower cardiac function post-MI
compared to mice receiving WT BM. Additionally, our analyses
of the BM cells and very small embryonic-like stem cells
(VSELs) circulating in peripheral blood (PB), indicate that βArrKO BM and PB contain fewer and less viable cardiac stem/
precursor cells compared to WT transplanted controls.
function (Ejection Fraction (MI vs. Sham) = 94.6±1.4% vs.
97.9±0.1%). Similar to the regenerative response of neonatal
mice to apical resection, the regenerative response of the
neonatal mouse heart to ischemic injury was associated
with robust and widespread activation of cardiomyocyte
proliferation (~11-fold), which peaked at day 7 post-injury. In
contrast, following LAD ligation at 7 days-of-age, the cardiac
regenerative response of neonatal mice was significantly
impaired and by 14 days-of-age, the cardiac injury response
was characterized by a fibrotic repair process that was
reminiscent of the adult heart’s response to ischemic injury.
Additionally, over-expression of miR-195, a potent inhibitor of
cardiomyocyte proliferation, impaired the cardiac regenerative
response of neonatal mice. Thus, the neonatal mammalian
heart is capable of complete regeneration following ischemic
myocardial necrosis and cardiomyocyte proliferation appears
to play an important role in this process.
E.R. Porrello: None. A.I. Mahmoud: None. E. Simpson: None. J.A. Hill:
None. J.A. Richardson: None. E.N. Olson: None. H.A. Sadek: None.
This research has received full or partial funding support from the
American Heart Association, South Central Affiliate (Arkansas, New
Mexico, Oklahoma & Texas).
P5 Direct Differentiation of Atrial and Ventricular Myocytes
from Human Embryonic Stem Cells
Yue Ma Sr, Inst of Biophysics; Chinese Acad of Sciences, Beijing, China
Although cell transplantation studies have suggested promising
therapeutic potentials for myocardial infarction, the leading
cause of death worldwide, the incapability to obtain relatively
homogeneous ventricular myocytes for transplantation is
one major obstacle to the development of clinical therapies
for myocardial repair1. Human embryonic stem cell (hESC)
is a promising source of cardiomyocytes. Here we show
that both Noggin and pan-retinoic acid receptor antagonist
BMS-1894532 (RAi) significantly increase the cardiac
differentiation efficiency of hESCs. Investigating retinoid
function by comparing Noggin+RAi-treated cultures with
Noggin+RA-treated cultures, we found that with 64±0.88%
(mean ±s.e.m) cardiac differentiation efficiency, 83% of the
cardiomyocytes in Noggin+RAi-treated cultures had embryonic
ventricular-like action potentials (AP); while, with 50±1.76%
cardiac differentiation efficiency, 94% of those in Noggin+RAtreated cultures had embryonic atrial-like APs. These findings
demonstrating that relatively homogeneous embryonic atrial
and ventricular myocyte populations can be efficiently derived
from hESCs by specifically influencing signaling cascades.
P6 A Potential Role for β-Arrestins in Cardiac Regeneration
Anna M Gumpert, Mai Chen, Henriette Brinks, Karsten Peppel, Erhe
Gao, Walter J Koch, Thomas Jefferson Univ, Philadelphia, PA
Development of chronic heart failure (HF) syndrome following
myocardial injury (MI) is characterized by an extensive loss
of myocytes due to substantial apoptosis and necrosis. As
bone marrow derived stem cells (BMSCs) are capable of
transdifferentiating, they also show potential for regenerating
the myocardium after infarction. Stem cell mobilization, egress
from the bone marrow and homing to the site of injury can
be regulated by signals through G protein-coupled receptors
(GPCRs). β-arrestins are known for their signalling and
scaffolding functions and as downstream regulators of GPCR
desensitization and endocytosis in particular. We have begun
to investigate whether β-arrestins play any role in cardiac
precursor cell function concentrating on the properties of
BMSCs. Using knockout (KO) mice, we investigated the role
β-arrestin1 (βArr1) and β-arrestin2 (βArr2) with respect to
modulation of regenerative competence of BMSCs and their
contribution to cardiac repair following ischemic injury. First,
.M. Gumpert: None. M. Chen: None. H. Brinks: None. K. Peppel:
A
None. E. Gao: None. W.J. Koch: None.
This research has received full or partial funding support from the
American Heart Association, Great Rivers Affiliate (Delaware, Kentucky,
Ohio, Pennsylvania & West Virginia).
P7 Cardiomyocyte Damage-Released Extracellular S100A1
Protein Promotes Regeneration of Infarcted Myocardium
by Modulating Cardiac Fibroblast Function
David Rohde, Inst for Molecular and Translational Cardiology, Univ
of Heidelberg, Heidelberg, Germany; Gang Qiu, Ctr for Translational
Med, Thomas Jefferson Univ, Philadelphia, PA; Nicole Herzog, Inst for
Molecular and Translational Cardiology, Univ of Heidelberg, Heidelberg,
Germany; Hugo A Katus, Angelika Bierhaus, Univ of Heidelberg,
Heidelberg, Germany; Karsten Peppel, Ctr for Translational Med, Thomas
Jefferson Univ, Philadelphia, PA; Patrick Most, Inst for Molecular and
Translational Cardiology, Univ of Heidelberg, Heidelberg, Germany
Background: Similar to heart muscle-specific creatinkinase
(CK-MB), S100A1 protein is released from damaged human
cardiomyocytes in response to myocardial infarction (MI).
Since S100A1-knock out (SKO) mice display rapid post-MI
onset of adverse myocardial remodeling and accelerated
transition to heart failure, we assessed the hypothesis that
ischemia-related release of S100A1 protein modulates
myocardial regeneration. Methods and Results: After LAD
ligation in C57/B6 mice, S100A1 serum levels peaked at 10
µg/ml 8 hours post-MI, precisely mirroring the time course
previously observed in MI patients. RT-PCR analyses in
post-MI whole heart samples revealed significantly lower
I-CAM (-50%) and IL-10 (-75%) mRNA abundance as well as
heightened Collagen-1 (+40%) and VEGF (+80%) expression
in SKO vs. WT mice (p<0.05, n=6 in each group). Interestingly,
injection of an S100A1-neutralizing antibody prior to MI in WT
mice mimicked the abnormalities observed in post-ischemic
SKO animals. To further elucidate extracellular S100A1
biological activity, cardiomyocytes, cardiac fibroblasts (CF),
endothelial and smooth muscle cells were exposed to S100A1
in vitro. A rapid internalization of S100A1 was exclusively
found in CF, resulting in a phosphorylation of ERK1/2,
JNK, and p38 with subsequent activation of NF-kappaB as
assessed by Western Blot (WB) and EMSA. RT-PCR and WB
analyses revealed significant alterations in CF gene expression
in response to S100A1, including an increase in I-CAM (3,5fold) and IL-10 (20-fold) mRNA levels and diminished Col-1
(-80%) expression. Similar effects were observed after direct
injection of S100A1 protein into the left ventricular apical
region of WT mice in vivo (S100A1- vs. PBS-injection, n=6).
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
Y. Ma: None.
21
Poster Presentations (continued)
In SKO mice, intraperitoneal application of S100A1 prior to
MI largely normalized the adverse gene expression pattern
towards WT animals. Conclusions: Our study provides first
evidence for cardiomyocyte damage-released S100A1 to
act as an endogenous mediator of post-MI inflammation and
tissue repair. Considering today´s unability to manipulate these
molecular mechanisms, extracellular S100A1 might represent
a promising target for future therapies of MI.
D. Rohde: None. G. Qiu: None. N. Herzog: None. H.A. Katus: None.
A. Bierhaus: None. K. Peppel: None. P. Most: None.
P8 Fibronectin Enhances Survival and Proliferation via Pim-1
and β1 Integrin in Cardiac Progenitor Cells
Mathias Konstandin, Mirko Völkers, Grady Gastelum, Natalie Gude,
Mark A Sussman, SDSU Heart Inst and Biology Dept, San Diego, CA
Background: Cardiac Progenitor Cells (CPC) are pivotally
involved in cardiac repair. Extracellular matrix (ECM)
components also contribute decisively in cardiac remodeling
after myocardial infarction (MI). However, impact of ECM on
CPC function and the involved signaling pathways have not
been clearly elucidated. This study examines the relationship
of ECM to cardioprotective signaling mediated by Pim-1, a
serine/threonine kinase downstream of Akt, in CPC. Methods:
Isolated mouse CPCs were plated on fibronectin (FN) or
albumin coated dishes. FACS analysis and fluorescence based
cell quantification have been used to determine viability and
proliferation under stress and growth conditions. Signaling
pathways were analyzed by immunoblotting, qRT-PCR and
siRNA. Immunhistochemistry (IHC) has been used for in
vivo studies after MI in mice. Results: FN inhibits starvation
and staurosporine induced cell death in CPCs and promotes
proliferation in conjunction with induction of Pim-1 expression.
Protective and pro-proliferative effects of FN are abrogated by
inhibition of Pim-1. Fibronectin signaling operates through the β1
integrin receptor that is crucial for FN-mediated signaling. Ongoing
studies correlate these observations with in vivo responses to
cardiomyopathic injury such as infarction challenge. Conclusion:
FN provides pro-survival and pro-proliferative effects in CPCs
in a Pim-1 kinase and β1 integrin dependent manner. Involved
molecules colocalize in vivo in an infarction injury model. The
causal contribution of FN in regeneration and wound healing
after MI is to be confirmed by studies of a conditional FN knockout mouse that is under construction.
ABSTRACTS
M. Konstandin: None. M. Völkers: None. G. Gastelum: None. N.
Gude: None. M.A. Sussman: None.
22
P9Notch Signaling Regulates Pluripotent Gene Expressions
in Cardiac c-Kit+ Cells
Liudmila Zakharova, Mohamed A Gaballa, Banner Sun Health Res
Inst, Sun City, AZ
We and others have shown that transplantation of explantderived cells (EDCs) obtained from cardiac biopsies improved
cardiac function after myocardial infarction. The current
study was designed to examine the molecular mechanisms
regulating expression of pluripotent genes in these cells.
Toward this end, EDCs were separated based on expression
of c-Kit antigen after 21 days in culture. We found that
Notch signaling was activated mainly in c-Kit+ cells, but to
a lesser degree in c-Kit- cells. In addition, we found that the
pluripotency markers Sca-1, Nanog and Sox2 were mainly
expressed in c-Kit+ cells. Also, in c-Kit+ cells, forced activation
of Notch signaling via over-expression of Notch intracellular
domain (NICD) induced cellular and molecular changes typical
of epithelial-mesenchymal transition (EMT), evident by a
decrease in VE cadherin and increases in N-cadherin, MMP,
and ICAM. Suppression of Notch signaling was associated
with c-Kit+ cells exhibition of an epithelial/endothelial
morphology. This observation is further supported by the
increase in Nanog, Sca1, Sox2, VEGFR2 gene expressions
after Notch suppression. In contrast, overexpression of
NICD resulted in down-regulation of pluripotency gene
expressions. Furthermore, suppression of Notch signaling was
coincided with stabilization of β-catenin and accumulation of
phosphorylated glycogen synthase kinase 3 beta (pGSK3β)
suggestive of a crosstalk between Notch and canonical Wnt
pathway. Thus, Notch regulates expression of pluripotency
genes and mesenchymal transition of c-Kit+ cardiac EDCs.
L. Zakharova: None. M.A. Gaballa: None.
P10Analysis of Turnover in Human Heart Pathologies
Sofia M Zdunek, Olaf Bergmann, Karolinska Instt, Stockholm,
Sweden; Mehran Salehpour, Uppsala Univt, Uppsala, Sweden; Samuel
Bernard, Univ Claude Bernard Lyon 1, Lyon, France; Karl Håkansson,
Uppsala Univt, Uppsala, Sweden; Ramadan Jashari, European
Homograft Bank, Brussels, Belgium; Michaela Schwarz, Univ Clinic
Graz, Graz, Austria; Göran Possnert, Uppsala Univt, Uppsala, Sweden;
Stefan Jovinge, Lunds Univt, Lund, Sweden; Jonas Frisén, Karolinska
Instt, Stockholm, Sweden
We have previously reported that human cardiomyocytes
regenerate throughout life. Whether pathological conditions
might trigger cardiac renewal is not known. However, gaining
this knowledge would potentially lead to new strategies to
treat heart failure. Hence, we are investigating cell turnover
in ischemic heart disease and dilated cardiomyopathy. Our
strategy is to use the 14C cellular birth-dating technology.
This is based on the measurement of the turnover dependent
concentration of radiocarbon in DNA isolated from cell nuclei.
Cardiomyocyte nuclei are identified and isolated based on their
specific nuclear localization of pericentriolar material protein-1
(PCM-1). It is well recognized that cardiomyocytes undergo
polyploidisation during heart failure. In line with this we found
that the average ploidy, expressed as average DNA content/
nucleus, was 5.7n (compared to normal 3.6n). This increase
in ploidy was attributed to a decrease in the 2n fraction (from
28.9%±10.6% to 12.5%±7.4%) and an increase in higher
ploidies. Our preliminary analysis of cardiomyocyte turnover
indicates increased turnover in aged (age>50 years) ischemic
and dilated hearts. We also measured increased turnover in
the non-myocyte population. To shed light on the regenerative
response we will present an extended study, including an
analysis of younger dilated hearts.
S.M. Zdunek: None. O. Bergmann: None. M. Salehpour: None. S.
Bernard: None. K. Håkansson: None. R. Jashari: None. M. Schwarz:
None. G. Possnert: None. S. Jovinge: None. J. Frisén: None.
P11Endothelial Differentiation and Lineage Tracing in
Adipocyte-Derived Multipotent Cells
Medet Jumabay, Raushan Abdmaulen, Yucheng Yao, Kristina
Bostrom, UCLA, Los Angeles, CA
We previously showed that so-called de-differentiated fat
(DFAT) cells, which are derived from mature white adipocytes,
spontaneously differentiate into beating cardiomyocytes.
Our aim in this study was to investigate if DFAT cells also
differentiate into endothelial cells (ECs) in vitro, and to
further examine the cellular origin of DFAT cells as well as
adipose stromal cells (ASCs) using lineage tracing. First, we
examined DFAT and ASCs prepared from aP2-Cre+/+;LacZ
ROSA(R26R)+/+ double transgenic mice, which express
LacZ under the aP2 promoter. The results revealed that
99.9% of DFAT cells and 45% of the ASCs stained positive
for LacZ, supporting that the DFAT cells and part of the ASCs
are of adipocytic origin. Second we allowed newly isolated
DFAT cells to spontaneously undergo EC differentiation,
which was monitored by expression of EC lineage markers
as determined by real-time PCR, immunofluorescence, and
FACS. Expression of the EC markers CD31 and VE-cadherin
increased progressively during 2 weeks in culture, the
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Poster Presentations (continued)
percentage of CD31(+) cells increased from 0.0% to 8.3%,
and the cells formed multi-cellular tube structures when
placed in Matrigel™/Collagen gels. The data supported that a
fraction of the DFAT cells differentiate into ECs. Furthermore,
the EC differentiation could be enhanced in DFAT cells by
treatment with bone morphogenetic protein (BMP)-4 and
BMP-9. In addition to EC differentiation, the DFAT cells also
expressed markers of other cardiovascular lineages including
smooth muscle cells and pericytes. The multipotency of DFAT
cells suggests that cellular de-differentiation might be a way
for differentiated cells to regain stem cell-like properties. Thus,
white mature adipocytes maybe a new stem cell source for
cardiovascular regeneration.
M. Jumabay: None. R. Abdmaulen: None. Y. Yao: None.
K. Bostrom: None.
This research has received full or partial funding support from the American
Heart Association, Western States Affiliate (California, Nevada & Utah).
P12KDR Expression Enhances Commitment Toward the Cardiac
Lineage of hESC- and iPS-Derived Cd15+ Progenitors
Elisa Di Pasquale, Fondazione Multimedica Onlus, Milan, Italy;
Portararo Paola, IRCCS Multimedica, Milan, Italy; Roberto Rizzi, Italian
Natl Res Council, Milan, Italy; Monica Mancino, IRCCS Multimedica,
Milan, Italy; Gianluigi Condorelli, Univ of California, San Diego, CA
E. Di Pasquale: None. P. Paola: None. R. Rizzi: None. M. Mancino:
None. G. Condorelli: None.
P13Canopy 2 Encodes for a Novel Secreted Proangiogenic
Factor that Revascularizes and Regenerates Murine
Hearts Post Myocardial Infarction
Jian Guo, Zhuo Sun, Anton Mihic, Sharon Au, Shu Hong Li, Kota
Hatta, Yue Mei Zhang, Sanjiv Dhingra, Jun Wu, Richard D. Weisel, .
Ren-Ke Li, Toronto Medical Discovery Tower, Toronto, ON, Canada
J. Guo: None. Z. Sun: None. A. Mihic: None. S. Au: None. S. Li: None.
K. Hatta: None. Y. Zhang: None. S. Dhingra: None. J. Wu: None.
R.D. Weisel: None. R. Li: None.
P14Oxidative Stress Mediated Regulation of Antioxidants in
Cardiac Progenitor Cells
ABSTRACTS
Cell-based therapies represent exciting therapeutic options
for myocardial regeneration after ischemic injury. Although
many cell types have been proposed as a source of cardiac
myocytes, the type of progenitor as well as the optimal
conditions for their stimulation still represent major challenges.
Moreover, molecular characterization of cardiac progenitors
(CP) in vitro is still unsatisfactory: the identification of novel
surface markers for their selective isolation is of utmost
importance. In our study we employed different human ESCs
and induced pluripotent stem (iPS) cells lines to set up a
simple, reliable and efficient protocol to selectively induce
cardiac fate in vitro. At first, we comparatively applied different
methods and monitored the differentiation process by FACS
analysis, RT-PCR and immunofluorescence for early and late
markers. Our results show that sequential treatments with
ActivinA and BMP4 or BMP2, ascorbic acid and TGFβ1 in a
specific media formulation induce CD15+ cardiac progenitor
population with an enhanced cell vitality compared to other
methods. While we confirmed that CD15 is among the earliest
markers upregulated after differentiation, the definition of
other associated markers could better specify progenitors
committed toward cardiomyogenesis. For this purpose, we
systematically evaluated induction of other markers already
shown associated with cardiac differentiation or heart
development, that is PDGFRα, KDR, Sca1, CXCR4, cKIT.
Our result demonstrate that a specific population expressing
both KDR and CD15 surface markers is more committed
toward cardiovascular lineages. The induced KDR+/CD15+ cell
population exhibits higher levels of Gata4 and Isl1 expression
(2-fold induction), compared to the KDR⁻/CD15+ counterpart
and, after isolation, gives rise to 66.15% Troponin I positive
cells (43 out of 65 in a single representative experiment),
against the 26.5% (18 out of 68) detectable in the KDR⁻/
CD15⁺ population. In conclusion, our studies, though
preliminary, strongly indicate that KDR in CD15+ progenitor
cells is an earlier marker of cardiomyogenesis.
Through genomic screening of 256 novel genes by RTPCR in human smooth muscle cells, we discovered that
Canopy 2 (CNPY2), previously named as MSAP, TMEM4
or ZSIG9, actually encodes for a secreted pro-angiogenic
factor, which was differentially regulated by in vitro hypoxia.
Tissue distribution profile reveals that CNPY2 has the highest
expression levels in the heart, liver and lung. Surprisingly,
CNPY2 was dramatically downregulated (~2–5 fold) in
the murine heart at day 1–7 post myocardial infarction.
Recombinant CNPY2 proteins were purified both from E.coli
to generate rabbit (blocking) antibodies, and from mammalian
cells with proper folding and posttranslational modification to
study signaling cascades. We demonstrate that recombinant
CNPY2 protein binds to the extracellular domain of VEGFR2
and phosphorylates the intracellular Tyr1175 and Tyr1214 of
VEGFR2, activates FAK and ERK which resulting in pERK
nuclear translocation in human endothelial cells. On the other
hand, CNPY2 binds to adhesion molecule desmoglein 1,
activates Cdc42, PAK1 and FAK in human smooth muscle
cells. CNPY2 reorganizes the dynamic vimentin structure in
both cell types, resulting in an accelerated cell migration. In
vivo delivery of recombination CNPY2 protein enhanced the
blood capillary formation in a retinal ischemia/reperfusion
mouse model, and dramatically revascularized blood vessel
network, reduced infarct size and improved cardiac function
in a rat myocardial infarction model. In line with the paracrine
mechanism in (stem) cell-based therapy, our data suggests
that CNPY2 might be employed as a promising therapeutic
protein for ischemic tissue revascularization and regeneration.
Gokulakrishnan Iyer, Georgia Inst of Technology, Atlanta, GA; Michael
E Davis, Georgia Inst of Technology/Emory Univ, Atlanta, GA
Cardiac diseases are the leading causes of death throughout
the world and transplantation of endogenous myocardial
progenitor population with robust cardiovascular lineage
differentiation potential is a promising therapeutic strategy.
Therefore, in vitro expansion and transplantation of cardiac
progenitor cells (CPCs) is currently in early clinical testing
as a potential treatment for severe cardiac dysfunction.
However, poor survival and engraftment of cells is one of the
major limitations of cell transplantation therapy. Oxidative
stress is increased in the ischemic myocardium and indirect
inferences suggest the vulnerability of CPCs to oxidative
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23
Poster Presentations (continued)
stress. In this study, we show that in vitro, resident c-kit
positive CPCs isolated from rat myocardium are significantly
(p;0.05) resistant to superoxide-induced apoptosis compared
to cardiomyocytes as analyzed by the number of sub-G1
population following xanthine/xanthine oxidase treatment.
Interestingly, CPCs have two to four fold higher basal SOD1
and SOD2 activities (p;0.01) compared to cardiomyocytes and
endothelial cells. Superoxide treatment increased expression
of SOD1 (p;0.01), SOD2 (p;0.01), and glutathione peroxidase
(p;0.05) mRNAs within 6 h of treatment compared to control
cells. Recent studies suggest the involvement of AKT in
controlling cell death, survival and also expression of SOD
enzymes. Therefore, we investigated the involvement of AKT
in CPCs subjected to oxidative stress. Western blot analysis
revealed that the amount of phosphorylated AKT increased
significantly within 10 minutes of xanthine/xanthine oxidase
treatment. In addition, treatment with LY294002 — a PI3
kinase/AKT inhibitor, increased apoptosis in CPCs treated
with superoxide. Our studies demonstrate a novel finding in
which resident progenitor cells are protected from oxidative
injury by containing higher basal levels of antioxidants as
compared to myocytes. Moreover, under oxidant challenge
antioxidant levels are regulated, possibly in an AKT-dependent
manner. Further elucidation of this pathway may lead to novel
therapeutic opportunities.
G. Iyer: None. M.E. Davis: None.
P15Hypoxia Regulates Embryonic Cardiac Myocyte
Proliferation and Differentiation
ABSTRACTS
Alexander Zambon, Jennifer Stowe, UCSD, La Jolla, CA
24
Mammalian cardiac myocytes (CM) proliferate readily during
development but exit the cell cycle perinatally. The molecular
mechanism for this is poorly understood could lead to new
approaches for cardiac regeneration. We employed a novel
bioinformatics approach to identify transcription factors that
may control perinatal cell-cycle arrest and identified hypoxia
inducible factor 1α (Hif1α) as a potential regulator of CM
proliferation and differentiation. To test our hypothesis, e12.5d
embryonic heart cells were cultured in atmospheric (17%)
or hypoxic (3%) [O2] and then assessed for proliferation
over 4 days. Total cell numbers were significantly increased
(~2 fold, P<0.005) in cells cultured in 3% vs. 17% [O2]
after 4 days. Hypoxia increased numbers of cTn+ cells
labeled with p-Histone-H3 indicating an increase in CM
proliferation. Carboxyfluorescein succinimidyl ester staining
indicated that the majority (70%) of cells cultured in 3%
[O2] divided 2–3 times while the majority (73%) of cells
in 17% [O2] divided 0–1 times after 3 days of culture. As
expected, hypoxia stabilized the Hif1α protein and induced
the expression of Vegf. Messenger RNA for the G1-S phase
cyclin dependent kinase Ccnd2 was increased (>2 fold) in
hypoxic vs. atmospheric cultured cells indicating that hypoxia
my increase proliferation by regulating the expression of cell
cycle machinery. Furthermore, early mesodermal transcription
factors Brachyury and Mesp1 and the CM progenitor marker
Nkx2.5 were induced (>5 fold) in hypoxic vs. atmospheric [O2]
cultured cells suggesting that hypoxia positively regulates early
mesodermal and CM progenitor cell differentiation. To validate
this hypothesis, we differentiated mouse embryonic stem
cells in atmospheric and hypoxic [O2] and observed similar
increases in the expression of Brachyury, Mesp1 and Nkx2.5 in
hypoxic differentiated cells. Transient culture (2 d) of embryoid
bodies (EBs) in hypoxia increased the frequency of beating
EBs and the % of cTn+ cells compared to cells differentiated
in atmospheric O2. Taken together these data suggest that the
hypoxic environment in which CMs develop may serve as a key
signal that regulates mesodermal progenitor marker expression
and CM progenitor cell differentiation and proliferation.
A. Zambon: None. J. Stowe: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P16Human Mesenchymal Bone Marrow-Derived Stem Cells
Require Connexin 43 to Form Beating 3-Dimensional
Tubes When Cocultured with Neonatal Rat Cardiomyocytes
Cristina Sanina, Claudia O Rodrigues, Ivonne Hernandez Schulman,
Irene Margitich, Wayne Balkan, Joshua M Hare, Univ of Miami Miller
Sch of Med, Miami, FL
Mesenchymal stem cells (MSCs) have begun to manifest
themselves as a safe and beneficial therapy for restoring
cardiac function in failing hearts. However, the mechanism
underlying this function is unclear. The present study was
initiated to investigate the role of Connexin 43 (Cx43) in
hMSC integration, migration and differentiation in co-culture
with neonatal rat cardiomyocyte (NRVMs) in vitro. Cx43 is
a gap junction connexin which is required for proper heart
development and heart electrophysiology. We generated
lentiviral constructs for Cx43 knockdown and Cx43
overexpression and used them to alter the level of Cx43 in
hMSCs. The effectiveness of these vectors was confirmed
by assessing Cx43 levels in hMSCs by Western Blot analysis
and by Real Time-PCR. These hMSCs were co-cultured
with NRVMs for up to 1 month on poly-lysine and collagen
I coated glass cover slips. Co-cultures containing MSCs
overexpressing Cx43 exhibited coordinated beating and
three-dimensional tube formation in 10 days, whereas 14 days
were required for control cells. The hMSCs were integrated
into these beating three dimensional tubes. No tubes were
observed in co-cultures with Cx43 knockdown hMSCs,
rather we observed the formation of small, unconnected
beating spheres after 30 days in co-culture. Human MSCCx43 knockdown cells were integrated into these spheres
and immunofluorescence staining demonstrated that Cx43
expression in hMSC-Cx43 knockdown remained reduced
when compared to the control group. Real Time-PCR
analysis using human specific primers showed significant
upregulation of KDR, smooth muscle actin, Pecam1, CD34,
CDH2, and CaCNA1C. Several genes, not initially seen in
hMSCs, including Gata4, CDH5, SCN5A, SLC8A1, and
KCNQ1, appeared in co-cultures with hMSCs with normal and
elevated expression levels of Cx43. However, in co-culture of
cardiomyocytes with Cx43 knockdown MSCs we observed
downregulation of KDR, CHD2, SCN5A, SLC8A1, KCNQ1
compare to control. These results suggest a strong correlation
between the presence of Cx43 and the ability of hMSCs to
differentiate into cardiac and endothelial lineages.
C. Sanina: None. C.O. Rodrigues: None. I.H. Schulman: None.
I. Margitich: None. W. Balkan: None. J.M. Hare: None.
P17Plasmonic Photothermal Therapy of Atherosclerosis with
the Use of Silica-Gold Nanoparticles and Mesenchymal
Stem Cells Versus Ferromagnetic Approach
Alexander Kharlamov, Alexander Perrish, Jan Gabinsky, Ural Inst of
Cardiology, Yekaterinburg, Russian Federation; Eugene Ivanova, High
Sch of Arnhem and Nijmegen, Nijmegen, Netherlands
Background. Some modern angioplasty techniques generally
just manipulate the form of the plaque and have some
clinical and technical restrictions, relatively high complication
rate and restenosis risk. Methods. A total of 101 Yucatan
miniature swines were assigned to the three groups (34
pigs into 60/15–70/40 nm silica-gold nanoparticles (NPs)
nanogroup, 34 swines — into ferro-magnetic group with 100
nm NPs with delivery in hand of magnetic fields, and 33 — in
sirolimus stenting control). Animals in nanogroup were split
up to 4 subsets by delivery approach: 1) infused circulating
progenitor cells; 2) with infused ultrasound-mediated albumin-
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Poster Presentations (continued)
coated gas-filled microbubbles; 3) CD73+CD105+ progenitor
cells in the composition of bioengineered on-artery patch;
4) CD73+CD105+ progenitor cells transplanted by manual
subadventitial injection. Results. A change of the PV (mm3)
immediately after the laser irradiation/ in 6 months in groups
were -28.9/ -56.8%, -30.8/ -59.1% and 0/ +4.3% (p;0.01)
respectively, and in the subsets in 6 mns reached -54.5/
-45.9/ -74.7/ -61.8% (p;0.05) respectively. Some cases of
atherothrombosis (3 cases in S2 subset) and distal embolism
(8/34, 23.5% in nanogroup, 2/33, 6% in control) were revealed
while first 4 weeks. Restenosis confirmed in 3 (9%) swines of
stenting group only. An impact over the non-organic part of the
plaque was predominated in nanogroup (-43.4% vs -12.1%
and +3.7% respectively, p;0.005). Coronary flow-mediated
vasodilation was observed after hyperemia and injection of
nitroglycerine (+10.2 and +16.6%, +8.2 and +9.6%, +8.1 and
+9.8% in groups respectively, p;0.05). The highest level of the
infiltration with NPs was achieved in the liver that equal to 612
units per cm3 without signs of fibrosis or allergic responses.
There were 344, 201 and 99 units per cm3 in the heart, brain,
and lungs respectively without any morphological dynamics.
Conclusion. Nanoburning is the novel promising technique to
demolish and melt the plaque especially in combination with
stem cell technologies challenging functional restoration of the
vessel. Plasmonics is the high-effective and safe alternative to
stenting for angioplasty.
A. Kharlamov: C. Other Research Support; Modest; International
Biomedical Research Center ÄGiKO, Yekaterinburg, Russia. A. Perrish:
C. Other Research Support; Modest; International Biomedical Research
Center ÄGiKO, Yekaterinburg, Russia. J. Gabinsky: None. E. Ivanova:
A. Employment; Modest; International Biomedical Research Center
“AGiKO”, Yekaterinburg, Russia.
P18 Withdrawn
P19Exosomes from Human CD34+ Cells: Critical Mediators of
Proangiogenic Paracrine Effect of EPCs
Susmita Sahoo, Sol Misener, Tina Thorne, Meredith Millay, Kathryn M
Schultz, Ting Liu, Aiko Ito, Christine Kamide, Ekaterina A Klyachko,
Douglas W Losordo, Northwestern Univ, Chicago, IL
S. Sahoo: None. S. Misener: None. T. Thorne: None. M. Millay:
None. K.M. Schultz: None. T. Liu: None. A. Ito: None. C. Kamide:
None. E.A. Klyachko: None. D.W. Losordo: None.
P20A Bioengineered Neonatal Cardiomyocyte Scaffold
Promotes Cell Survival and Improves Left Ventricular
Function in Rats with Heart Failure
Jordan Lancaster, Elizabeth Juneman, Nicholle Johnson, Joseph
Bahl, Steven Goldman, Southern Arizona VA HealthCare System,
Tucson, AZ
Background: Cell-based regenerative therapies hold
promise as a new treatment for heart failure. Tissue
engineered scaffolds used for cell delivery enhance potential
improvements in cardiac function by providing the structural
and nutrient support for transplanted cell survival, integration,
and re-population of injured tissues. Previously, our laboratory
reported improvements in left ventricular (LV) function in
rats with chronic heart failure (CHF) after placement of
a neonatal cardiomyocyte (NCM) seeded 3-dimensional
fibroblast construct (3DFC). In brief, 3 weeks after implantation
of the NCM-3DFC, LV function improves by increasing
(p<0.05) ejection fraction 26% and cardiac index 33%, while
decreasing (p<0.05) LV end diastolic pressure 38%. The
current report focuses on NCM survival and LV improvements
out to 7 weeks post NCM-3DFC implantation. Methods and
Results: Cardiomyocytes were isolated from neonatal rat
hearts and seeded onto a 3DFC. We evaluated NCM-3DFC
in vitro for cellular organization and the presence of functional
gap junctions, which demonstrated extensive cell-to-cell
connectivity. At 5 days in culture, the seeded patch contracted
spontaneously in a rhythmic and directional fashion, beating
at 43±3 beats/min with a mean displacement of 1.3±0.3 mm
and contraction velocity of 0.8±0.2 mm/sec. The seeded
patch could be electrically paced at near physiological rates
(270±30 beats/min) while maintaining coordinated, directional
contractions. For in vivo evaluation, rats underwent coronary
artery ligation and allowed to recover for 3 weeks to establish
CHF. NCM-3DFC were implanted 3 weeks after ligation and
evaluated 3 and 7 weeks later (6 and 10 weeks after ligation
respectively). Live cell tracking of implanted NCM using
Q-Dots revealed ~9% survival of transplanted cells 3 weeks
after implantation. In addition, improvements in LV function
continued at 7 weeks after implantation of the NCM-3DFC
by increasing (p<0.05) ejection fraction 37%. Conclusion: A
multicellular, electromechanically organized, cardiomyocyte
scaffold, engineered in vitro can improve LV function when
implanted directly on the hearts of rats with CHF; the
transplanted cells survive and improve LV function chronically.
ABSTRACTS
Local transplantation of human CD34+ hematopoietic stem
cells has been shown to promote neovascularization in preclinical studies in models of myocardial and limb ischemia.
In early phase clinical trials, transplantation of CD34+ cells
has been associated with reduced angina, improved exercise
time and reduced amputation rates. Several studies have
suggested that paracrine effects by these pro-angiogenic
cells mediate the effects induced by cell transplantation. We
hypothesized that CD34+ cells secrete exosomes (Exo), which
mediate at least a part of the therapeutic function of the cells.
Methods and Results: We isolated Exo from the conditioned
media of adult human peripheral blood (PB) CD34+ cells.
The angiogenic and therapeutic potency of CD34+ Exo was
compared with the intact CD34+ cells and also with PB
mononuclear cell (MNC) Exo. Exo from both CD34+ cells and
MNC are 50–90nm in size, have cup shaped morphology, and
carry known Exo-marker proteins such as CD63, TSG101
and Annexin V as shown by electron microscopy, Western
blot and flow cytometry. Compared to CD34+ cells or MNC
Exo, CD34+ Exo significantly induces in vitro angiogenic
activities such as viability, proliferation and tube formation
of HUVECs on matrigel- in a dose dependent manner. In
vivo, CD34+ Exo stimulated significant neovascularization in
mouse corneal angiogenesis assay (14±4 mm v MNC Exo,
4±1 mm, p<0.01) and incorporation of endothelial (CD31+)
cells in mouse matrigel-plug assay (6±1.7% v CD34+ cells,
2±0.8%, p<0.01). Finally, in a mouse model of hind limb
ischemia (HLI), CD34+ Exo significantly improved perfusion
(ratio: 1.01±0.04 v 0.57±0.1, P<0.05), increased capillary
density (1.8±0.3/HPF v 0.9±0.1/HPF, p<0.001) and prevented
ischemic leg amputation (16% v 100%), as compared with
MNC Exo. Conclusions: These data demonstrate that CD34+
Exo induce angiogenic activity and ischemic tissue repair in
the absence of CD34+ cells, and suggest that Exo represent
important mediators of the therapeutic effects associated
with CD34+ cell therapy. We speculate that Exo derived
from CD34+ cells may represent a significant component
of the paracrine effect of progenitor-cell transplantation for
therapeutic angiogenesis.
J. Lancaster: None. E. Juneman: None. N. Johnson: None. J. Bahl:
None. S. Goldman: None.
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25
Poster Presentations (continued)
Expression of more than 18% of the 11981 quantified
unigenes was significantly altered in the infarcted hearts.
BMNC therapy restored expression of 2099 (96.2%) of
the genes that were altered by infarction but led to altered
expression of 286 new genes, considered to be a side
effect of the treatment. Transcriptional therapeutic efficacy,
a new metric calculated using a formula that incorporates
both recovery and side effect of treatment, was 74.5%. In
conclusion, our results confirm a beneficial role for bone
marrow-derived cell therapy and provide new information on
molecular mechanisms operating after BMNC transplantation
on post ischemic heart failure in mice.
P21Adipose-Derived Stromal Cell Therapy Stabilizes Cardiac
Function and Improves Border Zone Remodeling After
Coronary Occlusion in Rats
Antonio C Campos de Carvalho, Luiza Bagno, João Pedro Werneck
de Castro, Patricia Oliveira, Marcia Abreu, Nazareth Rocha, Tais Kasai
Brunswick, Vivian Miranda, Regina Goldenberg, Federal Univ of Rio de
Janeiro, Rio de Janeiro, Brazil
Recent studies have identified adipose tissue as a new
source of mesenchymal stem cells for therapy. The purpose
of this study was to investigate the therapy with rat adipose
derived stromal cells (ASC) in a rat model of healed myocardial
infarction (MI). ASC from inguinal subcutaneous adipose tissue
of male Wistar rats were isolated by enzymatic digestion
and filtration. Cells were then cultured until passage 3. Four
weeks after ligation of the left coronary artery of female rats,
a suspension of either 100µl with PBS + Matrigel + 2 x 106
ASC labeled with Hoechst (n=11) or 100µl of PBS + Matrigel
(n=10) was injected along the borders of the ventricular wall
scar tissue. A sham operated group (n=5) was submitted to
the same surgical procedure except permanent ligation of left
coronary artery. Cardiac performance was assessed by electro
and echocardiogram. Echo was performed prior to injections
(baseline-BL) and six weeks after injections (follow-up — FU),
and values after treatment were normalized by values obtained
before treatment. Hemodynamic measurements were
performed 6 weeks after injections. All data are expressed as
mean ± SEM. Student’s paired or unpaired T test was used to
compare the same group in two different times or two distinct
groups, while two way ANOVA was used to compare more
than two groups along different times and p was set at <0.05.
All infarcted animals exhibited cardiac function impairment.
Ejection fraction (EF), shortening fractional area (SFA) and
left ventricular akynesia (LVA) were similar between infarcted
groups before treatment. Six weeks after therapy, ASC group
showed significant improvement in all three Echo indexes in
comparison to vehicle group. In non-anesthetized animals dp/
dt+ was also significantly higher in ASC when compared to
vehicle. In agreement with functional improvement scar area
was diminished in the ASC group. We conclude that ASC
stabilize cardiac function in infarcted rats when administered
directly to the myocardium.
ABSTRACTS
A.C. Campos de Carvalho: None. L. Bagno: None. J. Werneck de
Castro: None. P. Oliveira: None. M. Abreu: None. N. Rocha: None.
T. Kasai Brunswick: None. V. Miranda: None. R. Goldenberg: None.
P22Functional and Transcriptomic Recovery of Infarcted
Mouse Myocardium Treated with Bone Marrow
Mononuclear Cells
Adriana B Carvalho, Stephan Lachtermacher, Bruno Esporcatte,
Nazareth Rocha, Patricia Costa, Federal Univ of Rio de Janeiro, Rio
de Janeiro, Brazil; Dumitru Iacobas, Sanda Iacobas, David C Spray,
Albert Einstein Coll of Med, New York, NY; Regina Goldenberg, Antonio
Carlos Campos de Carvalho, Federal Univ of Rio de Janeiro, Rio de
Janeiro, Brazil
Although bone marrow-derived mononuclear cells (BMNC)
have been extensively used in cell therapy for cardiac
diseases, little mechanistic information is available to support
reports of their efficacy. To address this shortcoming, we
compared structural and functional recovery and associated
global gene expression profiles in post-ischaemic myocardium
treated with BMNC transplantation. BMNC suspensions were
injected into cardiac scar tissue ten days after experimental
myocardial infarction. Six weeks later, mice undergoing
BMNC therapy were found to have normalized antibody
repertoire and improved cardiac performance measured by
ECG, treadmill exercise time and echocardiography. After
functional testing, gene expression profiles in cardiac tissue
were evaluated using high-density oligonucleotide arrays.
26
A.B. Carvalho: None. S. Lachtermacher: None. B. Esporcatte: None.
N. Rocha: None. P. Costa: None. D. Iacobas: None. S. Iacobas: None.
D.C. Spray: None. R. Goldenberg: None. A. Campos de Carvalho: None.
P23HDAC Inhibition Promotes Cardiogenesis and the
Survival of Embryonic Stem Cells Through ProteasomeDependent Pathway
Ting C Zhao, Hongping Chen, Megan DeNicola, Roger Williams
Medical Ctr, Boston Univ Medical Sch, Providence, RI; Yu Zhao, Brown
Univ, Providence, RI; Xin Qin, Roger Williams Medical Ctr, Boston Univ
Medical Sch, Providence, RI; Ling Zhang, Shugang Zhuang, Brown
Univ, Providence, RI; Paul Liu, Roger Williams Medical Ctr, Boston Univ
Medical Sch, Providence, RI; Julio Ma, Brown Univ, Providence, RI
Objectives: Histone deacetylase (HDAC) inhibition plays a crucial
role in mediating cardiogenesis and myocardial protection,
whereas HDAC degradation has recently attracted attention in
mediating the biological function of HDACs. However, it remains
unknown whether HDAC inhibition modulates cardiogenesis
and embryonic stem cell (ESC) survival through the proteasome
pathway. Methods and Results: Using the well-established
mouse CGR8 mouse ESC culture, we evaluated the impact of
HDAC inhibition and proteasome pathway on the cell death,
viability and apoptosis in ESCs in response to oxidant stress
(100μmol/L hydrogen peroxides). We demonstrated that HDAC
inhibitors, both TSA (50 nmol/L) and sodium butyrate (200
μmol/L) that causes the pronounced reduction of HDAC4 activity,
decreased cell death and increased viability of ESCs. HDAC
inhibition reduced the cleaved caspase 3, 6, 9, PARP and TUNEpositive ESCs, which were abrogated with MG132 (0.5 μmol/L),
a specific proteasome inhibitor. Furthermore, we employed in
vitro “hanging drop” methods to carry out two weeks of embryoid
bodies (EB) culture to assess the effect of HDAC inhibition
and proteasome pathway on cardiogenesis. HDAC inhibition
stimulates the growth of EB, which is associated with faster
spontaneous rhythmic contraction. HDAC inhibition increased
the up-regulation of GATA4, MEF2, NKX2.5, cardiac actin, and
α-SMA mRNA and protein levels that were abrogated by MG132.
Immunostaining analysis demonstrates that trichostatin A and
sodium butyrate resulted in a significant increase in cardiac
lineage commitments that were blocked by the proteasome
inhibition. Notably, HDAC inhibitors led to noticeable HDAC4
degradation, which was effectively prevented by MG132.
Luciferase assay demonstrates an activation of MEF2 cardiac
transcriptional factor by HDAC inhibition, which was repressed
by MG132, revealing that the degradation of HDAC4 allows the
activation of MEF2. Conclusions: Taken together, our study
is the first to demonstrate that HDAC inhibition through
proteasome pathway forms a novel signaling to determine the
cardiac lineage commitment and elicit the survival pathway, .
which is dependent on specific HDAC4 degradation and
subsequent MEF2 activation of ESCs.
T.C. Zhao: None. H. Chen: None. M. DeNicola: None. Y. Zhao: None.
X. Qin: None. L. Zhang: None. S. Zhuang: None. P. Liu: None. J. Ma:
None.
This research has received full or partial funding support from the
American Heart Association, National Center.
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Poster Presentations (continued)
arteries (2 mm; rabbits and 5 mm; dogs) of the respective
animals. They were evaluated after determined period of
implantation. Results: Irrespective of species, BIOTUBEs
had thin wall (ca. 0.1mm) and mainly consisted of autologous
fibroblasts and collagen fibers. Rats; After 12-week
implantation, other than the oriented endothelial layer and
smooth muscle layer, multilayered elastin fiber formation was
observed in the grafts. Rabbits; Little thrombus was formed on
the luminal surfaces completely covered with endothelial cells
within 2 weeks. During 2 year-implantation, neither formation
of aneurysms nor rupturing was observed in BIOTUBEs. Dogs;
Longest follow up is 3 years under arterial pulsatile pressure
condition without any degenerative changes in the grafts.
Conclusions: BIOTUBEs could be ideal small caliber vascular
prostheses that greatly facilitate healing process and exhibit
excellent biocompatibility.
P24Not published at presenter’s request.
P25Modulatory Effect of Angiotensin II on Endothelial
Differentiation and Vasculogenic Capacity of Human MSCs
Michael Bellio, Cristina Sanina, Wayne Balkan, Claudia O Rodrigues,
Samirah Abreu Gomes, Ian McNiece, Joshua M Hare, Univ of Miami
Miller Sch of Med, Miami, FL; Ivonne Hernandez Schulman, Univ of
Miami Miller Sch of Med and VA Medical Ctr, Miami, FL
Human bone marrow-derived mesenchymal stem cell (MSC)based therapy holds great promise as a new approach for
cardiovascular regeneration. In heart failure, there is activation
of the renin-angiotensin system and increased formation of
angiotensin (Ang) II. We tested the hypothesis that Ang II
impairs endothelial differentiation and vasculogenic capacity
of human MSCs. MSCs were cultured in endothelial growth
media (EGM) and treated with vehicle, 1µM Ang II, or 10µM
Ang II for 7 days to assess by real time RT-PCR the expression
of two markers of endothelial differentiation, platelet endothelial
cell adhesion molecule (PECAM) and von Willebrand factor
(vWF). Expression of PECAM and vWF increased similarly
in each treatment group at day 7, suggesting endothelial
differentiation of MSCs. To assess vasculogenesis, MSCs were
cultured in EGM for 7 days and then plated on growth factor
reduced Matrigel for 5 hours under the following conditions:
vehicle, Ang II (1µM), Ang II + type 1 receptor (AT1R) inhibitor
(10µM losartan), or Ang II + type 2 receptor (AT2R) inhibitor
(0.1µM PD123177). The vascular index (VI) was determined by
multiplying the average number of endothelial tubes formed by
the average tube length. MSCs treated for 5 hours with 1µM
Ang II exhibited a reduced VI (84.5 ± 2.6% of vehicle treated
cells, P<0.05). Treatment with Ang II+AT1R inhibitor increased
VI (115 ± 4.9% of vehicle, P<0.05) whereas Ang II+AT2R
inhibitor decreased VI (77.1 ± 4.1% of vehicle, P<0.05).
Chronic treatment for 7 days in EGM with 1µM or 10µM Ang
II also showed decreased VI compared to vehicle (75.4 ±
5.4% and 60.7 ± 2% of vehicle, respectively, P<0.01). These
results reveal that both acute and chronic treatment with Ang
II produces a negative effect on the vasculogenic potential
of endothelial-differentiated human MSCs and suggests
opposing effects of AT1R and AT2R on vasculogenesis. In
conclusion, we propose that selective modulation of AT1R and
AT2R mediated pathways may serve as a promising means
for enhancing the regenerative capacity of human MSCs in
cardiovascular disease.
. Bellio: None. C. Sanina: None. W. Balkan: None. C.O. Rodrigues:
M
None. S.A. Gomes: None. I. McNiece: None. J.M. Hare: None.
I.H. Schulman: None.
Masashi Yamanami, Taiji Watanabe, Keiichi Kanda, Kyoto Prefectural
Univ of Med, Kyoto, Japan; Tomonori Oie, Keiichi Takamizawa, Natl
Cerebral and Cardiovascular Ctr Res Inst, Osaka, Japan; Hatsue
Ishibashi-Ueda, Natl Cerebral and Cardiovascular Ctr Hosp, Osaka,
Japan; Hitoshi Yaku, Kyoto Prefectural Univ of Med, Kyoto, Japan;
Yasuhide Nakayama, Natl Cerebral and Cardiovascular Ctr Res Inst,
Osaka, Japan
Objectives: There are actually no small-caliber synthetic
vascular grafts (< 6 mm) with acceptable patency rate for the
use of coronary bypass or peripheral vascular repair below
the knee in case the autologous vessels are not available.
We developed autologous small-caliber vascular grafts
“BIOTUBEs” using simple, safe and economical in vivo tissue
engineering. In this study, we summarize the development of
BIOTUBEs. Methods: Silicone rod molds (diameter: 1.5~5
mm, length: 20~50 mm) were placed into subcutaneous
pouches of Wister rats, Japan white rabbits or Beagle dogs.
After 1 month, BIOTUBEs formed around the molds were
auto-implanted to the aorta (1.5 mm; rats) or the carotid
M. Yamanami: None. T. Watanabe: None. K. Kanda: None. T. Oie:
None. K. Takamizawa: None. H. Ishibashi-Ueda: None. H. Yaku:
None. Y. Nakayama: None.
P27Preconditioning Cardiosphere-Derived Cells by Hypoxia
and Prolyl Hydroxylase Inhibitors to Induce HIF-Related
Metabolic Changes and C-Kit Expression
Suat Cheng Tan, Carolyn A Carr, Kar Kheng Yeoh, Lisa C Heather,
Lucy Ambrose, Chris Schofield, Kieran Clarke, Univ of Oxford, Oxford,
United Kingdom
Cardiosphere-derived cells (CDCs) decrease loss of cardiac
function following infarction, but the potential of CDC therapy
is limited due to the low retention rate and the time required
to obtain sufficient cells for transplantation. AIM: Here, we
aimed to precondition CDCs by culturing under hypoxia, or
with prolyl hydroxylase inhibitors (PHIs), to prepare the cells for
the hypoxic environment within the infarcted heart. Methods:
Rat heart explants were cultured under hypoxia (2% O2) or
normoxia (21% O2) to generate explant-derived cells (EDC).
EDCs were isolated, cultured to form cardiosphere and
expanded into monolayer CDCs. At 80% confluency, normoxic
CDCs were treated with dimethyloxalyl glycine (DMOG), Ethyl
2-(2,3-dihyroxybenzamido) (KKC226) and FG-2216 for 24 h.
RESULTS: Hypoxia, 1 mM DMOG, 0.5 mM KKC226 and 30
μM FG-2216 treated CDCs showed significantly increased
HIF-1α protein expression (3.9-fold, 2.6-fold, 2.1-fold and 1.5fold, respectively) and reduced oxygen consumption (81%,
32%, 47% and 35%, respectively), compared with normoxic
CDCs. Hypoxia and PHI treatment increased protein levels of
GLUT-1 (6.7-fold, 3.2-fold, 4.0-fold and 2.1-fold, respectively),
resulting in significant increases in glucose uptake and
lactate accumulation in the culture medium, compared
with controls, commensurate with increased glycolytic
metabolism after hypoxia or PHI treatment. Further, hypoxia
and KKC226 increased c-Kit mRNA expression 5.1-fold and
1.5-fold, respectively. qRT-PCR confirmed increased CXCR-4
mRNA in all hypoxia and PHI-treated CDCs. EDC and CDC
proliferation were 1.7-fold faster under hypoxia, compared
with normoxia, but did not increase with PHI treatment.
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
P26Development of Autologous Tissue Small-Diameter
Vascular Grafts (BIOTUBEs).
27
Poster Presentations (continued)
Conclusion: Hypoxia and PHIs stabilized and activated HIF,
which induced metabolic changes in CDCs, including GLUT-1
upregulation and a switch to anaerobic glycolytic metabolism.
All preconditioned cells had reduced oxygen consumption and
were better adapted to survive within an hypoxic infarct scar.
HIF-induced upregulation of CXCR-4 may increase the homing
of these cells to the infarcted myocardium. Finally, increased
proliferation and expression of the cardiac stem cell marker,
c-Kit, could decrease the time required for cell expansion prior
to therapy by a week.
S. Tan: None. C.A. Carr: None. K. Yeoh: None. L.C. Heather: None.
L. Ambrose: None. C. Schofield: None. K. Clarke: None.
P28The Effect of Cotransplantation of Human Embryonic
Stem Cell-Derived Cardiomyocytes and Mesenchymal
Stem Cells on Ventricular Function and Remodeling After
Myocardial Infarction in Nude Rats
ABSTRACTS
Wangde Dai, Heart Inst of Good Samaritan Hosp, Los Angeles, CA;
Mary Kearns-Jonker, Children’s Hosp Los Angeles, Los Angeles,
CA; Paul Gerczuk, Heart Inst of Good Samaritan Hosp, Los Angeles,
CA; Mirja Gunthart, Nandini Girish, Children’s Hosp Los Angeles,
Los Angeles, CA; Martin Pera, Broad Inst for Stem Cell Res, Keck
Sch of Med, Univ of Southern California, Los Angeles, CA; Christine
Mummery, Leiden Univ Medical Ctr, Leiden, Netherlands; Robert A
Kloner, Heart Inst of Good Samaritan Hosp, Los Angeles, CA
We determined whether co-transplantation of human
embryonic stem cell-derived cardiomyocytes (hESC-CMs)
and mesenchymal stem cells (MSCs) had additive effects on
left ventricular (LV) function and remodeling compared with
hESC-CMs treatment alone in a rat myocardial infarction
model. One week after myocardial infarction induced by left
coronary ligation, nude rats received hESC-CMs (n=15),
hESC-CMs + MSCs (n=16), hESC-CMs + MSCs transduced
to over-express hemeoxygenase 1(HO-1) (n=14), or saline
(n=19). At 4 weeks after treatment, LV function was assessed
by left ventriculography, echocardiography and Millar catheter.
Some hearts were processed for histology. The LV ejection
fraction (LVEF) in sham noninfarcted hearts was 78.1±1.8%
(n=5) in the nude rat model. LVEF in the 3 cell treated groups
(hESC-CMs: 67.6±1.4%; hESC-CMs + MSCs: 67.2±1.6%;
and hESC-CMs + MSCs with HO-1: 66.3±1.7%) were
comparable, and significantly higher than in the saline group
(60.6±1.2%, n=19; p=0.0022). There was a trend for less
left ventricular akinesis and dyskinesis (expressed as %
of LV circumference) assessed by left ventriculography at
8.96±1.9% in hESC-CMs group, 8.37±1.67% in hESC-CMs
+ MSCs group and 4.57±1% in hESC-CMs + MSCs with
HO-1 group compared to 10.73±1.76% in the control group
(p=0.056). There was a nonsignificant trend for LV fractional
shortening assessed by echocardiography to be greater in
the 3 cell groups (32.1±3.9% in hESC-CMs; 30.2±2% in
hESC-CMs + MSCs; 31.0±1.9% in hESC-CMs + MSCs with
HO-1) compared to 24.8±2.2% in the saline group (p=0.18).
Expansion index reflecting thinning and dilatation of the infarct
was significantly worse in the control group at 0.71±0.05
versus the other 3 groups at 0.32±0.05 (p=0.0039). Thus, cell
therapy by hESC-CMs alone or combination transplantation
of hESC-CMs and MSCs (with or without HO-1) significantly
improved LV function assessed by left ventriculography
and reduced expansion index. However, co-transplantation
of hESC-CMs and MSCs did not provide better functional
improvement compared with hESC-CMs treatment alone after
left coronary artery occlusion in nude rats over a period of 4
weeks, suggesting that there may be a ceiling effect above
which LV function can not further improve after cell therapy.
W. Dai: None. M. Kearns-Jonker: None. P. Gerczuk: None.
M. Gunthart: None. N. Girish: None. M. Pera: None. C. Mummery:
None. R.A. Kloner: None.
28
P29Resolving Discordance of Human Cardiomyogenesis
Estimates in the Healthy Adult Heart
Jeremy Elser, Kenneth Margulies, Univ of Pennsylvania, Philadelphia, PA
Rationale: Estimates of the endogenous cell turnover rates
in healthy human hearts conflict, varying from 0% to over
30%. Turnover rate age-dependence is also controversial
with some studies indicating decreasing turnover with
advancing age and other studies suggesting that turnover
increases with age. Methods/Results: We created a hybrid
mathematical model encompassing two prominent analyses
of human adult cardiomyogenesis that concluded highly
disparate turnover rates. The model was programmed to
accept input parameters for age-dependent cardiomyogenesis
and apoptosis. High-turnover input parameters (10%–30%,
increasing with age), extracted from a recent publication
by Kajstura, et. al., were used to create 12 model patient
hearts with ages and birthdates corresponding to the 12
patients analyzed by a C14 fate-mapping technique in a
publication by Bergmann, et. al., which reported low turnover
rates of 1%–0.45%, decreasing with age. Modeled hearts
parameterized with high, age-increasing input turnovers
produced low (5%–0.5%), age-decreasing estimates of
turnover when the C14 analysis technique was applied. This
systematic underestimation reflects the inability of fatemapping models to account for turnover events in which
myocytes created after patient birth are destroyed prior to
patient death. This effect may underestimate true turnover
events by over 90% and is more severe for patients with
longer lifespans, predisposing such techniques to report
decreasing turnover with advancing age. We next evaluated
the effect of input parameter uncertainty on the hybrid model.
We observed non-linear sensitivity of fate-mapping algorithms
to polyploidization correction factors, with 20% variation in
correction factor leading to approximately 30% changes in
reported turnover. The Kajstura model was acutely sensitive to
estimates of myocyte half-life (Δ20% produced a 45% change
in average myocyte age) and stem cell expansion coefficient
(Δ20% produced a 2-fold change in reported turnover).
Conclusions: Accounting for short-lived myocytes in fatemapping models reconciles apparent differences between the
models. Recognition of realistic input parameter uncertainty
improves interpretation of these models.
J. Elser: None. K. Margulies: None.
P30Not published at presenter’s request.
P31Enhanced Cardiac Progenitor Cell Function and
Differentiation in a Naturally Derived Extracellular Matrix
Kristin M French, Emory Univ, Atlanta, GA; Jessica A DeQuach,
Karen L Christman, Univ of California, San Diego, San Diego, CA;
Michael E Davis, Emory Univ, Atlanta, GA
Cardiovascular disease, including myocardial infarction,
is a leading cause of death worldwide. Though several
pharmacological treatments for severe dysfunction exist,
much recent work has focused on the transplantation
of adult-derived stem and progenitor cells. Early acute
functional improvements have been noted, however longterm clinical efficacy is hampered by poor cell survival and
engraftment. While the current treatment is infusion into
the coronary artery, biomaterials may play an important
role in modulating implanted cell function. This work aims
to establish the role that a naturally derived extracellular
matrix plays in the differentiation of cardiac progenitor cells
(CPCs) and their potentially protective enzymatic systems.
To test this hypothesis, we cultured rat CPCs in a naturally
derived porcine ECM (pECM) and compared it to Collagen I.
Quantitative real-time PCR was used to assess expression of
cardiac, endothelial and smooth muscle markers. Additionally,
Become an AHA/ASA Professional Member and save up to $300 off next year’s registration. (Promo code MBJ050ZZ)
Poster Presentations (continued)
angiotensin receptor (AT1R and AT2R) and antioxidant gene
expressions were evaluated to determine the protective
qualities of pECM. Preliminary data at 2 days following LIF
removal demonstrate an increase in the expression of cardiac
lineage markers (Nkx-2.5, Gata-4, α-MHC, and troponin I)
in pECM compared to collagen. Smooth muscle markers,
smooth muscle α-actin and sm22α as well as the endothelial
marker Flk1 were also increased in pECM samples. Increased
expression was also seen for antioxidant genes GPX1, SOD1,
SOD2 and catalase in pECM cultured cells. Culturing in pECM
for 7 days demonstrated an increase in Flt-1 and α-myosin
heavy chain, indicating a potential increase in cardiogenesis.
Moreover a 60% reduction in AT1R gene expression was
observed with no significant change in AT2R expression. Our
data demonstrate that culturing CPCs in naturally derived
matrices may provide protection and enhance differentiation
compared to collagen (present in high amounts in scarred
myocardium). Future work will further elucidate this protective
effect of AT1R downregulation and antioxidant increases in
functional studies. In conclusion, pECM may be a potential
cell delivery scaffold in post-MI treatment given its protective
nature and improved differentiation influence.
K.M. French: None. J.A. DeQuach: None. K.L. Christman: None.
M.E. Davis: None.
P32Nanoparticle Mediated Nox2-siRNA Therapy for Preventing
Cardiac Dysfunction Following Myocardial Infarction
Inthirai Somasuntharam, Niren Murthy, Michael Davis, Georgia Inst of
Technology, Atlanta, GA
I. Somasuntharam: None. N. Murthy: None. M. Davis: None.
P33β-Adrenergic Signaling Mediates Cardiac Stem Cell Survival
Mohsin Khan, Sadia Mohsin, Daniele Avitabile, Natalie Gude, Silvia
Truffa, San Diego State Univ, San Diego, CA; Walter J Koch, Thomas
Jefferson Univ, Philadelphia, PA; Mark A Sussman, San Diego State
Univ, San Diego, CA
Rationale: Adrenergic activity serves to increase acute
cardiac contractility but chronic adrenergic drive is
detrimental. Conventional therapy relies heavily on blockade
of the β-adrenergic system; however, the cellular basis for
improvement resulting from the treatment with β-Blockers
remains obscure. Objective: Demonstrate that the beneficial
effect of long term β-blockade therapy is dependent, in part,
upon restoration of regenerative capacity for the endogenous
cardiac stem cell (CSCs) population. Methods and Results:
Mouse CSCs express only β2 adrenergic receptor (β2-AR) in
conjunction with stem cell marker c-kit as assessed by qRTPCR and immunoblot. Activation of β2-AR signaling by the
specific β2-agonist fenoterol (1µM) improves proliferation and
increases protective signaling associated with elevated levels
of pAkt/Akt, eNOS, cyclin D and decreased levels of GRK2.
Conversely, CSCs treated with β2-AR antagonists (siβ2-AR,
ICI118, 551) exhibit impaired proliferation. Although CSCs
lack β1-AR under normal proliferative conditions, co-culture of
CSCs with neonatal rat cardiomyocytes promotes expression
of β1-AR concurrent with loss of c-kit and expression
of lineage commitment cardiac markers such as cTnT,
SERCA2a, MLC-2v, SMA as measured by species-specific
primers with qRT-PCR. Co-cultured CSCs became sensitized
to isoproterenol-induced cell death that is abrogated by
administration of the specific β1-antagonist (metoprolol, 1µM).
Involvement of β1-AR in catecholamine-induced apoptosis
was further confirmed by genetically engineering CSCs with
βARK-ct-GFP, a GFP fused version of the peptide shown
to rescue cardiac function by normalizing β-adrenergic
signaling. βARK-Ct over-expressing CSCs acquired β1-AR
after co-culture but were protected against isoproterenolinduced cell death. Conclusion; β2-AR signaling leads to CSC
activation and proliferation that is beneficial, but induction
of cellular differentiation leads to expression of β1-AR that
is proapoptotic when stimulated with adrenergic treatment.
The apoptotic effect of β1-AR signaling in CSCs can be
mitigated by engineering of cells with βARK-ct, providing
a novel molecular strategy to promote regeneration in the
pathologically injured myocardium.
. Khan: None. S. Mohsin: None. D. Avitabile: None. N. Gude:
M
None. S. Truffa: None. W.J. Koch: None. M.A. Sussman: None.
P34Bone Marrow-Derived Cells Are Not an Essential
Component in Cardiosphere Formation
Jianqin Ye, Andrew Boyle, Yerem Yeghiazarians, Univ of California,
San Francisco, San Francisco, CA
Background: Cardiospheres (CS) are composed of
heterogeneous population of cells but it is unknown whether
bone marrow derived cells are an essential cell component
in CS formation. Methods: Chimera mice were generated by
transplantation of bone morrow cells from GFP transgenic
mice to irradiated C57BL mice. Mice were randomized into 3
groups 5 months after transplantation: 1) myocardial infarction;
2) sham operated; 3) un-operated (n=5/group). Hearts were
harvested 2-weeks post-surgery. Cardiac explants were
cultured and putative cardiosphere forming cells (CFCs)
(small cells migrating out from the explants) were collected
14 days later and reseeded on new culture dishes for CS
formation. The number of CS from each heart was counted
at 3 days. CS cell composition was analyzed by FACS. To
further analyze the role of bone marrow derived CD45+ cells
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
Myocardial infarction (MI) is the most common cause of heart
failure in the developed world. Following MI, reactive oxygen
species (ROS) play a key role in the pathogenesis of cardiac
remodeling leading to impaired ventricular function. Oxidative
stress at high levels lead to many of the injury associated
changes: proinflammatory cytokine release, myocyte
apoptosis, cardiac fibrosis and hypertrophy. Nicotinamide
adenine denucleotide phosphate (NADPH) oxidase with Nox2
as the catalytic subunit, is a major source for cardiac ROS
production. After MI, Nox2 expression is significantly increased
in the infarcted myocardium. Moreover, mice lacking the Nox2
gene are protected from ischemic injury. We used polyketals,
a new class of acid-degradable polymers, as delivery vehicles
for Nox2-siRNA to the post-MI environment. When engaged
by macrophages, present in high quantities during MI, these
particles have been shown to be taken up by macrophages
and contents released within cells in active form. Nox2-siRNA
was ion-paired to the cationic lipid DOTAP, and spherical
particles averaging 500nm diameter were made by a single
emulsion procedure with polyketal PK3 using PVA as
surfactant. While a commercially available transfection reagent
yielded 7% uptake as measured by flow cytometery, 81% of
macrophages were positive for fluorescently-labeled siRNA
in PK3-siNOX2 treated cells. These data were confirmed
with confocal microscopy. Macrophages treated with PK3siNOX2 demonstrated a significant 43% knockdown in Nox2
gene expression at 24 hours, while no reduction was seen
with scrambled siRNA or empty particles. Functional activity
was assessed by a fluorescent dihyroethidium dye based
HPLC quantification after phorbol 12-myristate 13-acetate
stimulation following 72 hours of particle treatment to measure
superoxide production. PK3-siNox2 treated cells exhibited
a 41% reduction in activity, with no significant changes seen
with scrambled siRNA or empty particle treatment. Currently,
the therapeutic potential of the Nox2-siRNA particles is
being evaluated in a mouse model of ischemia-reperfusion.
Successful completion of these studies could lead to a novel
treatment for post-infarction injury.
29
Poster Presentations (continued)
in forming CS, CD45+ cells was isolated from CFCs by CD45
antibody coated immunomagnetic beads. The number of CS
formed from 1x105 putative CFCs, CFCs without CD45+ cells
and CD45+ cells from CFCs (n=6-9/cell type) respectively
were also counted at 3 days in culture. Results: Compared
to sham (122± 23/heart) and un-operated hearts (18± 5/
heart), infarcted hearts formed more CS (357± 64/heart,
P<0.01). In all groups, irrespective of any surgery, 18.4± 4.5%
of cells in CS co-expressed GFP and CD45, indicating they
originated in bone marrow. Low percentage of bone marrow
stem/progenitor cells (3.9% Sca-1+GFP+CD45+ and 1%
c-Kit+GFP+CD45+ cells) were detected in CS, but a high
percentage of cells within CS were cardiac stem/progenitor
cells (26.3± 9.4% cells were Sca-1+GFP-CD45-, 0.10± 0.04%
c-Kit+GFP-CD45-). Depleting CD45+ cells from putative CFCs
actually increased the formation of CS (67±10 CS/1x105
cells) compared to un-depleted CFCs (51± 6 CS/1x105 cells,
P<0.0001). Purified CD45+ cells from CFCs did not form CS
in culture. Conclusion: Myocardial infarction increases the
formation of CS in culture. Bone marrow derived CD45+ cells
make up a small percentage of CS, but are not necessary for
CS formation.
J. Ye: None. A. Boyle: None. Y. Yeghiazarians: None.
P35The Nucleolar Protein Nucleophosmin Regulates Mitosis
Progression in Cardiac Progenitor Cells
ABSTRACTS
Daniele Avitabile, Balaji Sundararaman, Silvia Truffa, Mark A Sussman,
San Diego State Univ, San Diego, CA
Besides its notorious function during ribosome biogenesis,
emerging evidence suggests a central role for the nucleolus
in controlling many other important cellular processes such
as stress response, proliferation and mitosis. Here we show
that the nucleolar protein Nucleophosmin (NPM) is necessary
for the correct progression of mitosis in primary cultures
of cardiac progenitor cells (CPCs). Methods: CPCs were
cultured on Permanox chamber slides at high confluence
and fixed with 4% PFA. CPCs were blocked in cytokinesis
with 2μM cytochalasin B (CytB) for 18 hours. Silencing of
NPM (ShNPM) or scramble (ShSc) was performed by specific
shRNA expressing lenti viral vectors. Confocal microscopy,
co-immunoprecipitation and proximity ligation assay (PLA),
FACS analysis and Cyquant assay were employed to study
protein localization and interaction, cell death and proliferation
respectively. Results: ShNPM infected CPCs showed
reduced proliferation with no apparent cell death compared
to scramble. Cells in prometa and metaphase significantly
increased after NPM knock down. ShNPM-CPC showed
50% decrease in the number of cells that complete mitosis as
assessed by counting binucleated cells after CytB treatment.
NPM localized in the nucleolus during interphase as assessed
by co-staining with nucleostemin (NS), a highly expressed
nucleolar protein. NS and NPM decorate the chromosome
periphery from prometaphase to anaphase and return to
the newly forming nucleolus at the end of telophase. The
phosphorylated form of NPM at threonine 199 (pNPM) was
found to co-localize with spindle components such us NUMA
and α-tubulin specifically at methaphase. NPM was also
found to co-precipitate and directly interact with α-tubulin
as shown by in-situ protein interaction assay (PLA) in the
cytoplasm during interphase and at the centrosome and
spindle during mitosis. Conclusion: This is the first study to
detail the role of nucleolar proteins during CPCs mitosis. Our
findings demonstrate a central role for NPM during metaphase
transition, possibly thorough interaction with components of
the spindle and in particular with α-tubulin.
30
. Avitabile: None. B. Sundararaman: None. S. Truffa: None.
D
M.A. Sussman: None.
P36Regional Characterization of Myocardial Renewal
in Humans
Olaf Bergmann, Sofia Zdunek, Karolinska Instt, Stockholm, Sweden;
Mehran Salehpour, Uppsala Univ, Uppsala, Sweden; Samuel Bernard,
Univ Claude Bernard Lyon 1, Villeurbanne cedex, France; Staffan
Sjöström, Karolinska Instt, Stockholm, Sweden; Karl Håkansson,
Uppsala Univ, Uppsala, Sweden; Kanar Alkass, Henrik Druid,
Karolinska Instt, Stockholm, Sweden; Göran Possnert, Uppsala Univ,
Uppsala, Sweden; Jonas Frisén, Karolinska Instt, Stockholm, Sweden
We and other groups have reported cardiomyocyte renewal
in adult human hearts. However, there is no consensus
about magnitude and regional heterogeneity in myocardial
turnover. Important requirements to quantify cellular turnover
are the accurate identification of cardiomyocytes and ploidy
level. We present a regional analysis of turnover in the adult
myocardium, using the integration of atmospheric radiocarbon
(14C) into genomic myocardial DNA. Cardiomyocyte nuclei
were labeled with pericentriolar protein 1 (PCM-1) and
isolated by flow cytometry. In order to improve estimates on
cardiomyocyte turnover, nuclei were isolated according to their
DNA content. A substantial fraction of cardiomyocyte nuclei
in the right and left ventricle were polyploid: 28.9%±10.6%
diploid, 58.9%±8.5% tetraploid and 12.1%±7.4% octaploid
in the left ventricle and 47.9%±10.6% diploid, 46.6%±8.3%
tetraploid and 5.6%±3.2% octaploid in the right ventricle.
Cardiomyocyte turnover was analyzed according to their
ploidy distribution in the left and right ventricle. Data from
this ongoing study indicates a limited regenerative potential
in both ventricles. A comprehensive mathematical analysis
of regional turnover distribution will be presented. Noncardiomyocytes were in average 18.0±2.3 years younger than
the respective subjects, indicating a substantial turnover in
the whole myocardium. The human heart has the capability
to regenerate cardiomyocytes. However, the magnitude of
this process does not allow for the renewal of the whole
myocardium under homeostatic condition in a lifetime.
Understanding the underlying mechanisms of cardiac renewal
will open up new avenues to treat cardiac injuries.
O. Bergmann: None. S. Zdunek: None. M. Salehpour: None. S. Bernard:
None. S. Sjöström: None. K. Håkansson: None. K. Alkass: None. H.
Druid: None. G. Possnert: None. J. Frisén: None.
P37Viable Autologous Umbilical Cord Patch for Aortic
Reconstruction in Newborn Piglets
James M Hammel, Haili Lang, Univ of Nebraska Medical Ctr, Omaha,
NE; Rebecca Siecke, Children’s Hosp and Medical Ctr, Omaha, NE;
Stan Radio, Univ of Nebraska Medical Ctr, Omaha, NE
Background: Aortic surgery in neonates often uses a patch
of cryopreserved allogeneic artery. Drawbacks include
alloimmunization in infants who may later need heart
transplant. We hypothesized that the umbilical cord (UC)
may be a non-immunogenic, viable vascular patch suited to
neonatal aortic surgery. This was evaluated in ex vivo studies
of human UC, and in vivo study in piglets. Method: Human
UC (20) were collected after vaginal birth, were dilated and
measured. Swab cultures were taken. For final culture UC
was put in Thiol, and held for 4 wk. Disinfection was in dilute
hypochlorite for 30 sec to 5 min. UC segments were stored in
HTK solution with antibiotic/antifungal agent at 4 deg for 3 to
9 d. Samples were fixed for histology. At 9 d, samples were
put in tissue culture to check viability. Then, 18 farm piglets
in three litters were delivered by hysterotomy. Each UC was
sanitized, cultured, and stored as described for human UC.
At 7 d, each piglet underwent laparotomy. A defect made
in the abdominal aorta was patched with autologous UC or
with PTFE. Piglets were killed after 8 and 12 wk to examine
the patched aorta. Result: Human UC diameter was 8–11
mm. Cultures of 5 UC in Dakins for 30 sec, 1, 2, 3, and 4 min
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Poster Presentations (continued)
were all negative. All later UC were treated for 30 sec only.
One culture from one UC segment after 9 d was positive; final
culture from this UC wwas negative. Histology did not differ at
9 d from day of delivery (n=3). Cells grew from 9 d UC in tissue
culture (n=2). Piglet UC at 7 d also held viable cells. Piglet UC
patch worked well in surgery. Hemostasis was rapid with UC,
and was longer with PTFE. Three piglets were killed before
8 wk due to hernia. There was no dehiscence, infection, or
aneurysm in either group. UC-patched aorta lengthened
114%, PTFE grew 21%. Patched aorta was non-stenotic
in both groups, 92% of normal in UC vs. 95% in PTFE. By
histology the UC did not remain viable, but calcified and
broke into fragments. Beneath both UC and PTFE patches,
fibroblast proliferation spanned the aortic defect. In UC piglets,
this layer enlarged after UC fragmentation. Conclusion: UC
can be sterilized and kept viable after vaginal birth . UC patch
worked well for repair of an aortic defect in piglets, but UC
viability and growth were not seen. UC needs further study
before trial in human aortic surgery.
J.M. Hammel: None. H. Lang: None. R. Siecke: None. S. Radio: None.
P38Cardiomyocyte Proliferating Chemicals: Activation of
Proliferation of ESC/iPSC-Derived Cardiomyocytes
Hideki Uosaki, Jun K Yamashita, Kyoto Univ, Kyoto, Japan
H. Uosaki: None. J.K. Yamashita: None.
P39Encapsulation of Human Mesenchymal Stem Cells in
Alginate Microspheres to Enhance Paracrine-Mediated
Vascular Recovery
Natalia Landázuri, Rebecca D Levit, Susan A Safley, Collin J Weber,
Emory Univ, Atlanta, GA; Athanassios Sambanis, Georgia Inst of
Technology, Atlanta, GA; W Robert Taylor, Emory Univ, Atlanta, GA
Bone marrow cells migrate to ischemic areas and facilitate
revascularization mostly through paracrine mechanisms. As a
result, therapeutic strategies for vascular repair have focused
N. Landázuri: None. R.D. Levit: None. S.A. Safley: None.
C.J. Weber: None. A. Sambanis: None. W. Taylor: None.
P40Mst1 Physically Interacts with and Phosphorylates Beclin1,
Thereby Negatively Regulating Autophagy in the Heart
Yasuhiro Maejima, Shiori Kyoi, Peiyong Zhai, Junichi Sadoshima,
Univ of Med and Dentistry of New Jersey, Newark, NJ
Autophagy mediates protein degradation and participates
in protein quality control (QC). Inhibition of autophagy below
physiological levels induces protein aggregation and cellular
dysfunction. Beclin1 is a BH3-only protein that binds to Bcl-2.
Bcl-2 negatively regulates autophagy through interaction
with Beclin1. Here we show that the interaction between
Bcl-2 and Beclin1 is regulated by mammalian STE 20 likekinase-1 (Mst1), a proapoptotic kinase, in the heart. In Mst1
transgenic mice (Tg-Mst1), a model of dilated cardiomyopathy,
accumulation of aggresomes was significantly increased and
p62/SQSTM1, a protein degraded by autophagy, accumulated
markedly in the myocardium. Autophagosome formation, as
evaluated by GFP-LC3 dots, was suppressed in Tg-Mst1. The
activity of Mst1 was suppressed by amino acid deprivation
(AD) in cultured cardiomyocytes (CMs), and restoration of
Mst1 activity attenuated AD-induced autophagy, as evaluated
by LC3-II/ LC3-I and accumulation of p62/SQSTM1. Mst1
inhibited the kinase activity of the Beclin1-Vps34 (Class III
PI3K) complex in CMs. ABT-737, a BH3 mimetic compound,
reversed the Mst1-induced suppression of autophagy. Mst1induced suppression of autophagy was alleviated when Bcl-2
were downregulated in CMs. Mst1 physically interacts with
Beclin1 and stimulation of Mst1 enhances the interaction
between Beclin1 and Bcl-2 in CMs. In vitro kinase assays
using recombinant Beclin1 as substrate showed that Mst1
phosphorylates Beclin1. Using mass spectroscopy, we found
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ABSTRACTS
Cardiomyocytes derived from embryonic stem cells (ESCs)
and induced pluripotent stem cells (iPSCs) are promising cell
sources for cardiac regeneration. Previously, we established
a novel systematic cardiovascular cell induction system with
mouse ESCs and iPSCs. ESC/iPSC-derived cardiomyocytes
rarely proliferate after differentiation, similar to neonatal and
adult cardiomyocytes. Cardiomyocyte proliferation is highly
restricted and the regulating machineries of proliferation
and growth arrest of cardiomyocytes are long-standing
mysteries of cell biology. In order to establish cardiac
regenerative medicine, it is critical to dissect and manipulate
the machineries involved. Here we devised a novel approach
using small molecules in an attempt to unravel the mystery
and to manipulate ESC/iPSC-derived cardiomyocyte
proliferation. We screened a chemical library containing
well-established kinase inhibitors with high content screen to
enhance mouse ESC-derived cardiomyocyte proliferation, and
identified two novel chemical groups, extracellular signalregulated kinase activators and Ca2+/calmodulin-dependent
protein kinase II inhibitors, and two previously reported
chemical groups, glycogen synthase kinase-3 inhibitors and a
p38 mitogen-activated protein kinase inhibitior. Each chemical
increased actual cardiomyocyte cell numbers two- to threefold compared to control. An optimal combination of these
chemicals strongly enhanced proliferation of ESC-derived
cardiomyocytes and ESCM number was reached up to 14fold. Expanded cells retained various functional and structural
features of cardiomyocytes. These chemicals are robustly
effective on cardiomyocytes from various sources including
human iPSCs. Efficient combination of stem cell and chemical
biology demonstrated a novel molecular mechanism of
cardiomyocyte proliferation and offered a critical technological
basis for cardiac regeneration.
on increasing the concentration of these cells at the site of
ischemia by direct injection. These strategies have led to
encouraging results in clinical trials, yet the number of cells
that remain at the site of delivery is very low. The goal of this
study is to design an effective cell delivery system to enhance
revascularization by maintaining large numbers of cells at the
ischemic site, thus increasing the local delivery of angiogenic
factors. To achieve this goal, we encapsulated bone marrow
derived human mesenchymal stem cells (hMSCs) in nondegradable, barium-gelled alginate spheres prior to delivery
to the ischemic site. Our microspheres are biocompatible,
do not generate a fibrotic response, and are stable for
extended periods of time. They prevent incorporation of
cells into the neighboring tissue and protect implanted cells
from immune mediated clearance or wash out by the host
capillary and lymphatic systems. The microspheres allow
diffusion of molecules as large as 80 kDa (which includes most
angiogenic factors) but not of larger molecules such as IgG.
To test this cell delivery system in vivo, we used a model of
murine hind limb ischemia in athymic nude mice. We delivered
encapsulated hMSCs, or, as a control, empty capsules, to
the ischemic muscle. At various time points, we retrieved
the capsules by gentle scooping, then assessed the viability
of the encapsulated cells and their protein secretion profile.
Approximately 70% of the cells remained viable within the first
week and 10–20% within the first month after implantation.
In addition, encapsulated cells secreted angiogenic factors
(VEGF, bFGF, HGF, angiogenin) in vitro and in vivo. We also
assessed recovery of perfusion in the hind limb and found that
delivery of encapsulated cells enhanced vascular recovery
as compared to empty capsules. The results obtained from
this study show that microcapsules are an effective delivery
vehicle of therapeutic cells for vascular repair. Encapsulation
represents a powerful and highly translatable tool to enhance
cell-based therapies in cardiovascular disease.
31
Poster Presentations (continued)
that Mst1 phosphorylates Beclin1 at Thr108 that located in
its BH3 domain. Transduction of the Beclin1 Thr108Asp
mutant adenovirus (Ad-T108D) attenuated AD-induced
autophagy in CMs, and injection of Beclin1 Thr108Ala
mutant adenovirus (Ad-T108A), but not Ad-T108D, restored
starvation-induced LV dysfunction (LVEF: Ad-LacZ=0.51±0.05,
Ad-T108D=0.53±0.04, Ad-T108A=0.66±0.05, p<0.05) in
beclin1-/+ mice. Collectively, these results suggest that Mst1
inhibits autophagy through phosphorylation of Beclin1,
enhancement of Beclin1-Bcl-2 interaction, and suppression
of Vps34. Activation of Mst1 by stress suppresses autophagy
below physiological levels and inhibits protein QC, which in
turn may contribute to cardiac dysfunction.
Y. Maejima: None. S. Kyoi: None. P. Zhai: None. J. Sadoshima: None.
This research has received full or partial funding support from the American
Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts,
New Hampshire, New Jersey, New York, Rhode Island, Vermont).
P41A Novel Preclinical Strategy for Identifying Cardiotoxic
Kinase Inhibitors and Mechanisms of Cardiotoxicity
Hui Cheng, Gabor Kari, Ulrich Rodeck, Adam Dicker, Thomas Force,
Thomas Jefferson Univ, Philadelphia, PA
ABSTRACTS
Rationale: 1) Despite intense interest in strategies to predict
which tyrosine kinase inhibitor (TKI) cancer therapeutics may
be associated with cardiotoxicity, current approaches are
inadequate. 2) Sorafenib is a TKI of concern since it inhibits
growth factor receptors and Raf-1/B-Raf, kinases that are
upstream of ERKs and signal cardiomyocyte survival in the
setting of stress. Objectives: 1) Explore the potential use of
zebrafish as a pre-clinical model to predict cardiotoxicity. 2)
Determine whether sorafenib has associated cardiotoxicity
and, if so, define the mechanisms. Methods and Results:
We find that the zebrafish model is readily able to discriminate
a TKI with little or no cardiotoxicity (gefitinib) from one
with demonstrated cardiotoxicity (sunitinib). Sorafenib, like
sunitinib, leads to cardiomyocyte apoptosis, contractile
dysfunction and ventricular dilatation in zebrafish. In cultured
rat cardiomyocytes, sorafenib induces cell death. This can be
rescued by adenovirus-mediated gene transfer of constitutively
active MEK1 which restores ERK activity even in the presence
of sorafenib. While growth factor-induced activation of ERKs
requires Raf, α-adrenergic-induced activation of ERKs does
not. Consequently, activation of α-adrenergic signaling virtually
abrogates sorafenib-induced cell death. Consistently, inhibition
of α-adrenergic signaling with prazosin augments sorafenibinduced contractile dysfunction in zebrafish. Conclusions:
1) Zebrafish may be a valuable pre-clinical tool to predict
cardiotoxicity. 2) We identify a here-to-fore unknown pathway
that bypasses Raf to activate ERKs, thereby limiting sorafenib
cardiotoxicity. Importantly, given that the majority of men
over the age of 60 have prostatic hypertrophy, for which
α-adrenergic antagonists are the primary therapy, our findings
suggest the consequences of the concomitant use of these
agents with sorafenib should be explored.
H. Cheng: None. G. Kari: None. U. Rodeck: None. A. Dicker: None.
T. Force: None.
P42Loss of Mcl-1 in the Heart Leads to Rapid Mitochondrial
Dysfunction and Development of Heart Failure
Robert L Thomas III, Youngil Lee, Univ of California, San Diego, CA;
Chengqun Huang, San Diego State Univ, San Diego, CA; Shivaji Rikka,
Univ of California, San Diego, CA; Kimberlee Fisher, San Diego State
Univ, San Diego, CA; Aleksander Andreyev, Anne N Murphy, Univ of
California, San Diego, CA; Mark A Sussman, San Diego State Univ, San
Diego, CA; Åsa B Gustafsson, Univ of California, San Diego, CA
Mcl-1 is an anti-apoptotic Bcl-2 family protein that is expressed
at high levels in the heart. However, the functional importance
of Mcl-1 in the heart has not been investigated. To study the
32
function of Mcl-1 in cardiac myocytes, we generated inducible,
myocyte-specific Mcl-1 knock-out mice using a tamoxifendriven loxP/α-MHC-Cre-recombinase system. We found that
deletion of Mcl-1 by tamoxifen injections resulted in rapid
development of cardiac hypertrophy, pulmonary edema and
cardiac dysfunction in adult mice. Although Mcl-1 is known to
inhibit apoptosis in cells, Mcl-1 deficient hearts were TUNEL
negative and showed no activation of caspase-3. The antiapoptotic proteins Bcl-2 and Bcl-XL have also been reported
to inhibit autophagy, but we found no increase in autophagy
upon loss of Mcl-1. Western blot analysis of other Bcl-2 family
members revealed that Bcl-2 was significantly increased
in Mcl-1 deficient hearts, suggesting a compensatory
mechanism. Interestingly, ultrastructural analysis revealed
the presence of swollen mitochondria, ruptured cells, and
intracellular contents in the extracellular space. These signs
are characteristics of necrotic cell death. Mcl-1 deficient
hearts also exhibited reduced mitochondrial respiration and
significantly lower cellular ATP levels compared to wild type,
suggesting that the loss of Mcl-1 impairs mitochondrial
function. Moreover, mitochondria isolated from Mcl-1-/hearts showed pronounced swelling at baseline that could be
alleviated by polyethylene glycol-induced shrinkage. Calcium
release experiments also showed higher baseline Ca2+ levels
in mitochondria from Mcl-1-/- hearts compared to wild type,
suggesting that Mcl-1 deletion increases permeability to Ca2+.
Our data suggest that loss of Mcl-1 in cardiac myocytes
leads to rapid mitochondrial impairment and necrotic cell
death. Thus, in addition to its anti-apoptotic role, Mcl-1 has
an essential but unidentified role in maintaining mitochondrial
function in cardiac myocytes.
R.L. Thomas: None. Y. Lee: None. C. Huang: None. S. Rikka: None.
K. Fisher: None. A. Andreyev: None. A.N. Murphy: None.
M.A. Sussman: None. Å.B. Gustafsson: None.
P43The COP9 Signalosome Regulates Autophagy
Huabo Su, Univ of South Dakota, Vermillion, SD; Faqian Li, Univ of
Rochester Medical Ctr, Rochester, NY; Mark J Ranek, Univ of South
Dakota, Vermillion, SD; Ning Wei, Yale Univ, New Haven, CT; Xuejun
(XJ) Wang, Univ of South Dakota, Vermillion, SD
By indiscriminately degrading portions of cytoplasm for
self-supply of nutrients, non-selective autophagy helps the
cell to survive starvation. Selective autophagy, however,
removes defective/surplus organelles and protein aggregates,
thereby playing an important role in quality control in the
cell. Autophagy is involved in the pathophysiology of a
variety of disease, including common forms of heart disease.
Mechanisms regulating autophagy, especially selective
autophagy, remain poorly understood. The COP9 signalosome
(CSN) is an evolutionarily conserved protein complex
consisting of 8 subunits (CSN1 through CSN8). CSN was
purported to regulate ubiquitin-proteasome system (UPS)
mediated proteolysis. We recently reported UPS malfunction
and the accumulation of ubiquitin positive aggregates in
the cardiomyocytes of mice with perinatal cardiomyocyterestricted Csn8 knockout (CR-Csn8KO), which displayed
massive cardiomyocyte necrosis, congestive heart failure, and
premature death. Here we report that Csn8/CSN is required
for the removal of autophagosomes in cardiomyocytes, an
exciting discovery that has not been reported in any types
of cells. CR-Csn8KO mouse hearts show marked increases
in LC3-II, indicative of increased autophagosomes. The
increase in LC-II is accompanied by a significant increase in
p62 protein levels, which is evident as early as 1 week of age,
long before the accumulation of a surrogate UPS substrate
becomes discernible. The increase in autophagosomes is
confirmed by probing with a transgenic GFP-LC3 and by
electron microscopy. Autophagic flux assessments reveal
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Poster Presentations (continued)
that the removal of autophagosomes in cardiomyocytes is
impaired by Csn8 deficiency and the defective fusion between
autophagosomes and lysosomes may be responsible. Rab7
transcript and protein levels in the heart are significantly
decreased by Csn8 deficiency. Confocal microscopy
reveals a striking reciprocal relationship between increases
in GFP-LC3 puncta and the decreased Rab7 expression.
Rab7 knockdown impairs the removal of autophagosomes
in cultured cardiomyocytes. Hence, Csn8/CSN is a central
regulator in not only the UPS but also autophagy. Csn8/
CSN supports autophagosome-lysosome fusion likely by
stimulating Rab7 expression.
. Su: None. F. Li: None. M.J. Ranek: None. N. Wei: None.
H
X. Wang: None.
This research has received full or partial funding support from the
American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa,
Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota,
South Dakota & Wisconsin).
P44Activation of Interferon Regulatory Factor-3 Prolongs
Stress-Induced Cardiomyocyte Apoptosis and Decreases
Survival Post Myocardial Infarction
Geoffrey de Couto, Univ of Toronto, Toronto, ON, Canada; Fayez
Dawood, Mei Sun, Manyin Chen, Univ Health Network, Toronto, ON,
Canada; Scott Heximer, William Stanford, Jeffrey Medin, Peter Liu, Univ
of Toronto, Toronto, ON, Canada
G. de Couto: None. F. Dawood: None. M. Sun: None. M. Chen: None.
S. Heximer: None. W. Stanford: None. J. Medin: None. P. Liu: None.
Yoshiya Monden, Yasuhiro Maejima, UMDNJ New Jersey Medical
Sch, Newark, NJ; Masaaki Komatsu, Tokyo Metropolitan Inst of
Medical Science, Tokyo, Japan; Mondira Kundu, St Jude Children’s
Res Hosp, Memphis, TN; Junichi Sadoshima, UMDNJ New Jersey
Medical Sch, Newark, NJ
Autophagy is an intracellular bulk degradation process in
which cytosolic proteins/organelles are sequestered into
double-membrane vesicles termed autophagosomes to
be fused with lysosomes for degradation. Recent evidence
suggests that an alternative form of autophagy, which utilizes
distinct sources of membrane for autophagosome formation,
may exist. Whether alternative autophagy exists in the heart is
unknown. Cardiac-specific Atg7-KO (Atg7-CKO) and systemic
Ulk1-KO mice were subjected to starvation for 48 hours.
LC3-II expression and p62 degradation were increased in
wild-type (WT) mice after starvation (ST), indicating increased
autophagy. In Atg7-CKO mice, LC3-II expression was reduced
and p62 was markedly increased at baseline and in response
to ST, indicating inhibition of LC3-dependent autophagy. In
contrast, in Ulk1-KO mice, neither LC3-II nor p62 was affected
at baseline or in response to ST. However, autolysosome
formation evaluated by Lamp2/Rab9 staining, indicators of
unconventional autophagy, was abolished. Furthermore, the
level of polyubiquitinated protein was increased after ST.
Echocardiographic analyses showed that fractional shortening
(FS) was maintained after ST in WT mice (38%). However, in
Atg7-CKO mice, FS deteriorated at baseline (22%) but was
not further affected by ST (20%). In contrast, in Ulk1-KO mice,
FS was relatively preserved at baseline (32%) but remarkably
decreased during ST (19%). After glucose deprivation (GD)
GFP-LC3 was co-localized with calnexin (ER marker) in Adsh-Control- and Ad-sh-Ulk1-transduced, but not in Ad-shAtg7-transduced, cultured cardiomyocytes (CMs) in vitro. In
contrast, Lamp2 was co-localized with TGN46 (Golgi marker)
in Ad-sh-Control and Ad-sh-Atg7-transduced, but not in Adsh-Ulk1-transduced, CMs after GD. These results suggest that
the autophagosomes are derived from ER in LC3-dependent
autophagy and Golgi in Rab9-dependent autophagy in CMs.
Atg7 is required for maintaining cardiac function at baseline
in an LC3-dependent manner, whereas Ulk1 is required
for preserving cardiac function during ST, possibly through
stimulation of Rab9-dependent autophagy. These results
support the presence of alternative autophagy in the heart.
Y. Monden: None. Y. Maejima: None. M. Komatsu: None. M. Kundu:
None. J. Sadoshima: None.
P46Hsp20 Promotes Cardiac Autophagy via Interaction
with Beclin-1
Hongyan Zhu, Xiaohong Wang, Xiaowei Zhang, Evangelia G Kranias,
Univ of Cincinnati Coll of Med, Cincinnati, OH; Qiangrong Liang, Univ of
South Dakota, Sioux Falls, SD; Guo-Chang Fan, Univ of Cincinnati Coll
of Med, Cincinnati, OH
ABSTRACTS
Background. The innate immune response is critical in
mediating host protection from foreign antigen or stress. It
is evident that multiple innate immune pathways also tightly
regulate the remodeling process post-myocardial infarction
(MI), however it is not clear how this process is mediated.
In order to further elucidate the underlying mechanism of
this process, we screened both mouse and human post-MI
microarray databases and identified a common, significantly
upregulated transcription factor of interest, interferon
regulatory factor-3 (IRF3). To assess its function in vivo,
we tested the hypothesis that activation of IRF3 post-MI
coordinates maladaptive repair by promoting cardiomyocyte
apoptosis. Methods/Results. We randomly allocated IRF-3
knockout (IRF-3-/-) mice and their wild-type (WT) counterparts
to either left anterior descending (LAD) coronary artery ligation
or sham operation. Four weeks post-MI, deficiency in IRF3
led to improved survival [88% (44/50) vs. 29% (14/49) in WT,
p;0.0001], preserved cardiac function via echocardiography
[Fractional Shortening: 37% vs. 22.9% in WT, LVEDD:
5.15mm vs. 5.85mm in WT, LVESD: 4.15mm vs. 5.14mm
in WT; p;0.05], and attenuated infarct size [27% vs. 44% in
WT, p;0.05]. Assessing the stress response 1-week postMI, we found that NFkB signaling (IKKa, Ikba, p65) as well
as pan-ubiquitination of proteins, were both significantly
downregulated in the border infarct zone of IRF3-/- mice.
These findings were complemented with increased protein
and mRNA expression of de-ubiquitinating enzyme A20.
Interestingly, in IRF3-/- mice these results were linked with
decreased apoptosis (Caspases, TUNEL staining), however
with a more specific attenuation of the mitochondrialspecific apoptotic pathway (Bid, Cytochrome C, Caspase9).
Conclusions. These data establish a novel link between the
innate immune transcription factor IRF3 and maladaptive
regulation of cardiomyocyte apoptosis through mitochondrial
apoptotic pathway activation. As an acute responder coordinating
maladaptive remodeling, IRF3 may prove to be an efficacious
therapeutic target to improve outcome following MI.
P45Two Forms of Autophagy in the Heart: Constitutive
Autophagy via an LC3-Dependent Pathway and StarvationInduced Autophagy via a Rab9-Dependent Pathway
Hsp20 has been shown to prevent stress-triggered cardiac
injury. However, it remains obscure whether Hsp20-elicited
cardioprotection is associated with the activation of autophagy.
We first assessed cardiac autophagy in a transgenic (TG)
mouse model with 10-fold overexpression of Hsp20 by
immunoblotting. Levels of two autophagy markers (LC3II and
Beclin1) were increased by 1.6- and 2.2-fold, respectively,
in Hsp20-hearts, compared to WTs. To examine whether
Hsp20 activates autophagic flux in the heart, mice were
i.p. injected with chloroquine (CQ, 50mg/kg), an inhibitor
for autophagosome-lysosome fusion, for 4h. Cadaverine
dye-binding analysis showed that autophagy levels were
increased by 1.5-fold in CQ-treated Hsp20-hearts compared
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33
Poster Presentations (continued)
to saline controls. To examine whether increased autophagy
levels contribute to Hsp20-induced cardioprotection,
3-methyladenine (3-MA, an inhibitor for autophgy) was i.p.
injected into TG mice (30mg/kg). One hour later, hearts
were subjected to global no-flow ischemia/reperfusion (I/R:
45min/1h), and showed that contractile function (+dP/dt)
was recovered by 93± 5 % in saline-treated Hsp20-hearts,
comparable to 74 ± 6 % recovery in 3-MA-treated TGs
(n=6, p<0.05), indicating that the activation of autophagy by
Hsp20 is beneficial for hearts upon I/R. To exclude in vivo
compensatory effects, Hsp20 was delivered into isolated adult
cardiomyocytes by adenoviral vector. Western-blotting analysis
indicated that acute expression of Hsp20 increased the levels
of LC3II and Beclin1 by ~2-fold, compared to GFP control.
Accordingly, overexpression of Hsp20 augmented myocyte
survival upon addition of H2O2, as related to GFP-control.
Furthermore, co-infection with AdBeclin1-siRNA attenuated
the protective effects of Hsp20 in myocytes upon H2O2 stress.
Excitingly, Hsp20 was found to localize at the LC3-labeled
autophagosome. Indeed, co-immunoprecipitation results
showed an interaction of Hsp20 with Beclin-1 in the heart.
Moreover, a competitive ELISA assay revealed that Hsp20
dose-dependently suppressed the interaction of Beclin1/Bcl-2,
a known complex negatively regulating autophagy. In summary,
Hsp20 activates cardiac autophagic flux via interaction with
Beclin1, thereby confering cadioprotection.
H. Zhu: None. X. Wang: None. X. Zhang: None. E.G. Kranias: None.
Q. Liang: None. G. Fan: None.
P47A Novel Antiapoptotic Function of Thioredoxin-1 Is
Mediated by Interaction with Caspase 3
ABSTRACTS
Anna Eckers, Joachim Altschmied, Judith Haendeler, IUF-Leibniz Inst
for Environmental Med, Düsseldorf, Germany
34
Thioredoxin-1 (Trx-1) is one of the most important redox
regulators in endothelial cells (EC). It is an oxidoreductase with
anti-oxidative and anti-apoptotic properties. In EC not only
the active site cysteines 32 and 35 but also the cysteine 69
are required for its anti-apoptotic function. Trx-1 has several
interaction partners, but this list is far from complete. The
underlying molecular mechanisms in apoptosis protection
by Trx-1 are not completely understood. The most important
apoptosis executor protein in EC is Caspase 3. Therefore,
the aim of this study was to investigate whether Trx-1
interferes with Caspase 3 function. We first analyzed if Trx-1 is
associated with Caspase 3. Coimmunoprecipitation revealed
an interaction between both proteins, which was only found
under non-reducing conditions, suggesting involvement
of a disulfide bridge. To get insights into the functional
relevance of the association between Trx-1 and Caspase 3,
we investigated whether Trx-1 can directly interact with the
catalytic subunit p17 of Caspase 3. After coexpression of p17
and Trx-1, coimmunoprecipitation experiments under nonreducing conditions revealed that p17 associates with Trx-1.
Analysis of Trx-1 mutants demonstrated that the interaction
depends on cysteine 32 and/or 69 in Trx-1. To assess the
functional relevance of the Trx-1/Caspase 3 interaction in
EC, we induced apoptosis by overexpression of p17 and
investigated the influence Trx-1 wildtype (wt) and mutants
in which single or multiple cysteine residues were replaced
by serines thereby preventing disulfide bridge formation.
Apoptosis induction by p17 was significantly reduced by
Trx-1 wt (p17+lacZ 16.99±1.35% vs. p17+Trxwt 8.60±1.31%
apoptotic cells). Interestingly, overexpression of Trx-1 with
mutations of cysteines 32 and 69 enhanced apoptosis
induction by Caspase 3 p17 (23.42±2.84% apoptotic cells).
On the contrary, no increase in apoptosis was observed when
only cysteine 32 or 69 were mutated, suggesting that both
residues can scavenge p17 via disulfide bridge formation
thereby preventing its association with the second Caspase
3 subunit p12 and thus activation of the enzyme. These data
suggest that Trx-1 has a novel anti-apoptotic function by
binding the apoptosis executing Caspase 3.
A. Eckers: None. J. Altschmied: None. J. Haendeler: None.
P48The Role of p62 in Cardiac Protein Quality Control
Qingwen Zheng, Huabo Su, Xuejun (XJ) Wang, Univ of South Dakota,
Vermillion, SD
Rationale: Two recent studies suggest an important role
of autophagy in protection against αB-crystallin-based
(CryABR120G) desmin-related cardiomyopathies (DRC) but this
has not been demonstrated in a different model of cardiac
proteinopathy. Moreover, mechanisms underlying the response
of cardiomyocytes to increased expression of misfolded
proteins remain incompletely understood. Objective: First,
to determine whether and how the autophagic activity is
changed in a mouse model of desminopathy; second, to
investigate the role of p62 in the protein quality control
of cardiomyocytes. Methods and Results: Using an
autophagosome reporter and determining changes in LC3-II
protein levels in response to lysosomal inhibition, we found
significantly increased autophagic flux in mouse hearts with
transgenic overexpression of a DRC-linked mutant desmin.
Similarly, autophagic flux was increased in cultured neonatal
rat ventricular myocytes (NRVMs) expressing a mutant
desmin. Suppression of autophagy by 3-methyladenine
increased, whereas enhancement of autophagy by rapamycin
reduced, the ability of a comparable level of mutant desmin
overexpression to accumulate ubiquitinated proteins in
NRVMs. Furthermore, p62 mRNA and protein expression
was significantly upregulated in cardiomyocytes by transgenic
overexpression of the mutant desmin or CryABR120G both in
intact mice and in vitro. p62 depletion impaired aggresome
and autophagosome formation, exacerbated cell injury, and
decreased cell viability in cultured NRVMs expressing the
misfolded proteins. Conclusions: Autophagic flux is increased
in desminopathic hearts, serving as an adaptive response to
overexpression of misfolded proteins. p62 is upregulated in
mouse proteinopathic hearts. p62 protects cardiomyocytes
against proteotoxic stress by promoting aggresome formation
and autophagy activation.
Q. Zheng: None. H. Su: None. X. Wang: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P49Molecular Mechanisms of PARP1-Mediated Necrosis
Diana L Douglas, Christopher P Baines, Univ of Missouri, Columbia, MO
Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme
associated with DNA replication, transcription, repair, and
cell death. Although, PARP1 is most commonly associated
with apoptosis, a specific PARP1-mediated caspaseindependent necrosis pathway has recently been described.
Two main models for this pathway have been proposed:
one involving RIP1 and JNK kinases and mitochondrial
permeability transition (MPT), the other involving activation
of Bax and subsequent release of apoptosis inducing
factor (AIF) from mitochondria. However, these models still
remain untested. Consequently, the purpose of this study
was to determine which (if any) of these molecules are truly
involved in PARP1-mediated necrosis. Murine embryonic
fibroblasts (MEFs) were treated with either N-Methyl-N’-NitroN-Nitrosoguanidine (MNNG) or β-Lapachone, 2 chemically
distinct PARP1 activators. To inhibit RIP1, CypD, and AIF,
MEFs were transfected with specific siRNAs for 48 hrs prior
to treatment. RIP1 and CypD-null cells were also used. To
block JNK activity, cells were incubated with the JNK-specific
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Poster Presentations (continued)
inhibitor SP600125. Inhibition of Bax was achieved using
either Bax/Bak-null MEFs or Bcl2 overexpression. Necrosis,
MPT, JNK activation, and AIF translocation were then analyzed
using a combination of cell death assays, western blotting,
and immunocytochemistry. Treatment of MEFs with MNNG
and β-Lapachone induced translocation of AIF from the
mitochondria to nucleus, and necrotic cell death. However,
inhibition of RIP1, CypD, and Bax had no significant effect
on MNNG or β-Lapachone- induced necrotic cell death.
Moreover, neither PARP1 activator induced MPT. In contrast,
the JNK inhibitor SP600125 significantly reduced MNNGand β-Lapachone-induced JNK activation, AIF translocation,
and necrosis. This suggests that JNK plays a key role in
the pathway and that it is upstream of AIF. To our surprise,
however, depletion of AIF did not influence PARP1-mediated
cell death, thus indicating that AIF is more of a marker, rather
than a key mediator, of this particular process. We conclude
that JNK plays a significant signaling role in PARP1-mediated
necrosis. However, RIP1, Bax, CypD, and AIF do not appear
to be essential components of the PARP1 death pathway.
D.L. Douglas: None. C.P. Baines: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P50Novel Mechanism of Ser-496 ERK5 Phosphorylation and
Association with p90RSK Is Critical for Regulating C
Terminus of Hsc70-Interacting Protein (CHIP) Ubiquitin
E3 Ligase Activity in Diabetes-Mediated Exacerbation
of Cardiomyocyte Apoptosis and Left Ventricular
Dysfunction After Myocardial Infarction
Nhat-Tu Le, Yuichiro Takei, Chang-Hoon Woo, Aab Cardiovascular
Res Inst, West Henrietta, NY; Tetsuro Shishido, Dept of Cardiology,
Pulmonology, and Nephrology, Yamagata, Japan; Yan Lu, Carolyn
McClain, Chen Yan, Aab Cardiovascular Res Inst, West Henrietta, NY;
Cam Patterson, Emory Univ Sch of Med, Chapel Hill, NC; Jay Yang,
Univ of Wisconsin, Madison, WI; Jun-ichi Abe, Aab Cardiovascular Res
Inst, West Henrietta, NY
. Le: None. Y. Takei: None. C. Woo: None. T. Shishido: None. Y. Lu:
N
None. C. McClain: None. C. Yan: None. C. Patterson: None. J. Yang:
None. J. Abe: None.
This research has received full or partial funding support from the
American Heart Association, Founders Affiliate (Connecticut,
Maine, Massachusetts, New Hampshire, New Jersey, New York,
Rhode Island, Vermont).
P51Induction of Cardiac Nitrosative Stress and Cell Death by
Chronic Alcohol Consumption in an Angiotensin II-, PKC-,
and NOX-Dependent Manner
Yi Tan Sr, Univ of Louisville, Louisville, KY; Zhanxiang Zhou Zhou,
Univ of North Carolina, Greensboro, NC; Lu Cai, Univ of Louisville,
Louisville, KY
Alcohol induced cardiac cell death is causative of
cardiomyopathy, but the mechanisms responsible for alcoholic
cardiac cell death remain largely unknown. We hypothesize
that alcohol-induced Ang II plays a critical role in cardiac
nitrosative stress, cell death and cardiomyopathy. To test this
hypothesis, male C57BL/6 mice were pair-fed an alcohol or
isocaloric maltose dextrin liquid diet for 2 months. Alcohol
feeding caused significant cardiac cell death and nitrosative
damage (shown by TUNEL positive cells, caspase-3 activation
and protein nitration) and cardiac remodeling (shown by
hypertrophy and fibrosis) along with increases in serum Ang
II and cardiac AT1 expression. Mechanistic study in which
cardiac H9c2 cells were treated with 100–400 mM alcohol
for 24h revealed that alcohol exposure induced cell death,
nitrosative damage and fibrosis, along with NADPH oxidase
p47phox (NOX) activation in a dose-dependent manner. Pretreatment of alcohol-treated cells with NOX inhibitor (apycinin),
peroxynitrite scavenger (urate), NOS inhibitor (L-NAME) and
superoxide dismutase mimic (MnTMPyP) significantly abolished
alcohol induced cell death. Furthermore, exposure to alcohol
significantly up-regulated the expression of angiotensin II (Ang
II) and its type 1 receptor (AT1) in these cultured cardiac cells.
Either PKCα/β1 inhibitor or AT1 blocker completely prevented
alcoholic NOX activation, and AT1 blocker inhibited the
expression of PKCβ1, implying that alcoholic NOX activation
is dependent of PKCα/β1 activation via AT1. To validate the
in vitro findings, alcohol-fed and pair-fed mice were treated
with MnTMPyP (5 mg/Kg) daily for 2 months, which did not
change blood pressure, alcohol-induced cardiac PKC and
NOX activation, but significantly prevented alcohol-induced
cardiac nitrosative damage and cell death, along with a
prevention of cardiac fibrosis and dysfunction. These results
suggest that alcohol-induced cardiac cell death is mediated by
up-regulated Ang II that via AT1 induces PKCα/β1-dependent
NOX activation. The cardiac cell death plays a critical role in the
development of alcoholic cardiomyopathy.
ABSTRACTS
ationale: Cardiac dysfunction is accelerated in DM patients
R
after MI. Previously, we reported the critical role of ERK5 and
CHIP association on CHIP Ub E3 ligase activity, which inhibits
inducible cAMP early repressor (ICER)-mediated apoptosis
and left ventricle (LV) dysfunction after MI in DM (DM + MI).
Yet the regulatory mechanism of ERK5-CHIP has not been
established. Objective: Since we found that p90RSK activation
was increased in DM heart, we investigated whether p90RSK
activation may inhibit ERK5-mediated CHIP activation, and
subsequent ICER induction and apoptosis. Methods and
Results: The inhibition of p90RSK activation prevented the
reduction of ERK5-CHIP binding, CHIP activity, as well as ICER
induction and cardiac apoptosis both in vitro after angiotensin
II (ang II) stimulation and in vivo after DM + MI. p90RSK and
CHIP share a same binding site with ERK5 C-terminal domain
(aa571-807), and overexpression of both p90RSK and ERK5
(aa571-807) fragment, but not kinase dead mutant of p90RSK,
inhibited ERK5-CHIP association, suggesting the critical role of
p90RSK activation on ERK5-CHIP interaction, and competitive
nature of p90RSK and CHIP against ERK5 association.
Furthermore. LC-MS/MS analysis identified ERK5-S496 as
being directly phosphorylated by p90RSK, and ERK5 S496A
mutant significantly impaired ang II-mediated inhibition of
CHIP Ub ligase activity, suggesting the critical role of Ser-496
phoaphorylation of ERK5 on CHIP activity. Therefore, p90RSK
activation is critical for both p90RSK-ERK5 association as well
as ERK5-Ser496 phosphorylation, and following disruption of
ERK5-CHIP interaction and subsequent inhibition of CHIP Ub
ligase activity. The reduction of CHIP Ub ligase activity and LV
dysfunction were accelerated both in cardio-specific ERK5
knock out and wild type p90RSK transgenic mice (WT-p90RSK-
Tg). Furthermore, double transgenic mice of WT-p90RSK and
constitutively active form of MEK5α (specific ERK5 activator)
inhibited single WT-p90RSK-Tg-medaited reduction of CHIP
Ub ligase activity, LV dysfunction, and improved mortality after
MI. Conclusions: These data strongly suggested that p90RSK
activation accelerated cardiac dysfunction and apoptosis after
DM + MI via inhibiting ERK5-CHIP module.
Y. Tan: None. Z. Zhou: None. L. Cai: None.
P52Parkin Deficiency Results in Disarray of the
Mitochondrial Network and Increased Susceptibility
to Myocardial Infarction
Dieter Kubli, Xiaoxue Zhang, Asa Gustafsson, Univ of California,
San Diego, La Jolla, CA
Parkin is an E3 ubiquitin ligase that was recently discovered
to be involved in mitochondrial turnover via autophagy.
Although Parkin has been characterized in the setting of
neurodegenerative diseases, it is expressed in many other
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
35
Poster Presentations (continued)
tissues including the heart. In this study, we have investigated
the functional role of Parkin in the myocardium using global
Parkin knockout (PKO) mice. We found that PKO mice
have no overt cardiac phenotypes under normal conditions.
Echocardiography showed normal left ventricular fractional
shortening (38.82 3.55% for PKO vs. 39.89 1.19% for WT)
and ejection fraction (68.53 4.21% for PKO vs. 71.27 1.60%
for WT) at 12 weeks of age. Cardiac mitochondria isolated
from PKO mice had normal respiratory rates and displayed
normal coupling. Additionally, Parkin deficient mitochondria
were as susceptible to mitochondrial permeability transition
pore (mPTP) opening as WT mitochondria. However,
mitochondria from PKO mice had higher baseline absorbance
values than WT mitochondria (0.66 0.02 for PKO vs. 0.59
0.02 for WT), suggesting a smaller morphology. Ultrastructural
analysis by transmission electron microscopy (TEM) confirmed
a disorganized mitochondrial network with the presence
of clusters of small mitochondria at 12 weeks of age in
PKO hearts. At 6 months, PKO mice displayed a decline
in cardiac function, and TEM revealed the presence of
degenerating mitochondria. Although PKO mice have normal
cardiac function at 12-weeks, they were very susceptible
to stress. The PKO mice developed more severe dilated
cardiomyopathies with reduced survival rates compared to WT
in response to myocardial infarction. These data demonstrate
that Parkin is involved in the maintenance of the mitochondrial
network in the myocardium, and that Parkin deficiency
exacerbates stress-induced heart failure. Given the role of
Parkin in mitochondrial turnover, Parkin deficiency may result
in a gradual decline of mitochondrial quality, the effects of
which may surface under conditions of acute stress.
D. Kubli: None. X. Zhang: None. A. Gustafsson: None.
P53Impaired Autophagosome Removal in Cardiomyocytes
Triggers Programmed Necrosis in Mouse Hearts
ABSTRACTS
Huabo Su, Zongwen Tian, Changhua Wang, Suleman Said, Jie Li,
Mark J Ranek, Xuejun (XJ) Wang, Univ of South Dakota, Vermillion, SD
36
Programmed cell death includes apoptosis and programmed
necrosis. The latter is also known as necroptosis. A wellrecognized feature of necrosis is the loss of membrane
integrity of the dying cell, resulting in inflammatory responses
at the surrounding tissue. Cells undergoing apoptosis,
however, maintain their membrane integrity very well and
hence do not trigger inflammation. These features are
effectively captivated to detect necrotic cells. By assessing
Evans Blue Dye (EBD) uptake and CD45+ leukocytes, we
previously detected massive cardiomyocyte (CM) necrosis,
before increased apoptosis was evident, in mouse hearts
deficient of COP9 signalosome (CSN) subunit 8 (Csn8) (Circ
Res 2011; 108:40–50). We show here that the detection of
necrosis is confirmed by immuno-detection of endogenous
IgG inside the CM. We found that virtually all EBD positive
cells were also mouse IgG positive whereas some of the
IgG positive CMs, as expected, did not show increased
EBD uptake, suggesting that probing intracellular IgG offers
a simpler and even more sensitive method for detecting
necrotic CMs in vivo. Notably, we were able to demonstrate
that both the EBD uptake assay and the immuno-detection
of intracellular IgG clearly have a resolution at the single cell
level. We then explored the mechanism by which Csn8/
CSN deficiency causes necrosis. Autophagosomes are
accumulated due to a defect in their fusion with lysosomes
in mouse hearts with perinatal CM-restricted Csn8 knockout
(CR-Csn8KO) but the accumulation is heterogeneous among
CMs likely due to a temporal heterogeneity of Csn8 gene
ablation and/or a spatial difference in the microenvironment
and functionality of different CMs. A remarkably higher
percentage of CMs with greater autophagosome accumulation
were undergoing necrosis than those with less or without
autophagosome accumulation. Importantly, chronic lysosomal
inhibition caused CM necrosis in wild type mice. Moreover, the
protein levels of RIP1, RIP3 kinases and the RIP1-bound RIP3
were all significantly increased in the CR-Csn8KO heart. Taken
together, these novel findings strongly suggest that impaired
autophagosome removal causes CM necrosis in Csn8/
CSN deficient hearts through likely activating the RIP1-RIP3
mediated necroptotic pathway.
H. Su: None. Z. Tian: None. C. Wang: None. S. Said: None. J. Li:
None. M.J. Ranek: None. X. Wang: None.
This research has received full or partial funding support from the
American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa,
Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota,
South Dakota & Wisconsin).
P54Crosstalk Between Toll-like Receptor 4 and Lipid
Metabolism Modulates Macrophage Survival
Joel Schilling, Heather Machkovech, Jean Schaffer, Washington Univ
Sch of Med, St Louis, MO
Diabetes and obesity are well-established risk factors
for cardiovascular disease, including atherosclerosis and
adverse cardiac remodeling after myocardial infarction (MI).
Abnormalities of lipid metabolism are a hallmark of diabetes
and are strong predictors of the risk of future cardiac events.
Macrophages are cells of the innate immune system that play
a vital role in the host response to infection and tissue injury. In
the cardiovascular system, macrophages are key participants
in the pathogenesis of atherosclerosis and post-MI infarct
remodeling. Toll-like receptor (TLR)-4 is highly active on
macrophages and has been shown to contribute to the injury
response in the heart and vasculature. Thus, understanding
how lipids modulate TLR4-mediated macrophage responses
is particularly relevant to diabetic cardiovascular disease. To
investigate this question, we studied the effect of palmitate;
an abundant dietary saturated fatty acid, on macrophage
viability and inflammatory signaling after stimulation with the
TLR4 ligand LPS. Interestingly, LPS or palmitate alone failed
to trigger significant cell death; however, the combination of
these stimuli markedly reduced macrophage viability. The
synergistic death induced by LPS and palmitate required
functional TLR4, yet occurred independently of ER stress,
reactive oxygen species generation, ceramide production,
caspase activation and autophagy. Instead, lysosomal
integrity is compromised early and followed by the loss of
mitochondrial membrane potential and ultimately cell death.
Interestingly, inhibition of the lipid remodeling enzyme iPLA2
attenuates lysosomal instability and cell death following
treatment with palmitate/LPS. Thus, we describe a novel cell
death response in macrophages that occurs via a mechanism
involving iPLA2 activation and lysosomal destabilization. These
data provide evidence that the lipid environment modulates
macrophage function following inflammatory stimuli. Given the
critical role of macrophages in the response to tissue injury,
these findings are highly relevant to understanding cardiac
injury and repair in diabetics.
J. Schilling: None. H. Machkovech: None. J. Schaffer: None.
P552-Deoxy-Glucose Reduces Doxorubicin Cardiotoxicity in
Tumor-Bearing Mice
Derek Timm, Michelle Phillips, Allison Haugrud, Yongxian Zhuang,
Keith Miskimins, Qiangrong Liang, Sanford Health, Sioux Falls, SD
Doxorubicin (DOX) is a potent, commonly used anthracycline
antibiotic for a wide range of cancers, but its dose is limited
by its strong cardiotoxic effects. The glucose analog 2-deoxyD-glucose (2-DG) has been shown to enhance the antitumor
efficacy of DOX in tumor cell lines and in animals. However,
the cardiac effects of 2-DG in these experiments were not
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Poster Presentations (continued)
determined. In the current study, we examined whether 2-DG
could protect from DOX cardiotoxicity in a BALB/c mouse
breast cancer model. A total of 32 mice were injected with
105 4T1 mouse mammary epithelial tumor cells in the right
hind leg. The tumor was allowed to progress for 1 week
before dividing the mice into four groups: saline, 2-DG,
DOX, and 2-DG with DOX treatment. 2-DG was given 150
mg/kg i.p. Monday through Friday; DOX was given 5 mg/
kg i.p. Wednesday. The study was conducted for 3 weeks
and echocardiography was performed the day before
sacrifice. Two of the eight mice in the DOX group died before
echocardiography, while none died in the 2-DG with DOX
group. Echocardiography indicated similar cardiac function
between saline and 2-DG groups as measured by fractional
shortening (36.3±1.31% vs 37.7±0.942%, p>0.05). DOX
markedly reduced cardiac function, which was almost
completely recovered by 2-DG (25.1±1.06% vs 36.7±1.76%,
p<0.01). Serum CK-MB content, a common clinical marker
for heart damage, was increased ~6 fold in the DOX group
(67.5±19.4 vs 400±145, p<0.023). 2-DG tended to reduce
DOX-triggered CK-MB release, but it did not reach significant
levels likely due to the small sample size and high variation
(279±68.7 vs 400±145, p>0.05). Nonetheless, 2-DG largely
eliminated DOX-induced apoptosis in the heart as shown by
a DNA laddering assay. 2-DG or DOX alone increased cardiac
autophagic flux as measured by the difference in LC3-II protein
levels in the absence and presence of the lysosomal inhibitor,
bafilomycin A1. Paradoxically, autophagic flux was not
notably elevated when mice were treated with 2-DG and DOX
simultaneously. These results demonstrate the ability of 2-DG
to reduce DOX cardiotoxicity without affecting its antitumor
activity, suggesting that 2-DG can improve the therapeutic
window for DOX, allowing greater flexibility in designing
different regimens for treating cancers.
D. Timm: None. M. Phillips: None. A. Haugrud: None. Y. Zhuang:
None. K. Miskimins: None. Q. Liang: None.
P56RalGDS-Dependent Cardiomyocyte Autophagy in LoadInduced Ventricular Hypertrophy
Oktay F Rifki, Brian O Bodemann, Michael A White, Joseph A Hill,
UT Southwestern Medical Ctr, Dallas, TX
O.F. Rifki: None. B.O. Bodemann: None. M.A. White: None. J.A. Hill: None.
This research has received full or partial funding support from the
American Heart Association, South Central Affiliate (Arkansas,
New Mexico, Oklahoma & Texas).
P57 Not published at presenter’s request.
P58Necessity of mTOR Inhibition and Autophagy
Induction for Maintaining Cardiac Homeostasis
During Caloric Restriction
Kai Chen, Derek Timm, Tyler Jepperson, Xianmin Xu, Satoru
Kobayashi, Qiangrong Liang, Sanford Res/USD, Sioux Falls, SD
Caloric restriction (CR) is a robust dietary intervention known
to delay age-related heart disease. CR is able to antagonize
cardiac remodeling in animal and human under various
pathological conditions. However, the direct effect of CR on
the heart at basal physiological state is not well characterized.
In this study, we examined the impact of CR on cardiac
homeostasis and explored the signaling mechanisms that
mediate the cardiac effect of CR. Male C57BL/6J mice
underwent 20% CR, i.e. 20% less food than ad libitum (AL),
for 2 weeks and 40% CR for another 2 weeks. This CR
regimen caused a roughly 20% decrease in heart weight and
myocyte cross-sectional area. CR diminished the activity of
the mammalian target of rapamycin (mTOR) and accelerated
autophagic flux. To determine the functional role of mTOR
inhibition in CR-induced heart size reduction, we used
transgenic mice that express a constitutively active mTOR
(mTORca) in the heart. CR did not inhibit cardiac mTOR
signaling in mTORca mice as it did in wild type (WT) mice. As
a result, the ability of CR to reduce heart size in WT mice was
severely impaired in mTORca mice. Specifically, CR reduced
the heart weight by 23.1% in WT mice (p<0.01 vs AL), but
only tended to reduce it in mTORca mice (13.1%, p>0.05
vs AL). Strikingly, when animals were treated with the mTOR
inhibitor rapamycin, CR decreased heart weight in both WT
and mTORca mice to the same extent, strongly suggesting
that mTOR inhibition is required for CR to exert its cardiac
effect. Autophagy is a cellular degradation pathway that may
contribute to CR-induced heart size reduction. Surprisingly,
CR can still decrease heart weight in 2 different autophagydeficient mouse models, namely, beclin 1 heterozygous
knockout mice and ATG16L1 hypomorphic mice.
Nonetheless, cardiac function in these mice was markedly
deteriorated during CR as indicated by decreased fractional
shortening (Beclin 1+/-: AL 37.9±2.8 vs CR 29.6±2.1, p<0.01;
ATG16L1-HM: AL 36.8±1.8 vs CR 30.1±2.1, p<0.05). This
was accompanied by increased oxidative injury, suggesting
that autophagy is indispensible for a normal cardiac function
during CR. In conclusion, both mTOR inhibition and
autophagy induction are essential for maintaining cardiac
homeostasis in response to CR-triggered mild stress.
ABSTRACTS
ackground: Recent work has demonstrated that autophagy,
B
a phylogenetically conserved, lysosome-mediated pathway
of protein degradation, is a key participant in pathological
cardiac remodeling. One common feature of cell growth and
autophagy is membrane biogenesis and processing. The
exocyst, an octomeric protein complex involved in vesicle
trafficking, is implicated in numerous cellular processes, yet its
role in cardiomyocyte plasticity is unknown. Here, we set out
to explore the role of small G protein-dependent membrane
trafficking in stress-induced cardiomyocyte remodeling and
autophagy. Methods and Results: Hearts from mice lacking
RalGDS (Ralgds-/-), a guanine exchange factor (GEF) for
the Ral family of small GTPases, were similar to wild-type
(WT) littermates in terms of ventricular structure, contractile
performance, and gene expression. However, Ralgds-/- hearts
manifested a blunted growth response (p<0.05) to TACmediated pressure-overload stress as determined by heart
weight to body weight ratios (HW/BW; WT, sham: 5.00 ± 0.21
mg/g, n=9; TAC: 6.93 ± 0.29, n=13; Ralgds-/- sham: 4.87 ±
0.19, n=6, TAC: 5.86 ± 0.10, n=6). Ventricular chamber size
and contractile performance were unchanged in response to
TAC in both genotypes. Interestingly, TAC-induced activation
of the fetal gene program was similar in both genotypes
despite the relative lack of hypertrophic growth in mutant
hearts. Ralgds-/- mice also exhibited diminished load-induced
cardiomyocyte autophagy. Consistent with the TAC findings,
Ralgds-/- mice manifested a blunted autophagic response
to 24-hour fasting, suggesting a generalized defect in
autophagy. To explore underlying mechanisms, we tested
in cultured neonatal cardiomyocytes two isoforms of Ral
that are downstream of RalGDS (RalA, RalB) and whose
actions are mediated by the exocyst. In these experiments,
mTOR inhibition was maintained in response to starvation
and rapamycin despite RalA or RalB knockdown; however,
autophagy was diminished only in NRCM’s with RalB
knockdown, implicating RalB as required for cardiomyocyte
autophagy. Conclusions: Together, these data implicate
RalGDS-mediated induction of autophagy as a critical feature
of load-induced cardiac hypertrophy.
K. Chen: None. D. Timm: None. T. Jepperson: None. X. Xu: None.
S. Kobayashi: None. Q. Liang: None.
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
37
Poster Presentations (continued)
μM Akt Inhibitor VIII. Cell death was induced by 500 μM H2O2
for 4 hours, and cells were stained by Sytox green to identify
dead cells and Bis-Benzimide to label all cell nuclei. Protein
expression as well as subcellular localization was determined
by Western Blot. The HK2 adenovirus resulted in 50–60%
increased HK2 expression. In WT MEFs, HK2 overexpression
resulted in roughly 50% less death compared to control
transfected cells. Surprisingly, although VDAC1 appears to
bind the greatest percentage of HK2, cells lacking VDAC1
were still protected by HK2 to a similar degree as WT cells.
However, cells with reduced levels of VDAC2, or cells lacking
VDAC3 were no longer protected from cell death by HK2.
Additionally, HK2 overexpression remained protective in cells
treated with the Akt inhibitor. In conclusion, association with
the less abundant VDAC2 and VDAC3 isoforms, not the more
abundant VDAC1 isoform, appears to be central to HK2mediated cytoprotection. It also appears that active Akt is not
essential to HK2-mediated protection against cell death.
P59Cardiomyocyte Unfolded Protein Response
Stimulates Autophagy
Zhao V Wang, Thomas G Gillette, Beverly A Rothermel, Joseph A Hill,
UT Southwestern Medical Ctr, Dallas, TX
ackground: Autophagy is an evolutionarily conserved
B
process of protein and organelle recycling. Under basal
conditions, autophagy is critical for protein and organelle
quality control. This cannibalization mechanism, however, can
be detrimental under certain conditions, and dysregulation
of autophagy has been implicated in numerous diseases.
Recently, activation of autophagic flux has been reported in
cardiac hypertrophy, heart failure, myocardial infarction, and
ischemia/reperfusion injury. The unfolded protein response
(UPR) is a cellular mechanism triggered by folding stress in the
ER. When protein folding capacity, governed by ER resident
chaperones, is overwhelmed by misfolded proteins, ER
stress ensues, stimulating chaperone protein expression, ER
associated degradation, and ultimately cell death if the stress
is not remediated. Recent studies in yeast suggest the UPR
can directly activate autophagy by phosphorylating ATG1, a
critical upstream kinase required during autophagy initiation.
However, whether and how ER stress, which is active in
cardiac disease, regulates autophagy in heart is unknown.
Methods and Results: Using neonatal rat ventricular
cardiomyocytes in culture, we found the classical ER stress
inducer, tunicamycin, triggers profound UPR signaling and
autophagy up-regulation. The processing of LC3-II, an
indication of autophagy activity, is dramatically increased. As
multiple pathways are involved in ER stress, we focused on
the IRE1/XBP1 branch. With cardiomyocyte-specific overexpression by lentivirus in vitro, we observed robust activation
of autophagy. Further, we found that in vivo over-expression of
XBP1s in cardiomyocytes triggered autophagy, as evidenced
by real-time PCR and immunoblotting assays. As autophagy
markers can accumulate due to blockage of lysosomal
degradation, we quantified the lysosomal proteins, cathepsin
D and LAMP1, finding each to be increased, suggesting
that autophagic activity and flux per se are enhanced.
Conclusions: Taken together, our data suggest that the
XBP1s arm of the UPR pathway activates autophagic gene
expression, autophagosome assembly, and autophagic flux.
K.S. McCommis: None. C.P. Baines: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P61The Possible Apoptosis Pathways Involved in Mechanical
Trauma-Induced Secondary Heart Injury
Zi Yan, Shanxi Medical Univ, Taiyuan, China; Li Guo, Cardiovascular
Disease Hosp of Shanxi Province, Taiyuan, China; Yongjin Wen,
Chengzhang Cao, Jie Wang, Yan Zhang, Yong Guo, Shanxi Medical
Univ, Taiyuan, China; Feng Liang, Steel General Hosp of Taiyuan,
Taiyuan, China
ackground: Trauma induces not only primary heart injury but
B
also secondary heart injury. Our previous study has demonstrated
that mechanical trauma could cause cardiomyocyte apoptosis
which contributes to posttraumatic secondary cardiac
dysfunction. The aim of the present study was to elaborate
the potential mechanism involved in secondary post-traumatic
cardiac dysfunction. Methods: Male adult rats were subjected
to Noble-Collip drum with a total of 200 revolutions at a rate of
40 rpm. The activities of cardiomyocyte Caspase 3, 8, 9, 12
were detected respectively by Fluorometric assays. The cardiac
function in vitro was determined using a Langendorff isolated
heart perfusion system. Results: No direct mechanical traumatic
injury was observed in the heart immediately after trauma. (1)
Compared with the sham group, myocardial Caspase-3 activity in
the traumatized rats significantly increased 6h after trauma (51±4
vs. 16±2, P<0.01). 12h after trauma myocardial Caspase-3
activity reached the peak (69±4 vs. Sham, P<0.01), which still
remained at high level (46±3 vs. Sham, P<0.01) 24h after trauma.
(2) Administration of Z-VAD-FMK, a broad-spectrum caspase
inhibitor, could reverse post-traumatic cardiac dysfunction
evidenced by increased +dP/dtmax [(4111±189) mmHg/sec, vs.
sham (3414±208) mmHg/sec, P<0.01], and decreased -dP/dtmax
[(-4997±351) mmHg/sec, vs. sham group (-3301±458) mmHg/
sec, P<0.01] 24h after trauma. (3) Compared with the sham
group, myocardial Caspase-12 activity significantly increased
3h after trauma (66±8 vs. 27±10, P<0.01). 6h after trauma
Caspase-12 activity reached the peak (89±16, P<0.01 vs. Sham)
, but 12h after trauma it decreased (P>0.05 vs. Sham). Both
myocardial Caspase-8 activity and Caspase-9 activity markedly
increased 24h after trauma (2312±648 vs. 1449±296, P<0.01 vs.
Sham; 875±460 vs. 470±222, P<0.01 vs. Sham, respectively).
Conclusion: Caspase-dependent apoptotic pathway played an
important role in post-traumatic secondary cardiac dysfunction.
Moreover, in the early period of trauma, cardiomyocyte apoptosis
was induced by activation the Caspase-12, an endoplasmic
reticulum (ER)-specific caspase, following by activation of
Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).
. Yan: None. L. Guo: None. Y. Wen: None. C. Cao: None. J. Wang:
Z
None. Y. Zhang: None. Y. Guo: None. F. Liang: None.
Z.V. Wang: None. T.G. Gillette: None. B.A. Rothermel: None.
J.A. Hill: None.
ABSTRACTS
This research has received full or partial funding support from the
American Heart Association, South Central Affiliate (Arkansas,
New Mexico, Oklahoma & Texas).
38
P60VDAC2 and 3, but Not VDAC1, Are Required for HK2Mediated Protection Against Cell Death
Kyle S McCommis, Christopher P Baines, Univ of Missouri,
Columbia, MO
The glycolytic enzyme hexokinase-2 (HK2) has been shown
to protect several cell types including cardiomyocytes against
death. However, the mechanisms of this HK2-mediated
protection have not been elucidated. HK2 is normally
associated with the mitochondrial voltage-dependent anion
channels (VDACs) on the outer mitochondrial membrane, but
it is unknown if association with VDACs is required for HK2’s
protective functions. In addition, Akt has been shown to
phosphorylate HK2, resulting in its translocation to the outer
mitochondrial membrane. The purpose of this study was to
investigate the role of the three VDAC isoforms, as well as
Akt signaling, on HK2-mediated protection. Studies were
conducted using cultured murine embryonic fibroblasts (MEFs)
from wildtype (WT), VDAC1-/-, and VDAC3-/- mice. WT
MEFs were also transfected with a VDAC2 siRNA to reduce
VDAC2 protein expression. Murine HK2 was overexpressed
by infection with adenovirus. Akt signaling was inhibited by 2
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Poster Presentations (continued)
pAKT was found in BMP-2 treated PASMCs. Increased
activities of caspase-3, -8 and -9 of PASMCs were found after
cultured with BMP-2 in both mediums. PTEN expression was
unchanged when Smad-4 expression was inhibited. However
pre-treat PASMCs with bpV(HOpic) and GW9662 (PPAR-r
inhibitor) inhibited PTEN protein expression and recovered
PASMCs proliferation rate. Conclusion: BMP-2 can increase
PTEN expression under hypoxia in a dose dependent pattern.
BMP-2 can increase apoptosis of PASMCs under hypoxia. The
increased PTEN expression may be mediated through PPAR-r
signalling pathway, instead of BMP/Smad signalling pathway.
P62Loss of Plasma Membrane Integrity Occurs in 2 Stages
During Early Myocardial Ischemia/Reperfusion Injury
Ming Zhang, Pradeepkumar Charlagorla, Junying Liu, Javi Balroop,
Eun Hee Ko, Danielle Green, Oghomwen Shaka-Idusuyi, Monaliben
Patel, SUNY Downstate Medical Ctr, Brooklyn, NY; Arlan Richardson,
Univ of Texas Health Science Ctr at San Antonio, San Antonio, TX; .
Julie Rushbrook, SUNY Downstate Medical Ctr, Brooklyn, NY
Myocardial ischemia is an acute health emergency. Reperfusion,
critical for salvaging ischemic tissues, contributes to the acute
tissue damage known as ischemia-reperfusion (I/R) injury.
While therapeutic approaches aimed at reducing I/R injury
have been promising in preclinical studies, clinical studies have
been inconclusive, suggesting an inadequate knowledge of
the mechanisms underlying I/R injury and their time-frame.
In the current work, myocardial necrosis was evaluated in a
murine I/R model using a propidium iodide-based fluorescent
method that evaluated loss of plasma membrane integrity
(LPMI) during the first 24 hours of reperfusion and permitted
histological analysis of other pathological events on adjacent
sections. LPMI was found to develop in two stages in WT
animals: a significant increase occurred between 0 and 1
hour of reperfusion and was maintained through 6 hours; an
additional increase was apparent at 24 hours. Complement
C3 deposition lagged LPMI by 2 hours and C3-/- mice were
protected from second-stage LPMI but not first-stage.
Transgenic mice overexpressing the anti-oxidant enzymes
superoxide dismutase or catalase were protected from
first stage LPMI but not second stage. The results provide
support for the development of therapeutic approaches
that are directed at the early stages of reperfusion and take
into account the two phases of ischemia-related myocardial
damage, designing strategies targeting the reactive oxygen
species of first stage and the complement-mediated events of
second stage.
W. Pi: None. L. Su: None. J. Zhu: None. W. Xu: None.
P64 Withdrawn
P65Hypercholesterolemia Induced Atherosclerosis via the
Mechanism of Autophagy Pathway
Chunjiang Tan, Wenlie Chen, Yanbin Wu, Ruhui Lin, Jiumao Lin, Fujian
Univ of TCM, Fuzhou City, China
bjectives: To investigate the mechanism of
O
hypercholesterolemia in the pathogenesis of atherosclerosis
(AS). Background: As the main risk factor of AS, the
mechanism of hypercholesterolemia (HC) in the pathogenesis
of AS remains unclear. Methods: White New Zealand male
rabbits were fed a high cholesterol diet (H) for 14 weeks
to induce AS with normal diet fed rabbits as a control(N).
In vitro, human aortic vascular endothelia (HAVED) and
smooth muscle cells (HSMC) were incubated with different
concentrations of HC in the comparison with the cells that
the specific gene of Beclin1 or LC3-I/II were knocked down
by siRNA transfection. Blood lipids, and vascular histologic
structure, mRNA and protein expressions of Becline1,
microtubule-associated protein 1 light chain 3(LC3-I/II) and
autophagosomes were determined in the rabbit abdominal
aorta or in the cultured cells. Results: Serum levels of total
cholesterol,triglyceride and low density lipoprotein cholesterol
were significantly increased in H than those in N (P<0.05).
Histologic structure of abdominal aorta showed more
aggravated in wall thickness, lumen narrowness and atheroma
in H than those in N (P<0.05). The expressions of Becline1,
LC3-I/II in mRNA or proteins were strongly increased in H
than those in N (P<0.05), which was consistent with the
expressions of autophagosomes in the two groups.At the cap
or the shoulder of atheromas, the expressions of Becline1,
LC3-I/II and autophagosomes were far more enhanced than
those in non-atheromatous areas in the same sample of H
group (P<0.05). In vitro, HAVED or HSMC that preincubated
with HC revealed more expressions of Becline1, LC3-I/II and
autophagosomes, which were relevant to the concentrations
and the preincubation time of HC, compared to the cells
that incubated with N serum. Further, the autophagosomes
in the cultured cells were correlated with the number of
survive cells. Compared to the intact cells, the expressions
of autophagosomes in the cells that the gene of Beclin1 or
LC3-I/II was knocked down were greatly suppressed, and the
number of survive cells was relatively increased, even they
were exposed to HC incubation. Conclusions: The current
experiments suggested that HC mediated the pathogenesis of
AS partially via the pathway of autophagy.
. Tan: B. Research Grant; Modest; Supported by the Key
C
Laboratory Foundation from Academy of Integrative Medicine, Fujian
TCM University (No.ZXY2008005), Chenkeji Integrative Medicine
Development Foundation (No.CKJ200800). B. Research Grant;
Significant; Natural Science and Education Department Foundation
of Fujian Province (No.2008J0326, No.JA08114). C. Other Research
Support; Modest; the Molecular Biology Centre Laboratory of Fujian
Academy of Integrative Medicine. W. Chen: None. Y. Wu: None.
R. Lin: None. J. Lin: None.
M. Zhang: None. P. Charlagorla: None. J. Liu: None. J. Balroop:
None. E. Ko: None. D. Green: None. O. Shaka-Idusuyi: None.
M. Patel: None. A. Richardson: None. J. Rushbrook: None.
P63BMP-2 Upregulates PTEN Expression and Induces
Apoptosis of Pulmonary Artery Smooth Muscle Cells
Under Hypoxia
Weifeng Pi, Xinhua Hosp, Shanghai, China; LiPing Su, Natl Univ
of Singapore, Singapore, Singapore; Jian Zhu, Nanjing First Hosp,
Nanjing, China; Weiguo Xu, Xinhua Hosp, Shanghai, China
ackground: Primary pulmonary hypertension is mainly
B
caused by increased proliferation and decreased apoptosis
in pulmonary artery smooth muscle cells (PASMCs). Aim:
To investigate the role of BMP-2 in regulation of PTEN and
apoptosis of PASMCs under hypoxia. Methods: Normal
human PASMCs were cultured in basal medium (BM) or
growth medium (GM) and treated with BMP-2 from 5–80 ng/
ml under hypoxia (5% CO2+ 94% N2+1% O2) for 72 hours.
Gene expression of PTEN, caspase-3, -8, and -9, AKT-1
and -2 were determined by quantitative RT-PCR (QRT-PCR).
Protein expression levels of PTEN, AKT and phosph-AKT
(pAKT) were determined. Apoptosis of PASMCs were
determined by measuring activities of caspases-3, -8.
and -9. siRNA-smad-4 was used to determine whether
Smad signaling pathway was involved in the regulation of
PTEN expression by BMP-2. bpV(HOpic) (PTEN inhibitor)
and GW9662 (PPAR-r antagonist) were also used. Results:
Proliferation of PASMCs showed dose dependence of BMP2, the lowest proliferative rate was achieved at 40–60ng/ml
under hypoxia (BM=77.2±4%, GM=80±2.8%). Increased gene
expression levels of PTEN, caspases-3, -8 and -9 were found,
while AKT-1, and -2 did not change. Consistently, the PTEN
protein expression also showed dose dependence of BMP-2.
Though AKT was unchanged in all treated samples, reduced
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ABSTRACTS
39
Poster Presentations (continued)
manner in cultured cells but this has not been tested in intact
animals or CMs. Here we sought to address these important
issues. Using the GFP-LC3 reporter, we demonstrate in both
cell cultures and intact mice that PSMI upregulates p62 and
increases autophagic flux in CMs. This is consistent with the
increased autophagic flux in proteinopathic mouse hearts in
which proteasome function is inadequate. We then tested
the effect of AI on the degradation of a surrogate proteasome
substrate (GFPu) in cultured CMs. GFPu was expressed
in cultured neonatal rat ventricular myocytes (NRVMs) via
adenoviral delivery. Compared with the vehicle treatment, a
24-hr treatment but not 6-hr treatment of autophagy inhibitor
3-methyladenine (3-MA) or lysosome inhibitor bafilomycin
A1 (BFA) significantly increased GFPu and p62 proteins in
cultured NRVMs. Next, we tested the effect of AI on UPSmediated protein degradation in intact mice. Transgenic mice
overexpressing GFPdgn, a previously validated surrogate
proteasome substrate, were treated with BFA (1.6 mg/
kg/12hr, i. p.). BFA induced lysosome inhibition was verified
by a significant increase in LC3-II and p62. Myocardial
GFPdgn protein levels were significantly increased 24hrs
but not 3hrs after the first BFA injection. The protein level of
representative proteasome subunits was not affected by AI.
Our data demonstrate that PSMI activates autophagy while
chronic AI impairs the degradation of proteasome substrates
in the heart. It will be interesting and important to test the role
of p62 in the interplay.
P66Imaging Autophagy in Living Mice
Roberta A Gottlieb, M R Sayen, Chengqun Huang, Jennifer Ramil,
Bruce Ito, Robert M Mentzer Jr, San Diego State Univ, San Diego, CA
Autophagy is a homeostatic response to cellular stress. It
has been shown to be potently upregulated in the heart in
response to a variety of interventions. However, to date, it has
not been possible to monitor autophagy without sacrificing the
animal. Here we report the use of the Caliper Life Sciences
Spectrum In Vivo Imaging System (IVIS) to image autophagy in
homozygous transgenic mice expressing mCherryLC3 under
control of the alpha myosin heavy chain promoter. Autophagy
was stimulated by the administration of rapamycin (2mg/
kg), and autophagosomal flux was blocked by administration
of chloroquine (10mg/kg) ip. Mice were imaged at baseline
and four hours later, using a protocol of 3 acquisitions of
15 seconds each. Total flux was 3.19+0.72 before drug
administration and 3.93+1.10 after 4 hr, p<0.01, n=14. These
results show for the first time imaging of autophagy in hearts
of live mice.
. Wang: None. Z. Tian: None. Q. Zheng: None. H. Su: None.
C
J. Li: None. X. Wang: None.
This research has received full or partial funding support from the
American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa,
Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota,
South Dakota & Wisconsin).
P70NEU3 Sialidase Overexpression Activates Cell Survival
in Murine Skeletal Myoblasts C2C12 and Protects Them
from Hypoxia
Luigi Anastasia, Brigham and Women’s Hosp, Boston, MA; Raffaella
Scaringi, Nadia Papini, Univ of Milan, Milano, Italy; Andrea Garatti,
Lorenzo Menicanti, IRCCS Policlinico San Donato, San Donato
Milanese, Italy; Pietro Allevi, Bruno Venerando, Univ of Milan, Milano,
Italy; Guido Tettamanti, IRCCS Policlinico San Donato, San Donato
Milanese, Italy; Cecilia Gelfi, Univ of Milan, Milano, Italy; Annarosa Leri,
Brigham and Women’s Hosp, Boston, MA
ABSTRACTS
.A. Gottlieb: None. M.R. Sayen: None. C. Huang: None. J. Ramil:
R
None. B. Ito: None. R.M. Mentzer: None.
P67Not published at presenter’s request.
P68Withdrawn
P69Interplay Between the Ubiquitin-Proteasome System and
Autophagy in the Heart
Changhua Wang, Zongwen Tian, Qingwen Zheng, Huabo Su, Jie Li,
Xuejun (XJ) Wang, Univ of South Dakota, Vermillion, SD
The ubiquitin-proteasome system (UPS) and autophagy are
responsible for the degradation of most cellular proteins.
Dysfunctions in the UPS and autophagy are both implicated
in cardiac pathogenesis. The UPS and autophagy, thought to
be two parallel pathways, seem to interact. Pharmacologically
induced proteasome inhibition (PSMI) was shown to increase
autophagosomes in cultured cardiomyocytes (CMs) but this
observation remains to be demonstrated in intact animals
and it is unclear whether the increased autophagosomes
are caused by an increased autophagic flux. A recent report
shows that chronic autophagic inhibition (AI) impairs the
degradation of proteasomal substrates in a p62-dependent
40
Membrane-bound sialidase NEU3 increase during skeletal
muscle differentiation has been shown to protect myoblasts
from apoptosis and drive the differentiation process [1].
Thus, the objective of this study was to assess whether
up-regulation of NEU3 would enhance the ability of murine
skeletal muscle cells to resist to hypoxia, ultimately opposing
cell death. We found that C2C12 myoblasts overexpressing
NEU3 (L-NEU3) became highly resistant to 1% oxygen or
200 mM deferoxamine induced hypoxia. Moreover, L-NEU3
myoblasts survived a seven-day treatment of combined
hypoxia and low serum (2% horse serum used to induce
myoblast differentiation), without any significant cell loss.
On the contrary, wild type C2C12 could not resist to these
culturing conditions and all died within 48h. Real Time
PCR showed NEU3 expression increase during all hypoxic
treatments both in C2C12 and L-NEU3 cells, suggesting
an endogenous NEU3 activation under these conditions.
Moreover, we found that NEU3 over-expression activated
pro-survival signalling pathways through up-regulation and
activation of EGF receptor. Overall, our data support the
hypothesis that NEU3 may play a critical role in the response
of skeletal myoblasts to hypoxia and the preservation of cell
viability by activating pro-survival signalling pathways. [1]
Anastasia L. et al. J.Biol.Chem. 2008, 283 (52): 36265–36271.
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Poster Presentations (continued)
L. Anastasia: None. R. Scaringi: None. N. Papini: None. A. Garatti:
None. L. Menicanti: None. P. Allevi: None. B. Venerando: None.
G. Tettamanti: None. C. Gelfi: None. A. Leri: None.
P71Not published at presenter’s request.
P72Blunted β-Adrenergic Response in R92L Cardiac Troponin
T Mutant Hearts Occurs via Decreased Accessibility to
PKA-Mediated Phosphorylation Sites at Serine 22/23
Residues of Cardiac Troponin I
Jesus Jimenez, Jil C Tardiff, Albert Einstein Coll of Med, Bronx, NY
While diastolic dysfunction due to impaired relaxation is
a classic finding in patients with Familial Hypertrophic
Cardiomyopathy (FHC), the primary cellular mechanisms
remain undefined. We have previously demonstrated impaired
relaxation in our transgenic mouse models of FHC carrying
the cTnT R92L mutation. We hypothesize that the impaired
relaxation in RL is a result of an allosterically mediated,
diminished structural accessibility to the PKA phosphorylation
sites on cTnI. Protein levels of phosphorylated TnI at baseline
and following stimulation with isoproterenol were significantly
lower for RL compared to non-transgenic (NT) mice while
protein levels of phosphorylated phospholamban (PLB), TnI,
and PLB were the same between both groups. These results
indicate that PKA signaling is intact and uncoupling occurs
at the myofilament level. Next, we crossed RL mice with
phosphomimetic mice that have had their cTnI residues S22S23
changed to D22D23 to generate the double transgenic RL/
DD. Preliminary isovolumic studies demonstrated improved
contractility and relaxation for RL/DD compared to RL alone
but did not reach levels observed for NT following graded
doses of dobutamine (Figures A and B). In Ca2+ transient
measurements of isolated adult cardiac myocytes, DD cells
demonstrated enhanced peak rates of Ca2+ rise and fall as
well as accelerated times to 50% and 90% Ca2+ declines
compared to NT and RL. However, RL/DD mice did not show
improvement in any of these parameters. Collectively, these
results indicate that the diastolic dysfunction observed in R92L
is caused by direct impairment of the myofilament axis in the
beta adrenergic signaling cascade.
P73Ca2+ Dishomeostasis and Unfolded Protein Response in
Idiopathic Dilated Cardiomyopathy: The Role of Presenilin 2
Cristina Balla, Beth Israel Deaconess Medical Ctr, Boston, MA; Davide
Gianni, UMASS Medical Sch, Worcester, MA; Khaushik Subramanian,
Bernhard Haring, Nicole Koulisis, Eduward Goihberg, Beth Israel
Deaconess Medical Ctr, Boston, MA; Massimo Volpe, Sapienza Univ .
di Roma, Rome, Italy; Federica del Monte, Beth Israel Deaconess
Medical Ctr, Boston, MA
Idiopathic dilated cardiomyopathy (iDCM) is a degenerative
disease characterized by abnormalities in the function and
integrity of cardiomyocytes, alterations in contractile proteins
and abnormal Ca2+ dynamics. Endoplasmic reticulum (ER)
plays an essential role in Ca2+ homeostasis, but it is also the
site for protein synthesis, post-translational modification and
folding. Changes in Ca2+ homeostasis and in the cellular
mechanism of protein quality control may reciprocally sustain
and contribute to the pathogenesis of the cardiac diseases.
Genetic variants of presenilin 2 (PSEN2), one of the major
genes involved in the pathogenesis of the best known disease
C. Balla: None. D. Gianni: None. K. Subramanian: None. B. Haring:
None. N. Koulisis: None. E. Goihberg: None. M. Volpe: None.
F. del Monte: None.
P74Loss of Function of TRPC4 Protects Against Cardiac
Dysfunction Progression After Myocardial Infarction
Hongyu Zhang, Catherine Makarewich, Temple Univ, Philadelphia,
PA; Erhe Gao, Thomas Jefferson Univ, Philadelphia, PA; Hajime Kubo,
Hui Gao, Fang Wang, Remus Berretta, Temple Univ, Philadelphia,
PA; Walter Koch, Thomas Jefferson Univ, Philadelphia, PA; Jeffery
Molkentin, Univ of Cincinnati, Cincinnati, OH; Steven Houser, Temple
Univ, Philadelphia, PA
The source of Ca2+ to hypertrophic signaling after myocardial
infarction (MI) is not clearly defined. Transient Receptor
Potential Canonical (TRPC) channels could be an important
source of hypertrophic Ca2+ after MI. The objective of this
study was to determine if TRPC 4 is a major source of Ca2+
influx mediating cardiac dysfunction after MI. Methods:
Cardiac-specific transgenic mice that express a dominantnegative (dn) TRPC4 that reduces the activity of the TRPC1/4/5
subfamily of channels in the heart were used. MI was
produced and in-vivo cardiac function was measured with
ECHO. Myocytes were isolated and isoproterenol (ISO) effects
on LTCC Current (ICa-L), fractional shortening (FS) and Ca2+
transients were measured 6 weeks after MI. Results: Baseline
ejection fraction (EF) and fractional shortening (FS) were
greater in (dn) TRPC4 vs. WT mice. Two weeks after MI, EF
and FS were significantly decreased in all animals (WT: 37.1%
and 18.2%; (dn) TRPC4: 41.7% and 20.5%), but there was
no significant difference between WT and (dn) TRPC4 mice.
Six weeks after MI, EF and FS were significant greater in (dn)
TRPC4 compared with WT mice (WT: 37.4% and 18.2%; (dn)
TRPC4: 52.2% and 27.4%). Heart weight and lung weight
were significantly increased after 2 weeks MI, but there were
significant lower heart and lung weight in (dn) TRPC4 vs. WT
mice after 6 weeks MI. ICa-L after 6 weeks MI was smaller than
that in sham myocytes, and there was no significant difference
between (dn) TRPC4 and WT myocytes. Contractions and
Ca2+ transients were significantly greater in sham and post-MI
(dn) TRPC4 vs. WT myocytes. ISO increased contractions
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
J. Jimenez: None. J.C. Tardiff: None.
of protein folding (Alzheimer disease), were described in iDCM
potentially contributing to the changes in Ca2+ homeostasis.
In this study, we evaluated the cellular response to the ER
stress (the unfolded protein response — UPR) in human
iDCM and, using a mouse model of misfolding disease — the
PSEN2 KO — we evaluated the role of ER Ca2+ homeostasis
in the UPR. Protein expression and mRNA levels for the
components of the UPR were measured in 9 iDCM and 7
donor hearts. In iDCM tissue, the expression of all UPR protein
tested was increased indicating an overall activation of the ER
stress response. PSEN2 KO mice showed a failing phenotype
in vivo (LVEDD p= 0.002; SW wall thickness p= 0.002; EF
p=0.005). In vitro, PSEN KO isolated cardiomyocytes showed
20% reduction in cell shortening at baseline. After infusion of
thapsigargin cells showed a 50% reduction in cell shortening
and shortening velocity, 80% reduction in all diastolic velocity
parameters, 25% and 60% increase in systolic and diastolic
Ca2+ respectively. The expression pattern of EC coupling
proteins showed a decrease in the expression of L-type
Ca2+ channels, Ryanodine Receptor and phospholamban.
Moreover the expression of UPR proteins demonstrated
an overall reduction. In conclusion, we described for the
first time a chronic activation of the UPR in human iDCM
induced, or further sustained, by the well-described ER
Ca2+ disequilibrium. Mice models of PSEN2 KO showed
that PSEN2 plays a role in the cardiac function and ER Ca2+
homeostasis. Further, changes in protein quality control in the
heart may be secondary to the changes in Ca2+ homeostasis.
41
Poster Presentations (continued)
and Ca2+ transients to a similar extent in all myocytes.
Conclusions: (dn) TRPC4 mice have greater baseline cardiac
and myocyte function. While initial effects of MI were similar
to control, there was improved function in these mice by 6
weeks. These results suggest that blocking TRPC4 after MI
may reduce pathological cardiac remodeling.
inhibited by KN93. In WT myocytes, KN93 decreased betaAR stimulated contraction (Ca2+ transients (Fluo-4) and
cell shortening). In NOS1-/- myocytes, beta-AR stimulated
contraction was blunted compared to WT, and KN93 had
no further effect on contraction. Furthermore, beta-AR
stimulated RyR activity was blunted in NOS1-/- compared to
WT myocytes. As with contraction, KN93 decreased beta-AR
stimulated RyR activity in WT myocytes, but had no effect
in NOS1-/- myocytes. Conclusion: These data suggest that
NOS1 is required for CaMKII-mediated RyR activation which
contributes to positive inotropy during beta-AR stimulation.
Further study of this pathway is warranted since CAMKII
expression and activity are increased in cardiac hypertrophy
and heart failure. A better understanding of the NOS1/CaMKII
pathway during beta-AR stimulation has beneficial therapeutic
potential for heart diseases.
H. Zhang: None. C. Makarewich: None. E. Gao: None. H. Kubo:
None. H. Gao: None. F. Wang: None. R. Berretta: None. W. Koch:
None. J. Molkentin: None. S. Houser: None.
P75Neuronal Nitric Oxide Synthase Contributes to the
Beneficial Cardiac Effects of Exercise
Steve R Roof, Joseph Ostler, Muthu Periasamy, Mark T Ziolo,
The Ohio State Univ, Columbus, OH
ABSTRACTS
Exercise results in beneficial adaptations to the heart. These
adaptations are observed at the level of the cardiomyocyte as
increased Ca2+ cycling through the sarcoplasmic reticulum
(SR) and greater fractional shortening. Interestingly, these
adaptations are similar to the contractile effects of neuronal
nitric oxide synthase (NOS1) signaling. Thus our objective is
to determine if the exercise induced adaptations at the level
of the cardiomyocyte are NOS1 mediated. After an 8 week
high-intensity aerobic interval training program, exercise
(Ex) mice had a higher VO2max, greater citrate synthase
activity, decreased weight, increased heart-to-body ratio
(P;0.05), but similar heart-to-tibia ratio compared to sedentary
(Sed) mice. Isolated ventricular myocytes from the Ex mice
exhibited larger Ca2+ transient and shortening amplitudes
and faster Ca2+ transient decline rates (RT50) compared
to Sed myocytes (P;0.05). There was also greater NOS1
expression levels in Ex ventricular myocytes (P;0.05 vs Sed).
The greater NOS1 expression was observed functionally in
myocyte contraction. That is, acute inhibition of NOS1 by
S-methyl-L-thiocitrulline (SMLT) resulted in a greater reduction
in Ca2+ transient amplitude, Ca2+ transient RT50, shortening
amplitude, SR Ca2+ load, and SR Ca2+ fractional release in
Ex versus Sed (P;0.05). Furthermore, acute NOS1 inhibition
(SMLT) normalized the Ex induced increase in contraction
(Ca2+ transients and shortening) and Ca2+ decline rates to
Sed levels. These data demonstrate that the enhanced Ca2+
cycling through the SR observed after a training period is, in
part, due to NOS1 signaling. Thus, NOS1 is a major contributor
to the increased contractile amplitudes and relaxation observed
with exercise. Hence, mimicking the beneficial effects of
exercise to the heart may be obtainable by enhancing NOS1
signaling. This pathway may be a novel therapeutic for cardiac
patients that are unable/unwilling to exercise.
42
Xiaoxu Zheng, Lingyun Zu, Zheqing P Cai, Johns Hopkins Univ,
Baltimore, MD
ackground: Ischemic preconditioning (IPC) increases
B
post-ischemic functional recovery. Although reduced infarct
size is associated with the better outcome, its underlying
mechanism is not fully understood. The phosphatase and
tensin homologue deleted on chromosome ten (PTEN)
promotes cell death and increases myocardial contractility.
We recently reported that IPC attenuates loss of PTEN
activity in post-ischemic hearts. In the present study, we have
investigated the hypothesis that IPC improves post-ischemic
cardiac function by preserving PTEN activity. Methods and
Results: Isolated mouse hearts were exposed to no ischemia
as control (CON) or IPC, consisting of 10-min ischemia
and 5-min reperfusion (I-10/R-5), followed by I-30/R-120.
bpV(phen) (10 µM), a PTEN inhibitor, or vehicle was added into
the perfusion line in IPC group following reperfusion. Isolated
hearts from muscle-specific PTEN knockout mice (Ptenloxp/
loxp
;ckm+/-) and control mice (Ptenloxp/loxp;ckm-/-) were exposed
to I-30/R-30. Left ventricular (LV) pressure was monitored by
a pressure catheter. Myocardial infarct size was measured by
triphenyltetrazolium chloride staining. After I-30/R-120, IPC
increased LV developed pressure (LVDP) and maximal +dp/
dt (+dp/dtm) compared with CON. bpV blocked the effects
of IPC (LVDP: 42±4 vs. 63±4 mmHg, p<0.01; +dp/dtm:
1209±101 vs. 1880±155 mmHg/sec, p<0.01). However, it
did not increase infarct size compared with vehicle. Musclespecific PTEN knockout modestly decreased LVDP and +dp/
dtm in isolated perfused hearts under basal conditions, but
it markedly impaired their recovery after I-30/R-30 compared
with control (LVDP: 21±4 vs. 45±5 mmHg, p<0.01; +dp/dtm:
784±146 vs. 1795±154 mmHg/sec, p<0.01). Myocardial
infarct size was decreased in PTEN knockout hearts after
I/R. Conclusions: PTEN inhibition blocks the improvement
of cardiac functional recovery induced by IPC. Loss of PTEN
exacerbates post-ischemic dysfunction in isolated hearts.
Therefore, our studies suggest that IPC attenuates postischemic cardiac dysfunction by preserving PTEN activity.
X. Zheng: None. L. Zu: None. Z.P. Cai: None.
S.R. Roof: None. J. Ostler: None. M. Periasamy: None. M.T. Ziolo: None.
P76Essential Role of Neuronal Nitric Oxide Synthase in Ca2+/
Calmodulin-Dependent Protein Kinase II Activation in
Cardiac Myocytes During β-Adrenergic Stimulation
Lifei Tang, Steve Roof, Mark Ziolo, The Ohio State Univ, Columbus, OH
ationale: Stimulation of the beta-adrenergic (beta-AR)
R
pathway leads to positive inotropy, and is the major regulator
of heart function. In addition to the traditional PKA pathway,
activation of Ca2+/calmodulin-dependent protein kinase II
(CaMKII) and neuronal nitric oxide synthase (NOS1) signaling
also play important roles in the positive inotropy by modulating
ryanodine receptor (RyR) activity. Objective: The upstream
activators of CaMKII during beta-AR stimulation are not
well defined. The purpose of this study is to investigate
if there is any cross-talk between the CaMKII and NOS1
signaling pathways. Methods and Results: Myocytes were
isolated from wildtype (WT, C57Bl/6) and NOS1-/- mice.
Ca2+ transients (Fluo-4) and cell shortening (edge detection)
were simultaneously measured. RyR activity was measured
using the SR Ca2+ leak/load relationship. CaMKII was acutely
L. Tang: None. S. Roof: None. M. Ziolo: None.
P77Ischemic Preconditioning Improves Postischemic Cardiac
Function by Preserving PTEN Activity
P78Dysregulated Calcium Handling in a Conditional
Cardiac Myosin Binding Protein C Knockout Model of
Hypertrophic Cardiomyopathy
E Michelle Capes, Peter P Chen, Daniel P Fitzsimons, Hector H.
Valdivia, Richard L Moss, Univ of Wisconsin — Madison, Madison, WI
Heritable cardiomyopathy (HCM) is the leading cause of
sudden cardiac arrest (SCA) in young people, affecting 1
in 500 individuals. HCM is chiefly caused by mutations in
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Poster Presentations (continued)
myofibrillar proteins of the cardiac sarcomere, and cardiac
myosin binding protein-C (cMyBP-C, encoded by MYBPC3) is
one of the most commonly affected. cMyBP-C, an accessory
protein that binds tightly to myosin, has an important role
in thick filament regulation. Mice with genetic ablation of
MYBPC3 exhibit cardiac hypertrophy, reduced ejection
fraction, and increased relaxation times in vivo. Experiments
with explanted hearts from these mice exhibit greater
susceptibility to arrhythmias compared to WT, suggesting
derangement of Ca2+ handling. The molecular mechanisms
underlying the progression of HCM are poorly understood,
and are difficult to tease apart in constitutive knock out
models due to potential compensatory changes that can
mask important aspects of the disease phenotype. We used
a tamoxifen-induced conditional MYBPC3 knock out mouse
to investigate the onset and progression of the disease
before development of overt hypertrophy. We recorded
Ca2+ transients in enzymatically isolated cardiomyocytes at
several pacing frequencies using a META LSM 510 confocal
microscope. We found that cell shortening in conditional CMs
(con-CMs) was reduced by as much as 54% (e.g., 5.46%
± 0.869 in con-CMs, vs. 11.91% ± 0.423 shortening in
WT-CMs). Ca2+ release was often dissynchronous, possibly
indicating remodeling at the diads. Ca2+ transient amplitude
was significantly lower in con-CMs across all pacing
frequencies (reduced by at least 22%; e.g., fluorescence
intensity ratios of 7.983 ± 0.537 in con-CMs vs. 10.332 ±
0.502 in WT CMs). Further, the time constant of transient
decay (tau) was dramatically increased in con-CMs (mean
217.27 ± 18.052 msec for con-CMs vs. 107.44 ± 4.045 msec
in WT CMs). In some cells (~5%), this prolongation of transient
decay was so profound (e.g., up to 600 milliseconds) that a
second Ca2+ release occurred before [Ca2+]i could return to
baseline. These data suggest that conditional knock out of
cMyBP-C produces not only contractile dysfunction, but also
pro-arrhythmic alterations in Ca2+ handling.
.M. Capes: None. P.P. Chen: None. D.P. Fitzsimons: None. H.H.
E
Valdivia: None. R.L. Moss: None.
P79Cardiac Contractility in Response to Isoproterenol from
Gravin Knockout (AKAP12) Mice
Bradley McConnell, Xing Yin, Univ of Houston, Houston, TX; Zhitao Li,
Johns Hopkins Univ Sch of Med, Baltimore, MD; Cori Wijaya, Andrea
Diaz, Muhammad Siddiqui, Abeer Rabaha’h, Ashley Guillory, Univ of
Houston, Houston, TX; Wei Dong Ga, Johns Hopkins Univ Sch of Med,
Baltimore, MD
. McConnell: None. X. Yin: None. Z. Li: None. C. Wijaya: None.
B
A. Diaz: None. M. Siddiqui: None. A. Rabaha’h: None. A. Guillory:
None. W. Ga: None.
P80Bestrophin: A Potential Mediator of Cardiac
Calcium Handling
Fiona C Britton, Steeven John, Kate E O’Driscoll, Martha Baring, Univ
of Nevada, Reno, Reno, NV
Bestrophins are a family of transmembrane proteins expressed
in heart. We reported that Bestrophin 1 and 3 cloned from
mouse heart function as calcium-activated chloride channels.
We hypothesized that bestrophin channels may function
in a macromolecule complex similar to other cardiac ion
channels. To identify proteins that bind Bestrophin 3 channel,
we screened a mouse heart cDNA library using 4 different
cytosolic cDNA fragments as bait. Yeast two-hybrid assays
were performed with the GAL4 system. Using bait consisting
of C-terminus amino acid residues 386 to 669 of the Best3
channel (Best3-C2), we obtained positive interactions with
the histidine-rich calcium binding protein (hrc). Hrc is a
component of the SR complex involved in calcium handling in
the heart. Eleven independent clones represented a fragment
encoding the cysteine rich domain of hrc previously shown
to interact with triadin. To further map the interaction sites
between Best3-C2 and hrc, we subdivided this region into
three fragments and tested these baits for interaction with
hrc. Two Best3 fragments (residues 541–600 and residues
601–669) displayed strong interaction with hrc, while Best3
bait (residues 386–540) failed to interact. This region of Best3
contains a KEKE protein binding motif. In a similar manner
C-terminal Best 1 fragments were confirmed to also interact
with hrc. In this study we report that Bestrophin channels
interact with hrc, a cardiac calcium handling protein. Calciumactivated chloride channels play a critical role in excitationcontraction coupling in the SR by balancing charge movement
during calcium release and reuptake. Recent evidence
confirms Bestrophin 1 is located in the ER membrane in
association with Stim1 and facilitates calcium cycling. Future
in vivo investigations will examine the role of bestrophins as
potential mediators of calcium handling in heart.
ABSTRACTS
Gravin (AKAP12), one of the A-Kinase-Binding-Proteins
(AKAPs), serves as a scaffold protein linking β2-adrenergic
receptor (β2-AR), phosphatase 2B and several kinases
including Protein Kinase A (PKA) and Protein Kinase C (PKC).
The presence of gravin facilitates signal transduction of β2-AR
and thus affects cardiac excitation-contraction coupling.
We previously showed decreased cardiac hypertrophy,
increased contractility and decreased arrhythmias in gravinKO mice, following chronic β-AR stimulation. This response
was associated with enhanced [Ca2+]i homeostasis. Here,
we test whether cardiac contraction is also affected in gravin
knock-out (gravin-KO) mice. Trabeculae or small papillary
muscles from the right ventricles were mounted between
a force transducer and a motor arm, and superfused with
K-H solution (pH 7.4) at room temperature. Developed
force increased as external Ca2+ ([Ca2+]o) was raised from 1
to 10 mM in both gravin-KO and wide-type (WT) muscles.
Developed force and intracellular Ca2+ transient (Ca2+i)
increased in a dose dependent manner as [Ca2+]o was raised.
Gravin-KO muscles had lower Ca2+i at any given [Ca2+]o,
but had similar force at [Ca2+]o >4.0 mM; at higher [Ca2+]os,
developed forces was significantly lower. Isoproterenol (ISO,
0.05-200nM) increased force and Ca2+i in both groups of
muscles. However, force and Ca2+i started to blunt in gravinKO muscle at higher ISO doses and became significantly
lower at ISO does >50 nM. Both force and Ca2+i were lower at
a given rest interval in gravin-KO muscles with no changes in
recirculation fraction (RF). These results show that gravin-KO
muscles maintain their response to both Ca2+ and ISO with
reduced capacity at higher doses, and to rest potentiations
with lower magnitudes, but with no changes in recirculation
fraction of Ca2+ through the sarcoplasmic reticulum (SR).
These findings suggest: (1) gravin-KO muscles may likely have
smaller Ca2+ content in the SR; (2) gravin plays an important
modulatory role in the argumentation of force by Ca2+ and β2AR stimulation; (3) at physiological [Ca2+]o, gravin-KO increases
myofilament Ca2+ responsiveness. Ongoing experiments are
focused on changes myofilament Ca2+ responsiveness in
gravin-KO mice.
.C. Britton: None. S. John: None. K.E. O’Driscoll: None.
F
M. Baring: None.
P81Voltage-Gated TTX-Sensitive Nav1.1 Brain Sodium
Channels Are Important for Cardiac Contractile Function
Susan T Varghese, Stephanie Humphrey, An Xie, Andrew P Escayg,
Samuel C Dudley Jr, Univ of Illinois at Chicago, Chicago, IL
Background: Mutations in voltage gated brain sodium
channel Nav1.1 have been linked to many disorders, including
Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and
Severe Myoclonic Epilepsy of Infancy (SMEI). Recent studies
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43
Poster Presentations (continued)
have identified TTX- sensitive Nav1.1 brain sodium channels
in the SA node and ventricular T-tubules of the heart, though
their role in cardiac function is still controversial. We tested
the functional significance of Nav1.1 sodium channels in the
heart by creating a novel knock-in of human epilepsy GEFS+
mutation SCN1A-R1648H at the Scn1a locus of a C57BL/6J
X 129 mouse. Method: In vivo 2-D echocardiography was
performed on 2 week old (juvenile) and 8 week old (adult) wildtype and heterozygote (Scn1aRH/+) mice after extracardiac
neuronal block through intraperitoneal injections of atropine
and propranolol (2.5mg/kg each). Calcium and contractility
studies on adult ventricular cardiomyocytes isolated from
the wild type and Scn1aRH/+ mice paced at 0.5Hz were
followed by administration of TTX (100nM, a brain sodium
channel inhibitor) and pacing at 2Hz. qRT-PCR and Western
blot of the isolated cells and whole heart samples was also
done. Results: A decrease in Nav1.1 gene expression in the
Scn1aRH/+ juvenile (by 31%, 0.69 of 1) and adult (by 60%,
0.4 of 1) whole heart samples and isolated cells (p<0.05) was
seen. Echocardiography revealed concentric hypertrophy in
the juvenile Scn1aRH/+ mice by a significant increase in LV
mass, LV mass/body weight ratio, and relative wall thickness
(p<0.05). In the adult Scn1aRH/+ mice, systolic isovolumic
contraction time (IVCT) was reduced (p=0.03) and decrease
in diastolic function was evident through significant decreases
in isovolumic relaxation time (IVRT) and E’/A’ ratio, and
increase in E/E’ ratio. Isolated adult ventricular Scn1aRH/+
cardiomyocytes showed significant reduction in percent
sarcomere shortening, maximum rate of contraction and
relaxation, and time to peak contraction, exaggerated with
TTX and pacing at 2Hz (p<0.5). Conclusions: Our study
demonstrates the importance of voltage gated TTX-sensitive
Nav1.1 brain sodium channels in cardiac contractile function
and their possible role in cardiac complications in epilepsy.
S.T. Varghese: None. S. Humphrey: None. A. Xie: None.
A.P. Escayg: None. S.C. Dudley: None.
P82Cardiac Calcium Handling and Protein Phosphatase 2A
Activity Are Differentially Regulated by JNK1 and JNK2
MAP Kinases
ABSTRACTS
Honey B Golden, TAMHSC CVRI, Temple, TX; Linley E Watson, Scott
& White Memorial Hosp, Temple, TX; Donald M Foster, David E Dostal,
TAMHSC CVRI, Temple, TX
44
We previously identified the JNK-B56α-PP2A signaling axis
as a major target in anthrax lethal toxin (LT)-induced cardiac
dysfunction. Thus, we further tested whether LT-mediated loss
in cardiac function is a consequence of dysregulated calcium
handling resulting from JNK inactivation. To biochemically
recapitulate the signaling effects of LT, we infected NRVM
with HA-B56α adenovirus and determined PP2A activity as
well as Ca2+i measurements. Over-expression of HA-B56α in
NRVM did not induce a significant increase in cellular PP2A
activity, however, it did induce a significant increase (p<0.01)
in Ca2+i compared to virus control. Furthermore, PLB, PP2Ac
and Akt protein co-immunoprecipitated with HA-B56α, and
immunostaining revealed colocalization of B56α with PLB at
the SR. Since B56α over-expression alone was not sufficient
to induce PP2A activity or PP2A-mediated Ca2+i dysregulation,
we hypothesized that JNK may serve as a functional regulator
of Ca2+i handling through PP2A activation as well as B56α
protein levels. Adenoviral-mediated over-expression of MEK7
in NRVM resulted in a significant reduction in LT-mediated
Ca2+i dysregulation compared to virus control (p<0.01).
To further determine whether the protection of MEK7 is
mediated by JNK1 or JNK2, gain-of-function/loss-of-function
experiments were performed utilizing adenoviral constructs for
CA-MEK7, DN-JNK1 and DN-JNK2. Results confirmed that
the protective effect of active MEK7 over-expression on Ca2+i
was significantly lost with knock-down of JNK1 compared to
JNK2 (p<0.05), suggesting that JNK1 plays a more substantial
role in regulating PP2A activity than JNK2. Furthermore, the
selective knock-down of JNK1 also increased Ca2+i levels
(p<0.001) compared to MEK7 during LT treatment, which
reveals the importance of JNK1 in PP2A-mediated Ca2+i
dysregulation. Interestingly, immunoblotting of PLB did not
reveal a JNK-dependent difference in PLB phosphorylation
at Ser16, however, loss of JNK2 almost completely inhibited
p-PLB-Thr17. Thus, our results suggest that JNK1 and JNK2
may differentially regulate Ca2+i through PP2A activation and
PLB-Thr17 phosphorylation, respectively.
H.B. Golden: None. L.E. Watson: None. D.M. Foster: None.
D.E. Dostal: None.
P83Post-Cardiac Arrest Syndrome Is a Form of Cardiac
Memory that Leads to Impaired Actin-Myosin Crossbridge
Activity Despite Increased Calcium Release
Christopher Woods, Stanford, Stanford, CA; Fouad Taghavi,
Papworth Hosp NHS Fndn Trust, Cambridge, United Kingdom; James
Spudich, Euan Ashley, Stanford, Stanford, CA
Sudden Cardiac Arrest (SCA) is a major health problem lethal
to more than 300,000 Americans yearly. Survival post-arrest
is complicated by post-cardiac arrest syndrome (PCAS),
whereby the heart is left stunned leading to hypotension and
an associated 30% higher mortality in survivors of arrest. We
developed an in vivo model of PCAS involving rat hypoxic
cardiac arrest (HCA). Following HCA, and specific warm
anoxic times to mimic down time, cardio-pulmonary bypass
was used to resuscitate the animal.Invasive hemodynamic
measurements demonstrated that the end-systolic pressure
volume relationship was reduced from 1.72±0.1 to 0.86±0.2
in after HCA and resuscitation (p<0.001), recapitulating PCAS.
To explore the cellular basis for it, we examined excitationcontraction coupling (ECC) in isolated adult left ventricular
cardiac myocytes (LVMs) from HCA and control cells. After
loading the low affinity calcium indicator Fluo-5F, LVMs
robustly twitching to electrical pacing and without contracture
were identified and placed on the stage of an epifluorescence
microscope adapted for both electrical pacing and the twocarbon fiber stretch (2CFS) technique.Single cell force and
electrically evoked calcium induced calcium release (CICR)
were measured simultaneously.Peak systolic force (PSF) and
the frank-starling gain (FSG), a novel measure of the single cell
frank-starling effect, were both lower in HCA cells (3.1±0.6
vs 10±2 for FSG, and 2.4±0.4 vs 4.3±0.5 mN/m2 for PSF in
HCA vs control for both; p<0.05 for both).We hypothesized
a reduction in the calcium transient as an explanation since
the SR calcium ATPase activity is an energy intensive cellular
processes. Surprisingly, we found the peak CICR to be
robustly higher in HCA cells(1.7±0.1 vs 1.0±0.1 in HCA vs
control, p<0.001).Together, this data demonstrates that ECC
is impaired in PCAS at the level of myofilament activation,
and enhanced CICR does not sufficiently compensate
this, in similarity to an acidemic effect. Indeed, at times this
augmented CICR proved pro-arrhythmic, a phenomenon not
observed in control or sham. However, in distinction, PCAS
persists in HCA cells under control conditions for up to 6 hours
after isolation, and so represents a form of cardiac memory.
C. Woods: None. F. Taghavi: None. J. Spudich: None. E. Ashley: None.
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Poster Presentations (continued)
P84Cardiac-Specific Overexpression of Human Histidine-Rich
Calcium Binding Protein (HRC-S96A) Genetic Variant
Impairs Sarcoplasmic Ca2+ Handling, Resulting
in Arrhythmias
Vivek P Singh, Wenfeng Cai, Min Dong, Qian Liang, Chi Keung Lam,
Xiaoqian Gao, Shan Chen, Univ of Cincinnati, Cincinnati, OH;
Demetrios A Arvanitis, Biomedical Res Fndn, Acad of Athens, Athens,
Greece; Kobra Haghighi, Hong Sheng Wang, Univ of Cincinnati,
Cincinnati, OH; Do Han Kim, Chunghee Cho, Gwangju Inst of Science
and Technology, Gwangju, Korea, Republic of; Evangelia G Kranias,
Univ of Cincinnati, Cincinnati, OH
.P. Singh: None. W. Cai: None. M. Dong: None. Q. Liang: None.
V
C. Lam: None. X. Gao: None. S. Chen: None. D.A. Arvanitis: None.
K. Haghighi: None. H.S. Wang: None. D.H. Kim: None. C. Cho:
None. E.G. Kranias: None.
This research has received full or partial funding support from the
American Heart Association, Great Rivers Affiliate (Delaware, Kentucky,
Ohio, Pennsylvania & West Virginia).
P85Inhibition of Akt Reverses the Isoproterenol-Enhanced
Diastolic Calcium Leak in Rabbit Ventricular Myocytes
Sathya Velmurugan, Jerry Curran, Thomas Shannon, Rush Univ
Medical Ctr, Chicago, IL
Cardiac ryanodine receptor (RyR)-dependent diastolic SR
Ca2+ leak is increased in heart failure and during betaadrenergic (β-AR) stimulation. This increased SR Ca2+ leak
could limit the SR [Ca2+] thereby decreasing contractility in HF
and could also lead to arrhythmogenic Ca-dependent inward
depolarizing current resulting in HF. It has recently been shown
S. Velmurugan: None. J. Curran: None. T. Shannon: None
P86Serca1a Expression Exacerbates Cardiac Arrhythmia and
Leads to Heart Failure and Early Mortality in a Mouse
Model of Exercise-Induced Sudden Cardiac Death
Anuradha Kalyanasundaram, Veronique Lacombe, Andriy Belevych,
Ohio State Univ, Columbus, OH; Bjorn C Knollmann, Vanderbilt Univ,
Nashville, TN; Muthu Periasamy, Sandor Gyorke, Ohio State Univ,
Columbus, OH
Abnormal calcium (Ca2+) handling has been implicated in a
range of cardiac diseases, including heart failure, a disease
state characterized by compromised systolic and/or diastolic
myocardial function and pathological remodeling. Excessive
diastolic SR calcium release via the Ryanodine receptor
(RyR2) has been suggested to deplete the SR Ca2+ store
thereby reducing cardiac contractility. Although abnormal
RyR2 activity and elevated SR Ca2+ leak are characteristic
features of advanced heart failure (HF) in both humans and
animal models, clear evidence is lacking whether altered
RyR2 function plays a causal role in HF development. Indeed,
while nearly 80 mutations in RyR2 have been identified and
associated with SR Ca2+ leak, none of these mutations have
been reliably linked to HF. One possible explanation for this
is that the SR Ca2+ leak caused by the mutations is too small
and/or compensated by other processes involved in Ca2+
homeostasis. In the present study we sought to generate a
genetic model with chronically increased diastolic release by
combining overexpression of the fast skeletal muscle isoform
of SERCA, SERCA1a, with ablation of CASQ2. Strikingly,
the new hybrid strain (CASQ2 null/1a) exhibit early mortality
(avg dying age 39.30days) due to heart failure preceded by
pathological remodeling including eccentric hypertrophy,
significant dilation of the atrial and ventricular chambers,
necrosis induced cell death and widespread fibrosis. Animals
are susceptible to ventricular arrhythmias at baseline in the
absence of adrenergic triggers and show significant systolic
dysfunction. Similarly, cardiomyocytes display increased
SR Ca2+ load along with significant disturbances in RyR2
mediated Ca2+ release at baseline and are increasingly prone
to spontaneous calcium waves. Our results demonstrate
that excessive Ca2+ leak via the RyR2 can lead to severe
contractile dysfunction and heart failure. Since abnormal RyR2
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ABSTRACTS
Disturbed Ca-homeostasis in the cardiomyocyte is a hallmark
of human and experimental heart failure. The histidinerich Ca binding protein (HRC), an intraluminal SR protein
is an additional regulator of SR Ca2+ cycling. Previous
biochemical studies have shown that HRC can bind to triadin
(a component of ryanodine receptors complex) and SERCA2a,
suggesting that HRC may be involved in the regulation of
SR Ca2+ release and Ca2+ uptake. We recently identified
a human HRC genetic variant (S96A), which appears to
correlate with ventricular arrhythmias and sudden death in
dilated cardiomyopathy (DCM) patients. To gain insight into the
physiological and pathological significance of the S96A-HRC
variant, we generated transgenic mice with cardiac-specific
overexpression of human HRC wild-type (HRC-WT) and S96A
mutant (HRC-S96A) in the HRC null (HRC KO) background.
We obtained lines with similar expression levels of HRC-S96A
and HRC-WT for further characterization. Overexpression
of human HRC-S96A resulted in decreased fractional
shortening by 22% (7.7 ± 0.5% in HRC-WTs vs. 6.0 ± 0.4%
in HRC-S96A), rates of contraction by 20% (75 ± 5 µm/sec
in HRC-WTs vs. 60 ± 4 µm/sec in HRC-S96A) and rates of
relaxation by 20% (87 ± 6 µm/sec in HRC-WTs vs. 70 ± 4.5
µm/sec in HRC-S96A) compared with HRC-WT. Myocytes
isolated from HRC-S96A mice had diminished Ca2+ transient
amplitude and delayed half-decay time of the Ca2+ transient.
In addition, the frequency of Ca2+ sparks was significantly
higher although caffeine-induced SR Ca2+ release (SR load)
was reduced in HRC-S96A cells. To determine the effect of
S96A-HRC under stress conditions, 5 Hz field stimulation in
the presence of 1 µmol/L Isoproterenol was applied: aftercontractions were developed in 46% (16 of 35) of HRC-S96A
cardiomyocytes, compared to 12% (4 of 33) of HRC-WTs
mice. The findings of the present study demonstrate that the
human HRC-S96A variant results in impaired myocytes Ca2+
handling, associated with depressed SR Ca2+-uptake rate
and increased rate of SR Ca2+ leak, which may destabilize
the cells, promoting aftercontractions under stress conditions.
Thus, there appears to be a link between this genetic variant
and ventricular arrhythmias in DCM human carriers.
in our lab that this leak is dependent on calcium-calmodulindependent protein kinase II (CaMKII) and subsequent NOS
activation, and that it is independent of either protein kinase A
(PKA) activation or an increase in free Ca2+ concentration in
the cytosol ([Ca2+]i). Here we investigate whether Akt is also
involved in the pathway leading to increased diastolic leak
during β-AR stimulation. Methods: [Ca2+]i was measured
using fluo-4 in left ventricular myocytes isolated from rabbits.
To vary the SR load, myocytes were field-stimulated to
steady state at different frequencies. Tetracaine (1mM) was
used to rapidly and reversibly block the RyR. The tetracainedependent shift of Ca2+ from the cytosol to the SR (decrease
in [Ca2+]i and increase in SR Ca2+ content) is proportional to
SR Ca2+ leak. To activate β-AR, isoproterenol (ISO; 250 nM)
was added to perfuse the myocytes starting 5 min before leak
assessment through the end. Results: ISO-induced increase
in the SR Ca2+ leak is abolished by the treatment of myocytes
with Akt inhibitor X (AIX; 5μM; 30min pre-incubation and
perfusion throughout the experiment). When SR Ca2+ load
was matched in each group (ISO alone: 155.22 ± 5.1 μM; ISO
+ AIX: 153.92 ± 3.8 μM), myocytes treated with ISO alone
had significantly higher leak (12.43 ± 3.8 μM) vs. those treated
with ISO and AIX (1 ± 2.2 μM) (P = 0.01, t-test). This evidence
indicates that Akt activation may also be involved in the β-ARinduced SR Ca2+ leak possibly upstream from CaMKII and
NOS activation.
45
Poster Presentations (continued)
activity has been implicated in human HF, our results suggest
that up-regulation of SERCA activity as a therapy for HF must
be advocated with extreme caution.
was 51 kN.m-2 compared to 26kN.m-2 at half-maximal
activation. During the isometric phase, Pi release is at a steady
rate of 8.2 and 4.8 s-1 (assuming a myosin head concentration
of 120µM) at 32 and 1µM calcium, respectively. During stretch,
the rate of Pi release decreases markedly at both activation
levels, to1.0 and 1.4s-1 respectively. Activation from an initial
sarcomere length of 2.1µm produced an isometric force of
65.5 kN.m-2 with a steady Pi release rate of 8.1 s-1 at maximal
activation, and an isometric force of 49.2 kN.m-2 with a steady
Pi release rate of 4.8 s-1 at half-maximal activation. The rates of
Pi release during stretch and after the end of the stretch at low
activation are equal to that at high activation indicating stretchinduced activation which occurs for stretches applied prior to
activation, and during activation. The results demonstrate that
stretch has a dramatic effect causing an immediate reduction
in the rate of Pi release. Stretch contributes to activation of
the trabecula’s actomyosin ATPase in a calcium-independent
manner, although the effect is more marked at low activation
levels. Thus a direct effect of stretch on thin filament activation
contributes to the Frank-Starling law.
. Kalyanasundaram: None. V. Lacombe: None. A. Belevych: None.
A
B.C. Knollmann: None. M. Periasamy: None. S. Gyorke: None.
P87Hydralazine and Nitroglycerin Combination Improves
Excitation-Contraction Coupling in Failing Cardiomyocytes
Raul A Dulce, Daniel R Gonzalez, Joshua M Hare, Interdisciplinary
Stem Cell Inst, Miami, FL
ABSTRACTS
Although hydralazine and organic nitrates have proven
clinical benefits for heart failure, the underlying mechanism of
action of this combination remains obscure. Spontaneously
Hypertensive Heart Failure (SHHF) rats are a representative
heart failure model which shares common phenotypic features
with human failing hearts. We have recently shown that
SHHF cardiomyocytes exhibit depressed contractility and
calcium transient amplitude in response to increasing rate and
increased sarcoplasmic reticulum (SR) calcium leak, typical
characteristics observed in heart failure. The purpose of this
study was to test the hypothesis that hydralazine, alone or in
combination with nitroglycerin, restores calcium cycling and
contractile performance in cardiomyocytes from SHHF rats.
We measured sarcomere length (SL) shortening and calcium
transient amplitude (Δ[Ca2+]i) in isolated cardiac myocytes
from male SHHF rats (22–24 months old, n=4) and their
normotensive controls Wistar-Kyoto rats (18–22 months old,
n=4) during pacing (0.5–4 Hz). Both SL shortening and Δ[Ca2+]i
responses to increasing pacing in SHHF cardiomyocytes were
augmented by 10 μM hydralazine. The same concentration of
nitroglycerin alone did not significantly affect these parameters.
In response to the combination of these two drugs, there
was a strong trend toward improved contractility and normal
Δ[Ca2+]i. We also assessed SR Ca2+ leak by a brief incubation
with tetracaine following by a caffeine challenge, a regimen
designed to test SR Ca2+ load. At matched SR Ca2+ content,
Ca2+ leak was not significantly changed by nitroglycerin or
hydralazine alone. However, in combination they restored
the leak in SHHF myocytes to the normal levels seen in WKY
myocytes. Hydralazine improves cardiac excitation-contraction
coupling that is impaired in this model, whereas nitroglycerin
does not exert a significant effect on this response. However,
when administered together, nitroglycerin appears to modulate
the enhancing effect induced by hydralazine and helps to
restore normal SR calcium leakage. These data offer new
insights into the mechanisms underlying the actions of
hydralazine and organic nitrates on heart failure.
46
R.A. Dulce: None. D.R. Gonzalez: None. J.M. Hare: None.
P88The Rate of Inorganic Phosphate Release in Contracting
Rat Trabeculae Is Independent of Calcium After Stretch
Catherine Mansfield, Timothy West, Mike Ferenczi, Imperial Coll,
London, United Kingdom
We aim to reveal the molecular mechanism underlying the force
response to stretch in the heart by examining the dependence
of the rate of inorganic phosphate (Pi) release in response to
stretch on the degree of calcium activation. Permeabilized
trabeculae of rat heart are activated by photolytic release
of ATP, at 20°C, in the presence of saturating (32µM), or
half-saturating (1µM), calcium concentrations, at an initial
sarcomere length of 1.9 or 2.1 µm. For trabeculae with an
initial sarcomere length of 1.9 µm, the trabeculae are stretched
to 2.1 µm during the isometric plateau, held at a constant
length and returned to their original length. The rate of Pi
release is determined by the fluorescence change associated
with phosphate binding to a fluorescently-labelled phosphate
binding protein diffused into the preparation. At an initial
sarcomere length of 1.9µm, the isometric force at full activation
C. Mansfield: None. T. West: None. M. Ferenczi: None.
P89Estradiol Impairs Cardiac Contractile Function
in Male Rodents
Georgios Kararigas, Charite Univ Hosp, Berlin, Germany; Virginie Bito,
Univ of Leuven, Leuven, Belgium; Hanna Tinel, Barbara Albrecht, Bayer
HealthCare, Wuppertal, Germany; Karin R Sipido, Univ of Leuven, Leuven,
Belgium; Vera Regitz-Zagrosek, Charite Univ Hosp, Berlin, Germany
Several clinical and animal studies have challenged the notion
that 17beta-estradiol (E2) is cardioprotective. Recently, we
identified that in E2-treated male human cardiac tissues there
was an increased expression level of the myosin regulatory
light chain (Mrlc) interacting protein (Mylip) gene compared
to controls. Mylip has been shown to bind and target Mrlc
for proteasomal degradation in neuronal cells. Modulation of
contraction by Mrlc in the heart has been well documented.
Mutant mice for Mrlc phosphorylation show defects in heart
contraction. Here, we tested the hypothesis that the induction
of Mylip by E2 is associated with reduced Mrlc protein levels
and impaired contractile function. Eleven-month old male
C57BL/6J mice were injected intraperitonealy with E2 (n = 6) or
vehicle (Ctrl; n = 4). Five hours after injection, cardiomyocytes
(CMs) were isolated and were either frozen in TRIzol reagent
for RNA isolation or used for cell shortening measurements.
Quantitative real-time PCR revealed that E2-treated CMs had
higher Mylip levels than Ctrl CMs (49% induction, P; 0.05). In
addition, there was a decreased abundance of Mrlc protein
in E2-treated CMs compared to Ctrl CMs (74% reduction,
P; 0.05). Recordings of unloaded cell shortening at 1, 2 and
4 Hz demonstrated that the treatment with E2 impaired CM
contractile function compared to Ctrl CMs (1 Hz: 31%, adjusted
P; 0.001; 2 Hz: 30%, adjusted P; 0.01; 4 Hz: 25%, adjusted P;
0.01). Similarly, there was a significant decrease in the rate of
contraction of E2-treated CMs compared to controls. Next, we
assessed the effect of E2 on cardiac contractility ex vivo using
Langendorff-perfused rat hearts. Although at baseline there were
no significant changes, the response to isoprenaline was blunted
in E2-treated hearts. E2 levels in elderly and/or obese men
might increase considerably and they have been associated
with an increased risk and incidence of cardiovascular disease.
However, explanations for causal pathways and putative
mechanisms for this association have not been identified. Based
on our present findings, we suggest that MYLIP could contribute
to this association and we propose that MYLIP could become a
pharmacological target in this high-risk group.
. Kararigas: None. V. Bito: None. H. Tinel: A. Employment;
G
Significant; Bayer HealthCare. B. Albrecht: A. Employment; Significant;
Bayer HealthCare. K.R. Sipido: None. V. Regitz-Zagrosek: None.
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Poster Presentations (continued)
P90 T
he Contractile Effect of Angiotensin II Type 1 Receptor
Autoantibodies on Human Placental Vessels
umbilical vein endothelial cells (HUVECs) using differential gene
expression analysis with a false discovery rate of 5%, and
greater than two-fold change between groups. We compared
significantly enriched transcripts in NRP1+CD34- cells
against genes expressed in the primitive streak in Xenopus
embryos using hypergeometric testing. Functional annotationbased clustering was used to identify putative cell signaling
pathways. Results: A total of 785 transcripts were significantly
enriched in NRP1+CD34- vascular precursors vs. hESCs,
and 605 transcripts were unique to NRP1+CD34- vascular
precursors. NRP1+CD34- cells shared a significant number of
transcripts (46 of 357 human homologs) with ventral mesoderm
isolated from Xenopus embryos, (p ≤ 1 x 10–14). We then
identified CXCL14, Neuregulin 2, Leptin and Apelin receptors
as significantly upregulated in NRP1+CD34- vascular
precursors, potentially controlling human ventral mesoderm
and new vessel formation. Conclusions: NRP1+CD34vascular precursors have significant transcript identity with
ventral mesoderm from embryonic primitive streak, and a
unique transcriptional profile compared with HUVECs. These
findings suggest NRP1+CD34- human vascular precursors
significantly resemble ventral mesoderm, and provide a
model of human vascular development. The identification of a
comprehensive database of cell-surface receptors expressed
by the human ventral mesoderm analogs permits future
pharmacological approaches to induce vascular differentiation
from human pluripotent stem cells.
Li Song, Capital Medical Univ, Beijing, China; Ronghua Zheng, Linfen
Vocational and Technical Coll, Linfen, China; Suli Zhang, Shanxi
Medical Univ, Taiyuan, China; Kehua Bai, Capital Medical Univ, Beijing,
China; Lihong Yang, Shanxi Medical Univ, Taiyuan, China; Huirong Liu,
Capital Medical Univ, Beijing, China
. Song: None. R. Zheng: None. S. Zhang: None. K. Bai: None.
L
L. Yang: None. H. Liu: None.
P91Transcriptional Profiling of Pluripotent Stem Cell-Derived
Neuropilin-1+CD34- Vascular Precursors Reveals
Significant Overlap with Ventral Mesoderm and Identifies
Signaling Pathways in Differentiation from Vascular Cells
Jennifer K Lang, Fraser J Sim, Thomas Cimato, SUNY at Buffalo Sch
of Med, Buffalo, NY
Background: Pluripotent stem cells are a model to understand
the mechanisms of vascular differentiation in humans. We
previously showed that the onset of Neuropilin-1 (NRP1)
expression identifies vascular precursors in mouse and
human embryonic stem cells. Our goal was to determine the
degree of overlap between NRP1+CD34- cells and embryonic
primitive streak, and identify novel growth factor signaling
pathways controlling their differentiation. Methods: Human
pluripotent stem cells were differentiated, and NRP1+CD34cells were isolated. Following RNA extraction, whole genome
transcriptional profiling was performed. Microarray data
were compared with undifferentiated stem cells and human
J.K. Lang: None. F.J. Sim: None. T. Cimato: None.
This research has received full or partial funding support from the
American Heart Association, Founders Affiliate (Connecticut,
Maine, Massachusetts, New Hampshire, New Jersey, New York,
Rhode Island, Vermont).
P92Simplified Monolayer Differentiation of Human-Induced
Pluripotent Stem Cells to Functional Cardiac Myocytes
Jennifer K Lang, Stanley Fernandez, Thomas Cimato, SUNY at Buffalo
Sch of Med, Buffalo, NY
ackground: Human induced pluripotent stem cells (hiPSCs)
B
are an important model for cardiovascular research, drug
discovery, and translational research applications. Commonly
used methods to direct iPSCs to cardiac myocytes can
be technically demanding. Prior studies have shown that
both VEGF and endothelial cells promote differentiation of
stem cells to cardiac myocytes. Furthermore, DMEM/F12
with 10% fetal calf serum (DMEM-FCS) has been shown to
induce cardiac myocytes in an embryoid body (EB) system.
The objective of this study was to determine if differentiation
of hiPSCs using conditions that support endothelial cell
differentiation would promote cardiac myocyte colony
formation. Methods: Two hiPSC lines derived using nongenome integrating methods were maintained on Matrigelcoated surfaces under serum free conditions in mTeSR1
medium. We performed a comparison of monolayer myocyte
differentiation efficiency using DMEM-FCS and endothelial
cell medium (EC). Cells were maintained in iPSC medium
(mTeSR1) as a negative control. The number of beating
colonies derived under each growth condition was determined
using phase microscopy at 4 weeks. Cardiac myocyte
commitment was characterized using an α-MHC-GFP reporter
vector and electrophysiologic action potentials on isolated
beating colonies. Results: Differentiation of human iPSCs in
EC medium induced substantial numbers of beating colonies
4 weeks after differentiation (2.29 ± 0.3 beating colonies/
cm2 culture area, n=42). Unlike EB models of myocyte
differentiation, no beating clusters were observed in our
monolayer system with DMEM-FCS medium (n=14) (p<0.01).
As expected, mTESR1 (n=12) did not induce any cardiac
myocytes. All beating cell colonies expressed GFP driven by
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ABSTRACTS
ackground: Decreased placental perfusion induced
B
by abnormal placental vascular contraction is one of the
pathological basis of preeclampsia. It has been reported
that the sera titers of the autoantibody against the second
extracellular loop of angiotensin II type 1 receptor (AT1AA) were negatively correlated with placental blood flow in
preeclampsia. Our previous study has found that AT1-AA
could induce contraction of rat thoracic aorta and coronary
rings by activing angiotensin II type 1 receptor (AT1R).
However, there is no direct evidence for explaining whether
AT1-AA might cause vasoconstriction on human placental
blood vessels. Methods: The SD rats were immunized with
the synthetic peptide corresponding to the sequence of the
second extracellular loop of the human AT1 receptor (AT1RECII), and anti-AT1R antibody (AT1R-Ab) was extracted.
The expression of AT1R on human placental vessels was
determined by immunohistochemistry. The effects of AT1R-Ab
on placental vessels were measured with isolated vascular
ring technique. Results: (1) AT1R was highly expressed in the
human placental artery, vein and vascular endothelial cells. (2)
AT1R-Ab (10 mmol/L) respectively enhanced the contraction
of the placental arteries and veins (29.21% ± 3.7% vs. 21.35%
± 2.8%, P>0.05), which could be completely reversed when
AT1R was blocked by AT1R inhibitor. (3) AT1R-Ab (0.1, 1
and 10 mmol/L) induced placental vasoconstriction of normal
human. The percentage of maximal contraction was 2.73%
± 1.11%, 4.00% ± 3.2% and 33.30% ± 5.6%, respectively.
There was significantly difference between the three groups
for contraction amplitude induced by different concentrations
of AT1R-Ab (P<0.01). (4) AT1R-Ab (0.1, 1 and 10 mmol/L)
induced placental vasoconstriction of preeclampsia. The
percentage of maximal contraction was 1.74% ± 0.3%,
5.58% ± 1.41% and 3.73% ± 2.5%, respectively. There
was significantly difference between the three groups for
contraction amplitude induced by different concentrations
of AT1R-Ab (P<0.01). Conclusion: AT1R-Ab could induce
human placental vasoconstriction in a concentrationdependent manner, which suggested that AT1-AA might
be involved in the pathogenesis of preeclampsia by directly
contracted placental blood vessles.
47
Poster Presentations (continued)
the cardiac specific α-MHC promoter. Electrophysiological
studies confirmed the presence of action potentials with
ventricular phenotypes. Conclusions: Differentiation of human
iPSCs under monolayer conditions that support endothelial
cells facilitates efficient induction of functional human cardiac
myocytes. Our findings simplify the differentiation of iPSCs to
cardiac myocytes, making research with human iPSCs more
accessible to a broad range of cardiovascular investigators.
embryos and the high efficiency of pGBT-RP2 (RP2), a genebreaking transposon-based in vivo protein trap cassette.
By selectively raising up founder fish with Red Fluorescence
Protein (RFP) reporter expression in the heart, we successfully
generated 15 cardiac mutant lines. All tagged loci exhibit cardiac
expression and the overall knockdown efficiency at transcript
level is greater than 95%. One of the cardiac lines, RP2_ #135,
is a homozygous embryonic lethal line that is caused by a RP2
insertion in methionine adenosyltransferase 2a (mat2a). Taking
advantage of the LoxP sites inside the RP2 vector, we further
demonstrated that the cardiac phenotypes in RP2_ #135 is
due to a disrupted expression of mat2a in cardiomyocytes, as
indicated by a conditional rescue assay using Tg(cmlc2:CreYFP), a myocardium specific Cre transgenic line. Finally, we
show that the reduced cardiomyocyte number, but not size, in
the mat2a mutant is conveyed by the p53-mediated apoptosis
pathway. Together, our data prove the feasibility of a facile
approach to generate a vertebrate cardiac mutant collection with
the following two unique features. First, the expression dynamics
of each tagged gene is reported by RFP. Second, the causality
between the tissue-specific gene expression and the resulted
cardiac phenotypes can be attested genetically.
J.K.B. Lang: None. S. Fernandez: None. T. Cimato: None.
This research has received full or partial funding support from the
American Heart Association, Founders Affiliate (Connecticut,
Maine, Massachusetts, New Hampshire, New Jersey, New York,
Rhode Island, Vermont).
P93Not published at presenter’s request.
P94Anesthetic Preconditioning in Human Cardiomyocytes
Derived from Type II Diabetic Patient-Induced Pluripotent
Stem Cells in a Varying Glucose Environment
ABSTRACTS
Scott Canfield, Ana Sepac, Filip Sedlic, Maria Muravyeva, Xiaowen
Bai, Zeljko Bosnjak, Medical Coll of Wisconsin, Wauwatosa, WI
Volatile anesthetic-induced preconditioning (APC) has been
shown to pharmacologically precondition the myocardium,
providing protection from an ischemia-reperfusion injury.
However, APC is attenuated or even eliminated in diabetic
individuals and in the presence of acute hyperglycemia with
the underlying mechanism(s) being unknown. In this study,
we used ventricular cardiomyocytes (CMs) differentiated from
non-diabetic and type II diabetic-induced pluripotent stem
cells (N-iPSCs and DM-iPSCs, respectively) to investigate
the influence of a high glucose environment and genetic
background on APC. Differentiated CMs were identified using
cardiac-specific immunostaining and expression of green
fluorescent protein under the transcriptional control of cardiac
promoter myosin light chain-2v, a genetic construct delivered
by lentiviral vector. N-iPSC- and DM-iPSC-derived CMs were
exposed to varying glucose environments (11 mM and 25 mM).
Confocal microscopy was utilized to measure mitochondrial
membrane potential and mitochondrial permeability transition
pore opening (mPTP) in CMs. The volatile anesthetic
isoflurane depolarized mitochondria in N-iPSC-derived CMs
via opening of the mitochondrial adenosine triphosphatesensitive potassium channel; however, isoflurane depolarized
mitochondria to a significantly lower level in diabetic-derived
cardiomyocytes and in the presence of 25 mM glucose.
APC delayed mPTP opening in both N-iPSC- and DM-iPSCderived CMs in 11 mM glucose environment only. We have
established an in vitro model based on directed differentiation
of ventricular CMs from N-iPSCs and DM-iPSCs that allows
us the unique opportunity to conduct comparative studies to
address the inability of diabetic individuals to be preconditioned
with anesthetics. Our preliminary results indicate for the first
time that both a high glucose environment and a diabetic
background have detrimental effects on the efficiency of APC
to protect CMs from an ischemia-reperfusion injury.
S. Canfield: None. A. Sepac: None. F. Sedlic: None. M. Muravyeva:
None. X. Bai: None. Z. Bosnjak: None.
P95An Expression-Based in Vivo Protein Trap Screen
Enriches Cardiac Mutants and Reveals Cardiac Functions
of Methionine Adenosyltransferase 2A in Zebrafish
Yonghe Ding, Wei Huang, Yun Deng, Beninio Jomok, Alissa Caron,
Jingchun Yang, Xueying Lin, Stephen Ekker, Xiaolei Xu, Mayo Clinic,
Rochester, MN
The present forward mutagenesis screen strategies in
vertebrates are difficult to identify genes and mutants affecting
a specific organ of interest. Here, we report a strategy to enrich
cardiac mutants by leveraging the transparency of zebrafish
48
Y. Ding: None. W. Huang: None. Y. Deng: None. B. Jomok: None. A.
Caron: None. J. Yang: None. X. Lin: None. S. Ekker: None. X. Xu: None.
P96Eya4, a Transcription Cofactor Crucial in Acquired
Heart Disease
Tatjana Williams, Jost Schoenberger, Moritz Hundertmark, Martin Czolbe,
Franziska Panther, Oliver Ritter, Univ of Wuerzburg, Wuerzburg, Germany
Introduction: We previously identified a mutation in the human
transcriptional cofactor Eya4 (E193) as cause of familial dilated
cardiomyopathy (DCM) and heart failure. Upon interaction with
real transcription factors such as Six family members, Eya4 is
recruited to and interacts with target genes. One of the few
known Eya-Six targets expressed in the heart is the cyclindependent kinase inhibitor p27kip1, which has been shown
to inhibit hypertrophic growth in adult cardiomyocytes, is. We
therefore hypothesize that Eya4/Six1 regulates targets relevant
in normal cardiac function. Methods and Results: We
examined p27 expression in response to Eya4 in permanent
mammalian cell lines. Western blot analysis demonstrated that
an overexpression of Eya4 led to a significant downregulation
of p27, whereas the overexpression of E193 had no effect on
p27 levels. Studies using a p27 promoter fragment including
Six1 consensus sites cloned in front of a Luciferase reporter
gene proved Eya4 acts as a suppressor of p27 already at
the transcriptional level, whereas E193 could not sufficiently
inhibit p27 expression. Further transfection and knockdown
experiments revealed that under basal conditions an Eya4
overexpression decreased protein synthesis in primary cardiac
myocytes. We constructed transgenic mouse models with a
constitutive myocardial overexpression of E193 and Eya4 to
study the precise role of Eya4 in the heart. First analysis of
these animals using magnetic resonance imaging to visualize
cardiac structures in detail show that an overexpression of
E193 leads to an age related onset of DCM as seen in an
increase in LVEDV, whereas Eya4 overexpressing mice show
no signs of heart disease. Conclusion: In summary, we
identified a mutation in Eya4 to cause DCM. We now provide
evidence that the Eya4/Six1 signalling cascade is also relevant
in more common forms of acquired heart disease. Eya4/Six1
seems to regulate the expression of p27kip1, an important
inhibitor of the development of hypertrophy in postmitotic
cardiomyocytes. Studies using E193 overexpressing mice
support our hypothesis whereas tempering the Eya4/Six1
signalling cascade disturbs cardiac physiology.
T. Williams: None. J. Schoenberger: None. M. Hundertmark: None.
M. Czolbe: None. F. Panther: None. O. Ritter: None.
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Poster Presentations (continued)
calcification of aortic valve that occurs with inhibition of Notch
signaling. Methods and Results: The expression of Notch
signaling pathway members was validated in the aortic valve
cusps from adult mice, and examination of diseased human
aortic valves revealed decreased expression of NOTCH1 in
areas of calcium deposition. To identify downstream mediators
of Notch1 signaling, we examined gene expression changes
that occur with chemical inhibition of Notch signaling in rat
aortic valve interstitial cells (AVICs). We found significant
downregulation of many cartilage-specific genes that
constitute the valve extracellular matrix (ECM). Analysis of
these cartilage-specific genes demonstrated that several
were transcriptional targets of Sox9, a master regulator of
chondrogenesis, which has been previously shown to be
essential for proper valve development and maintenance.
Utilizing an in vitro porcine aortic valve calcification model
system, inhibition of Notch activity resulted in accelerated
calcification while stimulation of Notch signaling attenuated the
calcific process. Finally, utilizing transfection studies, addition
of Sox9 was able to prevent the calcification of porcine AVICs
that occurs with Notch inhibition. Conclusions: Loss of Notch
signaling contributes to aortic valve calcification by a Sox9dependent mechanism. Further elucidation of the Notch1Sox9 molecular pathway and its role in the maintenance of the
ECM will lead to an improved mechanistic understanding of
aortic valve calcification and development of novel therapeutic
strategies for CAVD.
P97Endothelial Overexpression of LOX-1 Decreases Arterial
Thrombosis and TF Expression in Vivo: Role of SIRT-1
and NFκB
Alexander Akhmedov, Giovani G Camici, Simona Stivala, Erik W
Holy, Alexander Breitenstein, Christine Lohmann, Cardiovascular Res,
Zurich, Switzerland; Juerg-Hans Beer, Kanton Hosp Baden, Baden,
Switzerland; Christian M Matter, Thomas F Luescher, Cardiovascular
Res, Zurich, Switzerland
ackground: The hallmark of the initiation of atherosclerotic
B
lesion is foam cell formation, and oxidized LDL (OxLDL) is
believed to play a key role in the initiation of the atherosclerotic
process. OxLDL is internalized by several receptors, such as
SR-AI/II, SR-BI, CD36, and CD68. OxLDL is also internalized
by endothelial cells, but this uptake depends on receptors
other than the classic scavenger receptors. In 1997, a lectinlike oxidized LDL receptor-1 (LOX-1, OLR1) was identified in
bovine aortic endothelial cells. LOX-1 is a type II membrane
glycoprotein with an apparent molecular weight of 50 kDa.
It has a C-terminal extracellular C-type lectin-like domain.
This lectin-like domain is essential for binding to OxLDL.
Binding of OxLDL to LOX-1 induces several cellular events
in endothelial cells, such as activation of transcription factor
NF-kB, upregulation of MCP-1, and reduction in intracellular
NO, which may trigger the onset of cardiovascular events or
accelerate the development of atherosclerosis. Methods and
Results: We generated endothelial-specific LOX-1 transgenic
mice using the Tie2 promoter (LOX-1TG). 12-week-old male
LOX-1TG and wild-type (WT) mice were applied for carotid
artery thrombosis model. LOX-1TG mice developed carotid
artery thrombosis within a mean occlusion time of 36.96±4.83
min, while WT control mice occluded within a mean time
period of 22.75±3.87 min (n=10, P < 0.05). Initial blood flow
in carotid artery did not differ between both groups of mice.
Decreased occlusion time was in LOX-1TG mice vascular cell
adhesion molecule-1 (VCAM-1) and E-selectin expression,
macrophage accumulation and aortic fatty streaks were
increased, while eNOS phosphorylation and endothelial
function were reduced. In endothelial cells of LOX-1TG mice,
reactive oxygen species were increased and the transcription
factors NF-κB and Oct-1 activated. In atherosclerotic LOX1TG/ApoE-/- mice, high cholesterol diet increased VCAM-1
expression, number of macrophages, T-cells as well as plaque
size. Conclusions: Thus, our data suggest that LOX-1 plays
a protective role in the arterial thrombosis when expressed
at unphysiological levels. Therefore, LOX-1 might represent a
novel therapeutic target for atherosclerosis.
P98Inhibitory Role of Notch1 in Calcific Aortic Valve Disease
Is Mediated by Sox9
Chetan P Hans, Nationwide Children’s Hosp, Columbus, OH; Asha
Acharya, Univ of Texas Southwestern Medical Ctr, Dallas, TX; Sara N
Koenig, Nationwide Children’s Hosp, Columbus, OH; Haley A Nichols,
Cristi L Galindo, Univ of Texas Southwestern Medical Ctr, Dallas, TX;
Harold R Garner, Virginia Bioinformatics Inst, Virginia Tech, Blacksburg,
VA; Walter H Merrill, Univ of Cincinnati, Cincinnati, OH; Robert B
Hinton, Cincinnati Children’s Hosp Medical Ctr, Cincinnati, OH; Vidu
Garg, Nationwide Children’s Hosp, Columbus, OH
Introduction: Aortic valve calcification is the most common
form of valvular heart disease; however the mechanism(s)
underlying calcific aortic valve disease (CAVD) are unknown.
NOTCH1 mutations are associated with aortic valve
malformations and adult-onset calcification in families with
inherited disease. The Notch signaling pathway is critical
for multiple cell differentiation processes, but its role in the
development of CAVD is not well understood. Objective:
To investigate the molecular changes associated with the
.P. Hans: None. A. Acharya: None. S.N. Koenig: None. H.A. Nichols:
C
None. C.L. Galindo: None. H.R. Garner: None. W.H. Merrill: None.
R.B. Hinton: None. V. Garg: None.
P99Whole Genome Sequencing Identifies a New Inherited
Ion Channelopathy
Frederick E Dewey, Matthew T Wheeler, Marco V Perez, Karim
Sallam, Young M Kim, Sergio P Cordero, Aleks Pavlovic, Stanford
Ctr for Inherited Cardiovascular Diseases, Stanford, CA; Dmitry
Pushkarev, Stanford Bioengineering, Stanford, CA; Thomas Cappola,
Univ of Pennsylvania, Philadelphia, PA; Steve R Quake, Stanford
Bioengineering, Stanford, CA; Euan A Ashley, Stanford Ctr for Inherited
Cardiovascular Diseases, Stanford, CA
Background: Arrhythmic sudden cardiac death (SCD) is a
significant cause of mortality in industrialized countries. Clinical
assessment and molecular diagnosis identifies a cause in only
~ 40% of patients. Whole genome sequencing (WGS) may
identify causative genetic variants with sensitivity superior to
targeted sequencing approaches. Methods: We performed
single molecule WGS of genomic DNA isolated from paraffin
embedded formalin fixed tissue from a previously healthy
19 year-old man who died of presumed arrhythmic SCD.
Using a combination of gene coexpression network analysis
and curated literature associations, we identified rare and
novel variants (allele frequency < 5%) in genes known to
be associated with sudden death and genes coding for ion
channel, sarcomeric, costameric, sarcolemmal and Z-disc
proteins. Variants of interest were confirmed with Sanger
sequencing and first-degree relatives were genotyped for
these variants. Results: Toxicology screen, family history,
gross autopsy, histological examination of myocardium,
and commercial testing for variants associated with SCD
were un-revealing. Using WGS to provide 48x coverage
of 90% of genomic positions, we identified 118 rare or
novel non-synonymous coding and splice site variants.
Eighteen genes associated with cardiovascular diseases or
important to cardiomyocyte function harbored potentially
damaging variants. Family genotyping revealed compound
heterozygous mutations in the gene KCNJ12, encoding the
inward rectifying potassium channel Kir2.2 that contributes
to resting membrane potential and late action potential
repolarization (IK1). Both variants were absent in 634 healthy
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ABSTRACTS
A. Akhmedov: None. G.G. Camici: None. S. Stivala: None. E.W. Holy:
None. A. Breitenstein: None. C. Lohmann: None. J. Beer: None.
C.M. Matter: None. T.F. Luescher: None.
49
Poster Presentations (continued)
individuals genotyped in the 1000 genomes project and in
our laboratory and were very highly conserved in a multiple
sequence alignment of 44 vertebrate species. Conclusion:
We present the whole genome sequence of a patient who died
of SCD and identified putatively damaging digenic mutations.
Experimental confirmation of the functional effects of these
variants may describe a new syndrome of inherited cardiac ion
channelopathy associated with SCD.
F.E. Dewey: None. M.T. Wheeler: None. M.V. Perez: None. K. Sallam:
None. Y.M. Kim: None. S.P. Cordero: None. A. Pavlovic: None.
D. Pushkarev: None. T. Cappola: None. S.R. Quake: G. Consultant/
Advisory Board; Significant; Helicos Biosciences. E.A. Ashley: None.
P100Not published at presenter’s request.
P101Genetic Variation of Phenylethanolamine-Nmethyltransferase Influences Cardiovascular
Function in Healthy Humans
Abdulaziz A Mohammed, Courtney M Wheatley, Nicholas A Cassuto,
Eric M Snyder, Univ of Arizona, Tucson, AZ
ABSTRACTS
Objectives: Genetic susceptibility to hypertension can be
caused by alterations in cardiovascular and/or renal function.
Previous work using genome-wide association studies have
demonstrated that the gene that encodes phenylethanolamineN-methyltransferase (PNMT) is a candidate for hypertension.
Follow-up studies confirmed that the gene that encodes PNMT
may alter susceptibility to hypertension in African Americans,
although no mechanistic data was assessed. Genetic variation
of PNMT alters the catecholamine response to exercise, and
variation of PNMT at position -182 has been associated with
susceptibility to multiple sclerosis. Methods: We sought to
determine the influence of genetic variation of PNMT (A-182G)
on renal Na+ handling and cardiovascular function in humans.
We collected serum epinephrine (Epi) and norepinephrine (NE),
24-hour urine, serum Na+, K+, and creatinine, and measured
cardiac output (Q, acetylene rebreathing), heart rate (HR,
12-lead EKG), blood pressure (systolic, SBP, diastolic, DBP,
and calculated mean arterial, MAP) and calculated fractional
excretion of Na+ (FENa), stroke volume (SV), and systemic
vascular resistance (SVR) in 20 normotensive subjects (n: AA=6
and GG=14). Results: We found that the GG genotype had
higher Epi and Epi/NE when compared to AA (Epi=98±21 vs.
53±10pg/l; NE= 344±56 vs. 462±79pg/l; Epi/NE= 0.31±0.07
vs. 0.13±0.03, for GG and AA, respectively, mean±SE, p<0.05
for Epi and Epi/NE). We found no differences between the
genotype groups in 24hr. renal Na+ handling (serum Na+,
urine Na+, or FENa). We found no differences in HR, SBP, or
SV between the groups. The GG group had a trend towards
a lower DBP, and MAP, but these did not reach statistical
significance (DBP= 70±3 vs. 75±2mmHg; MAP= 83±3 vs.
87±2mmHg, for GG and AA, respectively, mean±SE). The GG
group had a higher Q and a lower SVR when compared to
the AA group (Q= 6.0±0.5 vs. 4.8±0.4 l/min; SVR= 1020±64
vs. 1316±95 dynes*sec/cm5, for GG and AA, respectively
mean±SE, p<0.05). Conclusion: These results suggest that
genetic variation of PNMT can influence resting blood pressure
in normotensive individuals and that this may be the result of
cardiovascular function, rather than renal Na+ handling.
A.A. Mohammed: None. C.M. Wheatley: None. N.A. Cassuto: None.
E.M. Snyder: None.
P102Apelin Regulates the Function of Angiotensin II Type 1
Receptor Through APJ
Xiao Sun, Saitama Medical Ctr, Irumagun, Japan; Shinichiro Iida,
Saitama Intl Medical Ctr, Hidaka, Japan; Rina Senbonmatsu, Bates
Coll, Lewiston, ME; Kei Maruyama, Saitama Medical Univ, Irumagun,
Japan; Tadashi Inagami, Vanderbilt Univ Sch of Med, Nashville, TN;
Shigeyuki Nishimura, Saitama Intl Medical Ctr, Hidaka, Japan; Takaaki
Senbonmatsu, Saitama Medical Univ, Irumagun, Japan
50
Apelin and its G protein-coupled receptor (GPCR) APJ,
which is most closely related to the angiotensin II (Ang II)
type 1 receptor (AT1) but Ang II does not bind to APJ, are
potent regulators of the cardiovascular system. Although
recent studies have suggested that apelin-APJ reverses
the function of Ang II-AT1, the mechanism remains unclear
because the accumulating evidences indicates that apelinAPJ may contribute to both cardioprotection and pathological
progression. In APJ and AT1 co-expression human embryonic
kidney (HEK) 293 cells, we found that APJ and AT1 created
receptor heterodimers. Co-expression with APJ significantly
suppressed the phosphorylated extracellular signal-regulated
kinases 1/2 (pERK1/2) induced by Ang II-AT1, whereas apelin
eliminated this ligand-independent function of APJ out of
relation to its heterodimerization. The pharmacologically
non-activated APJ upon apelin stimulation elicited by the
Gi/o-specific inhibitor pertussis toxin (PTX) restituted the
pERK1/2 level similar to that of AT1 and APJ co-expression
without apelin stimulation. However, the co-expression of the
beta-2-adrenergic receptor (β2AR) or the pharmacologically
non-activated Ang II type 2 receptor (AT2) induced by the AT±specific antagonist, PD123319, did not suppress pERK1/2
through Ang II-AT1. Pretreatment with 30 nM of the AT1
blocker (ARB) TA-606A suppressed 50% of the AT1-mediated
pERK1/2, whereas this suppression raised to 75% when
the non-activated APJ was co-expressed. In contrast, 120
nM of TA-606A only reached 34% suppression when it was
co-expressed with activated APJ with apelin. Taken together,
we demonstrated that apelin may regulate the function of
AT1. Non-activated APJ may suppress Ang II-AT1 signaling,
whereas this ligand-independent function was diminished
with apelin activation. These results may contribute to ARB
treatment in the clinical setting.
X. Sun: None. S. Iida: None. R. Senbonmatsu: None. K. Maruyama:
None. T. Inagami: None. S. Nishimura: None. T. Senbonmatsu: None.
P103Genome-Wide Study of Gene Variants Associated with
Differential Event Reduction by Pravastatin Therapy
Dov Shiffman, Celera, Alameda, CA; Stella Trompet, Leiden Univ
Medical Ctr, Leiden, Netherlands; Judy Z Louie, Charles M Rowland,
Joseph J Catanese, Olga A Iakoubova, Celera, Alameda, CA; Todd
G Kirchgessner, Bristol-Myers Squibb, Princeton, NJ; David J Scott,
Naveed Sattar, Univ of Glasgow, Glasgow, United Kingdom; James J
Devlin, Celera, Alameda, CA; Christopher J Packard, Ian Ford, Univ
of Glasgow and Royal Infirmary, Glasgow, United Kingdom; Frank M
Sacks, Brigham and Women’s Hosp, Harvard Medical Sch, Boston,
MA; J W Jukema, Leiden Univ Medical Ctr, Leiden, Netherlands
ackground: Statin therapy reduces the risk of coronary
B
heart disease (CHD); however, the variability in response to
statin therapy is not well understood. We investigated the
effect of genetic variation on the reduction of CHD events
by pravastatin therapy. Methods: We genotyped 682 CHD
cases from CARE and 383 CHD cases from WOSCOPS,
two randomized placebo-controlled studies of pravastatin
using the Illumina OMNI1 bead array. Single nucleotide
polymorphisms (SNPs) that were associated with differential
CHD event reduction by pravastatin therapy were investigated
in PROSPER, a randomized placebo-controlled study of
pravastatin in the elderly. Results: A combined case-only
analysis of CARE and WOSCOPS identified 62 SNPs
associated with differential event reduction by pravastatin
therapy (Pint<0.0001 for interaction between treatment and
genotype in an additive model). We investigated 57 of these
SNPs in PROSPER. In an analysis that included cases as
well as non-cases of CARE, WOSCOPS, and PROSPER, we
found that for an intronic SNP in DNAJC5B (rs13279522),
CHD event reduction by pravastatin therapy according to
genotype differed in all 3 studies: Pint=0.001 in CARE, Pint=0.01
in WOSCOPS, Pint=0.002 in PROSPER, and Pint=3X10-7 in
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Poster Presentations (continued)
a combined analysis of CARE, WOSCOPS and PROSPER
(Figure). Conclusions: We have identified a SNP that is
associated with differential event reduction by pravastatin
therapy in CARE, WOSCOPS, and PROSPER. This SNP
merits investigation in additional randomized studies of
pravastatin and other statins.
stage. Intriguingly, we found that loss of MBNL1 or gain of
CELF1 activity, two major RNA binding proteins disrupted
in DM1, are not driving the miRNA misregulation since their
expression is indistinguishable between wild type, MBNL1
knock out and CELF1 over expressing mice. Moreover,
comparable decrease in ten out of ten primary miRNA
transcripts examined suggests loss of expression is not due
to a processing defect. Instead, we discovered that adult-toembryonic shift in expression of select micro- and messenger
RNAs in DM1 heart occurs due to specific inactivation of a
Mef2 transcriptional program. We are currently determining
causal contributions of this Mef2-miRNA circuitry in the
developmental reprogramming of gene expression in DM1 as
well as its direct role in cardiac manifestations of this disease.
A. Kalsotra: None. R. Singh: None. C. Creighton: None. T. Cooper: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P105 Not published at presenter’s request.
P106Polymorphisms in Candidate Genes and Early
Atherosclerosis: A Study of 115 Autopsy Cases of
Young Adults
Débora S Faffe, Wiliam R Miranda, José B Netto, Fabiane S Lima,
Leonardo Baumworcel, Rosane Silva, Turan P Urményi, José Carlos
P Esperança, Edson Rondinelli, Federal Univ of Rio de Janeiro, Rio de
Janeiro, Brazil
D. Shiffman: A. Employment; Significant; Celera. F. Ownership Interest;
Significant; Celera. S. Trompet: None. J.Z. Louie: A. Employment;
Significant; Celera. F. Ownership Interest; Significant; Celera. C.M.
Rowland: A. Employment; Significant; Celera. F. Ownership Interest;
Significant; Celera. J.J. Catanese: A. Employment; Significant;
Celera. F. Ownership Interest; Significant; Celera. O.A. Iakoubova: A.
Employment; Significant; Celera. F. Ownership Interest; Modest; Celera.
T.G. Kirchgessner: A. Employment; Significant; Bristol-Myers Squibb.
D.J. Scott: None. N. Sattar: None. J.J. Devlin: A. Employment;
Significant; Celera. F. Ownership Interest; Significant; Celera. C.J.
Packard: None. I. Ford: None. F.M. Sacks: None. J.W. Jukema: None.
P104Reactivation of Embryonic Gene Program due to CUG
Repeat RNA Expression in Myotonic Dystrophy
Auinash Kalsotra, Ravi Singh, Chad Creighton, Thomas Cooper,
Baylor Coll of Med, Houston, TX
ABSTRACTS
Myotonic dystrophy type 1 (DM1) is a dominantly inherited
disease that affects multiple organ systems. Cardiac
involvement, which is characterized by conduction defects
and arrhythmias, is the second leading cause of death in DM1
patients. The causative mutation is a CTG expansion in the
3’ untranslated region of DMPK gene resulting in aberrant
expression of CUG repeat RNA that accumulates into nuclear
foci and causes misregulation in alternative splicing. Here
we show that heart-specific and inducible expression of
CUG repeat RNA in a DM1 mouse model results in global
reactivation of embryonic gene expression program in adult
heart that is distinct from a general hypertrophic stress
response. Using q-PCR TaqMan arrays, we identified 54
miRNAs that were differentially expressed in DM1 mouse
hearts one week following induction of CUG repeat RNA.
Interestingly, 83% (45/54) of them exhibited a developmental
shift in expression towards the embryonic pattern. Because
over 90% (41/45) of them were down regulated within 72
hr after induction of repeat RNA and only 2/22 examined
decreased in two unrelated mouse models of heart disease,
we conclude their reduced expression is specific to DM1 and
not simply a general response to cardiac injury. Microarray
studies revealed a developmental switch not only in the
miRNA expression patterns but also a pervasive shift in
mRNA steady state levels of a number of genes to embryonic
Atherosclerosis, a major cause of death, is a complex
disease, involving both genetic and environmental
factors. Chronic inflammation plays a central role in its
pathogenesis; however, the influence of genetic variations
on early development of atherosclerosis has been poorly
investigated. We examined the relationship of five common
single-nucleotide polymorphisms (SNPs) with atherosclerotic
severity in the anterior descendent coronary artery (DA) in
115 consecutive individuals under 30 years autopsied at
the Legal Medical Institute of Rio de Janeiro. Histological
sections were stained with hematoxilin-eosin and classified
for atherosclerosis lesion accordingly to the American Heart
Association. DNA was extracted and genotyped for SNPs in
the angiotensin conversion enzyme, tumoral necrosis factoralpha (-308G/A and -238 G/A), interferon-gama (+874 A/T),
and metalloproteinase-9 (-1562 C/T) genes by automatic
sequencing. Fire weapon lesion was the major causa mortis
(72%). Population mean age was 26 years, 100 male and
15 female; 35% were slims, 58% regular weight and 7%
obese; 22% white, 51% non-white, and 17% blacks. Only 2
subjects had normal DA histology, while 17 presented level
II lesions, 74 level III, 7 level IV, 14 level V. and 1 level VI. 49
individuals presented genotype DD, related to high serum
levels of ECA, while 18 were II (related with low ECA levels)
and 48 DI (allele frequencies of D = 0,635; I = 0,365). For
TNF-α promoter polymorphisms (where allele A is associated
with high TNF-α level), 59 presented genotype GG, 21 GA,
and 8 AA at position -308 (G = 0,790; A = 0,210), while 84
were GG, 3 GA, and 2 AA at position -238 (G = 0,961; A =
0,039). The allele T at position +874 of INF-γ (related to high
INF-γ levels) was present in 45 subjects, while for MMP-9
SNP 49 individuals presented genotype CC, 14 CT, and 3 TT
(C = 0,848; T = 0,152). In conclusion, we observed a high
prevalence of early atherosclerotic lesions (level III) in young
adults, with high prevalence of DD genotype, associated with
high ECA serum levels.
D.S. Faffe: None. W.R.R. Miranda: None. J.B. Netto: None. F.S. Lima:
None. L. Baumworcel: None. R. Silva: None. T.P. Urményi: None.
J.P. Esperança: None. E. Rondinelli: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
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51
Poster Presentations (continued)
P107Interactions of the BDNF Val66Met Polymorphism,
Gender, and High-Carbohydrate Diets on Risk Factors
for Cardiovascular Diseases
Minshan Hu, Ronghui Li, Zhen Zhang, Yongyan Song, Yuanhao Li,
Ding Zhi Fang, Sichuan Univ, Chengdu, China
Background: Brain-derived neurotrophic factor (BDNF) is
a member of the “neurotrophin” family of growth factors, it
was lately found involved in cardiovascular diseases(CVDs),
somehow whether BDNF has any effect on dyslipidemia
has not been detected. We hypothesized that Val (Valine)
66Met (Methionine ) polymorphism of BDNF could change
risk factors of CVDs including serum lipids profile difference,
and interaction with a high-CHO diet could modified these
factors. Methods and Results: Fifty-six young and healthy
Chinese subjects were recruited. A stabilization diet of 54%
carbohydrate was first given to exclude these subjects’ own
various diet background interferences, and then a highCHO diet of 70% carbohydrate was followed to study its
intervention effect. The serum lipid profiles were analyzed
at baseline, after the stabilization diet and after the highCHO diet. After the stabilization diet, significant difference
among BDNF Val66Met polymorphism was found for the
females’ body mass index (BMI) with the means appeared
at Val/Val>Val/Met>Met/Met. After the high-CHO diet, most
of the cardiovascular risk factors were changed to a more
favorable direction and the effect has gender difference. But
interestingly, both TAG and food intake notably increased in
female Met carriers. Conclusions: The above results indicate
that although Met mutation could be protective to CVDs in
females, after interacting with high-CHO diet, due to the rising
triacylglycerol (TAG) and food intake, the risk for carbohydrateinduced hypertriacylglycerolemia happens in these female Met
carriers was actually increased.
M. Hu: None. R. Li: None. Z. Zhang: None. Y. Song: None. Y. Li:
None. D. Fang: None.
P108Direct Evidence of Postnatal Cardiomyocyte Generation in
Murine Models of Aging and Cardiac Injury
ABSTRACTS
Shah R Ali, Reza Ardehali, Stanford Univ Sch of Med, Stanford, CA;
Simon Hippenmeyer, Liqun Luo, Stanford Univ, Stanford, CA; Irving
Weissman, Stanford Univ Sch of Med, Stanford, CA
52
The dogma of the adult mammalian heart as a post-mitotic
organ has recently come under question. Radiolabeled
isotope studies have demonstrated that the human heart
exhibits a low rate of renewal of cardiomyocytes throughout
one’s lifespan. Furthermore, a recent study has elegantly
shown that a neonatal mouse can regenerate its ventricle if the
chamber is resected within the first week of birth. However,
a number of questions remain unanswered about the nature
of the cell type that gives rise to cardiomyocytes postnatally.
The field has yet to clonally address whether cardiomyocytes
divide symmetrically upon birth, or if a resident progenitor
differentiates into cardiomyocytes. Rather than rely on
proxies for cell division (e.g. BrdU incorporation studies), we
use genetic mouse models in which cell division results in
asymmetric, indelible labeling of the daughter cells (“Mosaic
analysis of double markers” (MADM)) and a stochastic multicolor Cre reporter in the Rosa26 locus akin to the “Brainbow”
mouse to investigate postnatal cell division clonally in order
to identify which cell type(s) generate cardiomyocytes. Our
studies have demonstrated limited symmetric division of
cardiomyocytes during normal aging up to six weeks, with
a significantly high rate of cardiomyocyte division during the
first postnatal week. While we provide evidence for limited
cell division in the peri-infarct region of a myocardial infarction
model in the 24 hours after the infarct, the extent of cell
division and proliferation over a longer time frame after the
infarct remains to be explored.
S.R. Ali: None. R. Ardehali: None. S. Hippenmeyer: None. L. Luo:
None. I. Weissman: None.
P109Withdrawn
P110EBP50 Regulates Vascular Smooth Muscle Cell Growth
by Skp2-Mediated Degradation of p21/cip1
Gyun Jee Song, Stacey Barrick, Kristen L Leslie, Nathalie M FiaschiTaesch, Alessandro Bisello, Univ of Pittsburgh, Pittsburgh, PA
The PDZ domain-containing scaffolding protein, EzrinRadixin-Moesin-binding phosphoprotein 50 (EBP50)
regulates vascular stenosis following endoluminal vessel
injury. Its expression in vascular smooth muscle cells
(VSMC) increases after wire injury, and neointima formation
is significantly reduced in EBP50 knockout (KO) mice. The
molecular mechanisms underlying EBP50 actions in VSMC
are unknown. Genetic ablation of EBP50 reduced VSMC
proliferation and was associated with increased (5-fold)
expression of the cell cycle inhibitor p21cip1 both in vessels
and in primary cells. No differences in mRNA levels of
p21cip1 were observed in WT and KO cells. However, the
half-life of p21cip1 in KO VSMC was significantly longer than
in WT VSMC (80 min vs. 45 min) and p21cip1 levels were
similar in WT and KO VSMC treated with the proteasome
inhibitor MG132. These observations suggest that EBP50
regulates post-translational degradation of p21cip1. The
S-phase kinase-associated protein 2 (skp2) is a component of
the E3 ligase complex that degrades p21cip1. The C-terminal
four amino acids of skp2 (ProSerCysLeu) are a canonical
PDZ-binding sequence. Indeed, co-immunoprecipitation and
in-gel overlay assays demonstrated the direct interaction
between EBP50 and skp2. Mutation of the C-terminal Leu
to Ala (L424A-skp2) abrogated the interaction with EBP50.
Skp2 expression was significantly lower in KO than in WT
cells and inhibition of EBP50 expression by an shRNA
lentivirus decreased skp2 expression in WT cells. Moreover,
expression of skp2, but not of the mutant L424A-skp2, in
WT cells reduced p21cip1 levels. Therefore, EBP50 regulates
both expression and activity of skp2 with attendant effects
on p21cip1 and VSMC proliferation. Collectively, these
experiments show that EBP50, by regulating skp2 and
p21cip1 expression, controls VSMC proliferation and the
progression of neointima formation. These studies identify a
novel function for EBP50 in the direct regulation of the cell
cycle and provide a mechanistic basis for the remarkable
effect of this scaffolding protein on vascular remodeling.
. Song: None. S. Barrick: None. K.L. Leslie: None.
G
N.M. Fiaschi-Taesch: None. A. Bisello: None.
This research has received full or partial funding support from the
American Heart Association, Great Rivers Affiliate (Delaware, Kentucky,
Ohio, Pennsylvania & West Virginia).
P111Rheb-mTOR Signaling Pathway Regulates Cardiomyocyte
Mass During Post-Neonatal Period
Takahito Tamai, Shungo Hikoso, Tomokazu Murakawa, Jota Oyabu,
Takafumi Oka, Manabu Taneike, Toshihiro Takeda, Osamu Yamaguchi,
Hiroyuki Nakayama, Kazuhiko Nishida, Issei Komuro, Kinya Otsu,
Osaka Univ Graduate Sch of Med, Suita-City Osaka, Japan
Rheb (Ras homologue enriched in brain) is a major activator
of mTOR. Rheb-mTOR pathway is a critical mechanism for
maintenance of homeostasis, cell growth and stress response
by regulating both protein synthesis and degradation. In
this study, we attempted to clarify the role of Rheb-mTOR
pathway in the heart using cardiac-specific Rheb-deficient
mice (Rheb-/-). We generated floxed Rheb mice and crossed
them with transgenic mice expressing Cre recombinase in
cardiac-specific mannner to generate Rheb-/-. Rheb-/- were
born in Mendelian ratio, but they started to die 8 days
after birth and all of them had died until 10 days after birth.
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Poster Presentations (continued)
Echocardiographic analysis revealed that chamber dimension
and contractile function of Rheb-/- were indistinguishable from
those of control mice (Rheb+/+) 5 days after birth. However,
Rheb-/- exhibited cardiac dilatation and reduced contractility 8
days after birth (LV end diastolic dimension, Rheb-/-: 2.5±0.2
mm vs. Rheb+/+: 2.1±0.2 mm, p<0.01, fractional shortening,
Rheb-/-: 19.7 ± 9.7 % vs. Rheb+/+: 48.6 ± 8.8 %, p<0.01).
These suggest that Rheb-/- died of cardiac dysfunction
and heart failure. Heart weight and cross-sectional area of
cardiomyocytes were significantly lower in Rheb-/- 8 days after
birth. Electron microscopic analysis revealed that the area of
sarcomere was significantly lower in Rheb-/- cardiomyocytes.
Expressions of sarcomeric proteins, such as myosin heavy
chain, actin or desmin, were decreased in Rheb-/-, while the
mRNA expression of desmin was significantly increased in
Rheb-/-. Thus impairment of cardiomyocyte growth observed
in Rheb-/- could be due to either increased degradation or
decreased translation. Although autophagic activity was
enhanced in Rheb-/- heart, ablation of Atg5, an essential
molecule for autophagy, could not prevent premature death of
Rheb-/-. On the other hand, polysome analysis revealed that
the mRNA translation activity had decreased in Rheb-/- heart
compared with Rheb+/+. Thus, we concluded that Rheb-mTOR
pathway in the heart is essential to regulate mRNA translation
activity and protein synthesis, thereby to cardiomyocyte
growth in neonatal period.
T. Tamai: None. S. Hikoso: None. T. Murakawa: None. J. Oyabu:
None. T. Oka: None. M. Taneike: None. T. Takeda: None. O. Yamaguchi:
None. H. Nakayama: None. K. Nishida: None. I. Komuro: None.
K. Otsu: None.
P112Estradiol Induces Physiological Hypertrophic Growth in
the Healthy Mouse Heart
Georgios Kararigas, Charite Univ Hosp, Berlin, Germany; Ba Tiep
Nguyen, Hubertus Jarry, Goettingen Univ Hosp, Goettingen, Germany;
Vera Regitz-Zagrosek, Charite Univ Hosp, Berlin, Germany
. Kararigas: None. B. Nguyen: None. H. Jarry: None.
G
V. Regitz-Zagrosek: None.
P113Protein Tyrosine Phosphatase-Like A Is a Unique
Regulator for Myogenesis
Xi Lin, Xiangsheng Yang, Inst of Biosciences and Technology,
Houston, TX; Qi Li, Yanlin Ma, Hainan Provincial Key Lab for Human
Reproductive Med and Genetic Res, Affiliated Hosp of Hainan Medical
Coll, Hainan, China; Xia Lin, Baylor Coll of Med, Houston, TX; Jiang
Chang, Inst of Biosciences and Technology, Houston, TX
Background: Protein tyrosine phosphatase-like A (PTPLa) is
highly expressed in skeletal and cardiac muscle. Therefore,
it has been implicated to play a role in skeletal myogenesis
and cardiogenesis. Mutations in PTPLa correlated with
arrhythmogenic right ventricular dysplasia in humans and
congenital centronuclear myopathy with severe hypotonia
in dogs. However, the molecular mechanisms of PTPLa in
myogenesis are unknown. Method and Results: Mouse
myoblast C2C12 cells were used to generate PTPLa deficient
and PTPLa overexpression cell lines and the cells were
cultured in proliferating and differentiating media, respectively,
to study regulatory role of PTPLa in cell proliferation and
differentiation. We demonstrated that PTPLa was required for
myoblast growth and differentiation. The cells lacking PTPLa
remained immature and failed to differentiate into mature
myotubes along with repressed MyoG expression. To define
the role of MyoG in PTPLa-mediated myoblast differentiation,
myoblast cell line lacking MyoG expression was generated.
Two cell lines, the PTPLa-deficient and MyoG-deficient
myoblasts were used to demonstrate that PTPLa-mediated
myoblast differentiation was through MyoG regulation.
Meanwhile, the impeded cell growth with an obvious S-phase
arrest was observed in PTPLa-deficient myoblasts. Further
study demonstrated that the upregulation of cyclin D1 and
cyclin E2 complexes along with a compromised Cdk1 activity
contributed to the mutant cell S-phase arrest and eventually
led to the retarded cell growth. Finally, the transcriptional
regulation of PTPLa gene was explored. We identified that
PTPLa was a new target gene of serum response factor
(SRF). Skeletal- and cardiac-specific SRF knockouts resulted
in significant decreases in PTPLa expression, suggesting a
conserved transcriptional regulation of PTPLa gene in mice.
Conclusion: PTPLa is a unique pro-myogenic factor regulated
by SRF. It regulates myoblast proliferation and differentiation
through MyoG and cell cycling signaling pathway.
X. Lin: None. X. Yang: None. Q. Li: None. Y. Ma: None. X. Lin: None.
J. Chang: None.
ABSTRACTS
Estradiol-17beta (E2) has been shown to exert antihypertrophic actions by either attenuating or blunting the
development of left ventricular hypertrophy. However, the
vast majority of these studies have been performed in
stressed or diseased hearts. Consequently, very little is
known about the actions of E2 in the stress- and diseasefree heart. The aim of our study was to identify and
characterize structurally and molecularly the role of E2 in the
healthy heart. Female C57Bl/6J mice were ovariectomized
at the age of two months. Mice were randomly assigned
into groups feeding on either an E2-containing (n = 19) or
soy-free (Ctrl; n = 19) diet for three months. Following this,
all mice were sacrificed and hearts were collected for weight
measurement. Left ventricles were analyzed structurally by
immunohistochemistry and molecularly by genome-wide
expression profiling. E2 led to an increase in the heart weight
(11%; P < 0.001) and the heart-to-body weight ratio (32%;
P < 0.001) compared to Ctrl mice. Cardiomyocyte crosssectional area revealed cardiomyocyte hypertrophy in E2 (n =
6) compared to Ctrl (n = 5) mice (32%; P = 0.004). Analysis
of the left ventricular transcriptome identified 1059 probe
sets (adjusted P ≤ 0.05) differentially expressed between
E2 (n = 5) and Ctrl (n = 5). Hypergeometric testing for Gene
Ontology showed most genes to be associated with cell
cycle, regulation of growth, cell and tissue development.
Pathway analysis revealed 140 pathways (adjusted P =
0.05) modulated between the two groups, such as the
DNA replication and Wnt signaling pathways. Next, we
tested the hypothesis that this hypertrophic effect of E2
is of the physiological type. To this extent, we identified
that angiogenesis was increased with cardiac growth as
determined by the microarray analysis and VEGF-A protein
levels assessed by Western blotting. Furthermore, the
embryonic gene program was not activated and no fibrosis
was observed in the E2-treated group. In conclusion, our
study is the first to demonstrate pro-hypertrophic actions
of E2 in the healthy heart through the modulation of
growth-related genes and pathways. Due to that we have
characterized the hypertrophic effect of E2 as physiological,
we expect this effect to be beneficial for the heart.
This research has received full or partial funding support from the
American Heart Association, South Central Affiliate (Arkansas, New
Mexico, Oklahoma & Texas).
P114Evidence of Transplacental Antibody Transfer in Proposed
Animal Model for Hypoplastic Left Heart Syndrome
Charles R Cole, Mitali Basu, R Scott Baker, Chris Lam, Cincinnati
Children’s Hosp, Cincinnati, OH; Adita Blanco, Madeleine Cunningham,
Univ of Oklahoma Coll of Med, Oklahoma City, OK; Pirooz Eghtesady,
Cincinnati Children’s Hosp, Cincinnati, OH
Background: The pathogenesis of Hypoplastic Left Heart
Syndrome (HLHS), a congenital heart disease with significant
morbidity and mortality, remains unknown. We previously
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
53
Poster Presentations (continued)
proposed a hypothesis wherein HLHS represents a type of
rheumatic heart disease in the fetus; trans-placental passage
of maternal anti-strep or anti-cardiac myosin (CM) antibodies
are postulated to play a key role in the pathogenesis of
disease. This is a first report of an animal model that we
have developed to assess our hypothesis. Methods: Female
Lewis rats (~ 8 weeks old) were immunized with either
streptococcal antigen M type 5 S. pyogenes (PepM5; n=6),
rat CM (n=8) or saline (controls; n=5) with three booster
injections administered at 2-week intervals. Serum titers of
acquired PepM5 or CM antibodies were determined by ELISA
assays every 7–14 days. No boosters were administered
during gestation. Trans-uterine echocardiography was
performed near term (E19–21) to determine fetal number
and viability then cesarean section was performed under
anesthesia to deliver the progeny. Maternal and fetal serum
and hearts were harvested for analysis. Results: All rats
immunized with PepM5 had elevated serum anti-PepM5
antibody titers (>1:12800) and two of these animals also had
elevated anti-CM titers (1:800). The offspring of these PepM5
immunized animals had elevated anti-PepM5 antibody titers
(≥1:6400), but no CM elevation. Rats immunized with CM
had a variable response ranging from anti-CM titers of 1:1600
to >1:12800; there were two non-responders. Their fetuses
had anti-CM titers that ranged from 1:100 to 1:800. None
of the controls had detectable serum titers. Fetal CM titers
of ≥1:200 correlate with maternal peak CM titers of ≥1:6400
and/or maternal harvest titers of ≥1:800. Thus far, 6 of the
CM treated fetuses have evidence of left-sided morphologic
abnormalities along a variable spectrum; all of these fetuses
had CM titers of ≥1:200. Conclusion: We have documented
maternal antibody response and trans-placental antibody
transfer from maternal rats immunized with CM or PepM5
prior to pregnancy. Anti-CM antibody does not cross the
placental as readily as PepM5. Preliminary histologic findings
demonstrate probable HLHS phenotype, which appears to
correlate with fetal antibody titer.
C.R. Cole: None. M. Basu: None. R. Baker: None. C. Lam: None.
A. Blanco: None. M. Cunningham: None. P. Eghtesady: None.
P115Selective Vasoconstriction of the Ductus Arteriosus in the
Rat by Stimulation of Thromboxane A2 Receptor
Tomohiro Yokota, Takashi Aida, Waseda Univ, Tokyo, Japan;
Utako Yokoyama, Yokohama City Univ, Kanagawa, Japan; Susumu
Minamisawa, Waseda Univ, Tokyo, Japan
ABSTRACTS
54
bjective: Patent ductus arteriosus (PDA) is a common
O
life-threatening complication of premature infants.
Cyclooxygenase inhibitors are frequently used for
pharmacologic therapy of PDA to inhibit the synthesis of
prostaglandin E2 (PGE2) that is the most potent vasodilator
in the DA. However, they often cause severe side-effects.
Thromboxane A2 (TXA2), a vasoconstrictive lipid mediator,
induces vascular contraction via TXA2 receptor (TP). TXA2 is
also known to counteract prostacycline, a vasodilative lipid
mediator. Therefore, we hypothesized that TXA2-TP stimulation
can be an alternative strategy to induce DA closure by
counteracting PGE2. Methods and Results: Quantitative RTPCR analysis revealed that the expression of TP mRNA was
higher in the DA than in the aorta during fetal periods. When
the selective TP agonist U46619 was intraperitoneally injected
into Wister rat fetuses at embryonic day 19th (e19) and e21,
we found that U46619 selectively constricted the fetal DA in a
dose-dependent manner even though the level of circulating
PGE2 was supposed to be high. The vasoconstrictive effect of
U46619 was weaker at e19 than at e21 in the DA, suggesting
that immature DA is less sensitive to TP stimulation.
Importantly, U46619 also showed vasoconstrictive effect on
two different types of postnatal PDA models: immature PDA
and hypoxia-induced PDA. These effects of U46619 were
stronger than the effect of indomethacin that is the most
common cyclooxygenase inhibitor. In addition, we found that
U46619 exhibited little vasoconstrictive effect on other vessels
such as the aorta, pulmonary arteirs, carotid arteries, renal
arteries, and portal veins. Furthermore, U46619 was not likely
to induce microthrombosis in the pulmonary capillary arteries.
These results suggest that TP agonists could selectively
promote the DA constriction without severe complications and
be an alternative potent vasoconstrictor for patients with PDA.
T. Yokota: None. T. Aida: None. U. Yokoyama: None.
S. Minamisawa: None.
P116The Physiological Ventricular Growth Signal Can Be
Determined Using Infants with Congenital Heart
Disease as Models
John E Foker, James M Berry, Brian A Harvey, Lee A Pyles, Univ of
Minnesota, Minneapolis, MN
Objectives: The signal for ventricular growth has not been
defined. This basic developmental question was studied
using infants with congenital heart lesions as models. Our
first hypothesis was that clinical ventricular hypoplasia is a
developmental rather than a primarily genetic defect and,
therefore, catch-up growth can be induced. Our clinical
observations also led to the hypothesis that forward flow
across the atrioventricular (AV) valve (mitral or tricuspid) is
what generates the growth signal. To test these hypotheses
we analyzed clinical data from infants with a variety of
congenital defects including three groups of patients with a
hypoplastic ventricle in whom a procedure was carried out
to increase flow across the AV valve. Methods: Infants with
one of several congenital heart problems had right and left
ventricular volumes (RV, LV) assessed by biplane echo and
indexed to body surface area (m2). The degree of hypoplasia
was calculated using nomograms to determine the number
of standard errors of the mean (SEM) below the expected
volume (Table 1). Hypoplasia was considered significant
when the SEM < -2.0. The three groups were studied before
and after (3–6 months) procedures which increased AV flow.
Results:
Infants assessed by biplane echo and indexed
to body surface area (m2)
Pre-Operative
Post-Operative
Unbalanced AV
canal defects
9.8–19.4 cc/m2
-7.1 to -4.2 SEM
Hypoplastic
32–45 cc/m2
-1.2 to -0.1
SEM Normal
Hypoplastic left
heart subsets
6.0–13.8 cc/m2
-6.8 to -5.2 SEM
Hypoplastic
24–51 cc/m2
-1.8 to +0.1
SEM Normal
Pulmonary
atresia with
intact septum
2.5 + 1.1 cc/m2
-5.1 + 2.5 SEM
Hypoplastic
9.4 + 4.6 cc/m2
-1.5 + 1.9 SEM
Normal
LV growth
RV growth
Generally observed clinically: Patients with isolated ASDs have
increased TV flow and enlarged RVs Patients with isolated
VSDs have increased MV flow and enlarged LVs
Other possible growth mechanisms were assessed. (1)
High wall stress with systemic or supra-systemic pressures
produced no net cavitary growth unless AV valve flow was
increased. (2) Significant retrograde flow from semilunar valve
regurgitation did not increase ventricular size until failure
developed. Therefore, no evidence was found for other growth
mechanisms. Conclusions 1) Patients with congenital heart
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Poster Presentations (continued)
synthesis by more than 75% (±12%, n=9, p<0.01), as well
as expression of the proliferation markers PCNA and KI-67
(by more than 70% (±15%, n=9, p<0.01). Reduced S100A6
levels in HUVEC lead to an increase in cellular senescence,
as measured by the expression of senescence associated
β-galactosidase expression (by more than 6-fold, n=4,
p<0.01) and a reduction of EC tube formation on matrigel
matrix (by more than 90%, n=3, p<0.01). Reduction of
S100A6 increased the expression of the cell cycle control and
DNA repair-associated gene BRCA2, but did not change in
the expression of BRCA1, cyclins or p53 (by RT-PCR array).
We conclude that Ca2+ regulation by S100A6 is essential for
multiple aspects of EC physiology.
disease have a variety of defects, some of which can serve
as models to answer basic developmental questions. 2)
Increased AV valve flow provides the signal which induces
ventricular growth. 3) Operations which increased AV valve
flow induced catch-up growth of hypoplastic ventricles and
allowed beneficial two-ventricle repairs in these patients.
J.E. Foker: None. J.M. Berry: None. B.A. Harvey: None.
L.A. Pyles: None.
P117Does Mammalian Target of Rapamycin Complex-2
Regulate Protein Degradation Pathways in the Heart?
Pankaj S Shende, Christian Morandi, Marijke Brink, Univ of Basel and
Univ Hosp Basel, Basel, Switzerland
Background: Mammalian target of rapamycin (mTOR)
occurs in the cell in two distinct multiprotein complexes
called mTOR complex 1 (mTORC1) and mTORC2, which
contain raptor and rictor, respectively. We have recently
demonstrated that mTORC1 activity is required for the
hypertrophic response to aortic constriction and for the normal
cardiac homeostasis. Moreover, we showed that raptor
deletion causes Akt hyperphosphorylation and lower gene
expression of Atrogin-1 and MuRF1, two muscle specific E3
enzymes part of the proteasomal degradation pathway. These
results suggested that, as a counter-regulatory response to
mTORC1 inactivation, mTORC2 reduces protein degradation
via phosphorylation of Akt at Ser473. It has previously
been shown that the phosphorylation state of Akt regulates
Atrogin-1 and MuRF1 gene expression at the transcriptional
level via FoxO. In the present study, we have tested whether
mTORC2 inactivation induces the ubiquitin-proteasomal
degradation pathway. Methods and Results: In 10 week-old
male mice, transgenic for MerCreMer driven by the α-MHC
promoter and homozygous for floxed rictor, deletion of the
rictor gene was induced by tamoxifen. Protein and RNA
extracts were analyzed at three weeks after tamoxifen by
Western blotting and qPCR, respectively. The rictor gene was
efficiently ablated from the heart as its protein levels were
reduced. Phosphorylation of Akt and PKC-α, direct targets of
mTORC2, was abolished, identifying these signaling molecules
as downstream targets of mTORC2 in the heart. However,
the reduced Akt phosphorylation was not associated with
any changes in the mRNA levels of Atrogin-1, MuRF1, and
MuRF3. Conclusion: Our study suggests that mTORC2induced phosphorylation of Akt is not required for the
maintenance of low expression levels of these genes. Further
studies are ongoing to identify the factors that modulate
Atrogin-1 and MuRF1 gene transcription in the heart.
P.S. Shende: None. C. Morandi: None. M. Brink: None.
P118Withdrawn.
P119S100A6 Regulates Endothelial Cell Physiology
Shumei Ren, Karsten Peppel, Patrick Most, Thomas Jefferson Univ,
Philadelphia, PA
Endothelial cell dysfunction is central to the development
of cardiovascular pathologies. While Calcium cycling is of
recognized importance to myocyte physiology and cardiac
perfomance, the role of Ca2+ signaling in endothelial cell (EC)
physiology remains relatively unexplored. In this study we
investigated the role of S100A6 (calcyclin), an EF-hand type
Ca2+ -binding protein that modulates target protein function
dependent upon intracellular Ca2+ signaling. S100A6 displays
cytoplasmic distribution in quiescent, confluent HUVEC, but
translocates to the nucleus upon serum stimulation. Oxidative
stress, induced by incubation of HUVEC with H¬2O2 (150 uM)
leads to redistribution of nuclear S100A6 to the cytosol and
cell cycle arrest. Knockdown of S100A6 by siRNA transfection
reduced S100A6 levels by over 70% and diminished DNA
P120Increased [ca2+]e Enhances Adipocyte Development In
Bone Marrow Stroma
Ryota Hashimoto, Juntendo Univ Sch of Med, Tokyo, Japan; Youichi
Katoh, Juntendo Univ Sch of Med Urayasu Hosp, Urayasu, Japan;
Seigo Itoh, Takafumi Iesaki, Hiroyuki Daida, Juntendo Univ Sch of
Med, Tokyo, Japan; Yuji Nakazato, Juntendo Univ Sch of Med Urayasu
Hosp, Urayasu, Japan; Takao Okada, Juntendo Univ Sch of Med,
Tokyo, Japan
Background: Bone marrow stroma contains adipocytes,
osteoblasts, and lymphohematopoietic donor cells. With
age, fatty marrow gradually predominates in bone marrow
stroma and is a factor underlying age-related fracture and
anemia. Thus, it is important to understand the mechanism
of adipocyte development in bone marrow stroma. Bone
marrow Ca2+ levels can reach high concentrations of 8 to 40
mM, while circulating plasma Ca2+ levels normally range from
2.3 to 2.6 mM. However, the effects of a high extracellular
calcium concentration ([Ca2+]e) on adipocyte development in
bone marrow stroma remain largely unknown. Methods and
Results: We studied the effects of high [Ca2+]e on adipocyte
development in bone marrow stroma. First, we used the fura-2
method to examine whether a change in [Ca2+]e alters [Ca2+]i
levels in bone marrow stromal cells. Changes of [Ca2+]e
from 1.8 mM to 5.4 mM and 10.8 mM significantly increased
[Ca2+]i by 1.1 and 1.3 times, respectively. Next, bone marrow
stromal cells were cultured for 14 days in high [Ca2+]e (5.4
mM and 10.8 mM) and normal [Ca2+]e (1.8 mM) conditions.
Adipocyte development was monitored by Oil Red O
staining of cytoplasmic lipids and by the activity of glycerol3-phosphate dehydrogenase (GPDH). In 5.4 mM and 10.8
mM [Ca2+]e, Oil Red O-stained cells increased significantly by
1.4 and 2.3 times, respectively, and GPDH activity increased
significantly by 1.7 and 2.3 times, respectively, compared with
the respective values in 1.8 mM [Ca2+]±. Conclusions: These
results indicate that high [Ca2+]e induces an increase of [Ca2+]i,
which enhances adipocyte development in bone marrow
stroma. Further studies are required to determine the influx
pathway of Ca2+, since prevention of Ca2+ influx into bone
marrow stromal cells might suppress development of fatty
marrow and reduce age-related fracture and anemia.
ABSTRACTS
S. Ren: None. K. Peppel: None. P. Most: None.
. Hashimoto: None. Y. Katoh: None. S. Itoh: None. T. Iesaki: None.
R
H. Daida: None. Y. Nakazato: None. T. Okada: None.
P121Cardiac Deletion of Aryl Hydrocarbon Nuclear
Translocator (Hypoxia-Inducible Factor 1-β) in the Adult
Heart Results in Cardiomyopathy
Rongxue Wu, Feinberg Cardiovascular Res Inst, Chicago, IL
Rongxue Wu, Kusum Chawla, Yoshihiko Ichikawa, Mohsen
Granefar Hossein Ardehali Background: Aryl hydrocarbon
receptor nuclear translocator (ARNT) is a member of basic
helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins and
serves as a binding partner for a number of other family
members. ARNT is also a required dimerization partner of
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55
Poster Presentations (continued)
HIF1a. Although HIF1a is known to be required for normal
cardiac development, the role of ARNT in basal cardiac
function in the adult heart is not known. We hypothesized
that ARNT is required for normal cardiac physiology, and its
deletion in adult heart results in cardiomyopathy. Methods
and Results: The deletion of ARNT gene in the heart of 3
month old mice was achieved by crossing ARNTflox/flox mice
with αMHC-MCM (tamoxifen-inducible heart specific Cre)
transgenic mice followed by administration of tamoxifen
chow. MHC-MCM/ARNT+/+ (WT) littermates were used as a
control. The ARNT knock out (KO) mice exhibited enlarged
left ventricle with a significant reduction in ejection fraction
(KO vs. WT: 35.8 ± 3.6% vs. 61.2 ± 2.8%, n=12, p<0.01)
and fractional shortening (KO vs. WT: 18.3 ± 1.5 % vs. 30.1
± 1.1%, p<0.01), assessed by Echocardiography. Closedchest catheterization also demonstrated reduced +dP/dt (KO
vs. WT: 4355 ± 538 vs. 9426 ± 180 mmHg, p<0.01) and
increased left ventricular end diastolic pressure (KO vs. WT:
3.12 ± 1.1 vs. 8.47± 1.2 mmHg, P<0.05) in KO mice but
not in WT mice. The worsened cardiac function in the KO
mouse heart was associated with increase in ANF and BNP
expression, interstitial fibrosis, and apoptosis as determined
by TUNEL staining. Furthermore, electron microscopy reveals
a variety of degenerative changes and some lipid droplet
in the KO hearts, and histological studies demonstrated
intramyocardial lipid accumulation in the perivascular area
in the KO mouse hearts. Analysis of gene expression in the
KO heart revealed up-regulation of peroxisome proliferatoractivated receptor alpha. Conclusion: ARNT is essential for
the maintenance of structural and functional homeostasis
in the adult heart, and its inactivation leads to cardiac
contractile dysfunction. Our findings implicate a novel critical
transcriptional requirement for ARNT in the maintenance of
adult cardiac function.
R. Wu: None.
P122Characterization of Ventricular Assist Device Mediated
Sensitization in the Bridge to Heart Transplant Patient
ABSTRACTS
Murray H Kwon, Jennifer Q Zhang, Ani Abrahamyan, Zilu K Zhang,
David W Gjertson, Abbas Ardehali, UCLA, Los Angeles, CA; Jon
Kobashigawa, Cedars Sinai Heart Inst, Los Angeles, CA; Richard J
Shemin, Elaine F Reed, UCLA, Los Angeles, CA
56
Purpose: To clarify patterns of anti-HLA antibody expression
(sensitization) occurring in patients bridged to transplantation
(BTT) with ventricular assist devices (VADs). Methods: The
study is a retrospective review of 68 patients undergoing BTT
with either Heartmate II (HMII) axial flow LVAD or paracorporal
BIVAD from January 2007 to July 2010 at UCLA Medical
Center. Results: Five of 15 (33.3%) HMII pts became
sensitized during treatment compared to 29 of 53 (54.7%)
BIVAD patients, p=0.24. Table 1 shows common etiologies
for patient sensitization of which only PRBC transfusion
was statistically significant. [table 1] Multiple variable
analysis comparing BIVAD vs. HMII while controlling for
previous cardiac surgery, pregnancy, and PRBC transfusion
demonstrated an Odds Ratio of 5.20, p=0.029 (robust
variance estimator). Of sensitized patients, all 5 (100%) of
the HMII patients had pre-existing antibodies prior to VAD
placement compared to 11 of 29 (62.1%) BIVAD patients,
p=0.016. Maximum cumulative MFIs for BIVAD were 46,259
± 66,349 vs. 42540 ± 12840 for HMII, p=0.90. Time to
maximum antibody expression was shorter for the HMII group
(34 ± 28 days vs. 5.8 ± 9 days, p=0.04). Conclusion: BIVADs
were associated with a five fold increased risk for sensitization
when accounting for other risk factors. HMII patients
required pre-sensitization to express antibodies during their
treatment interval whereas BIVAD patients developed de novo
antibodies. Although the peak cumulative MFIs were similar for
both VAD types, the days to reach this peak were significantly
less in the HMII group. These data suggest that sensitization in
HMII patients may not be due to antigenic stimulation from the
device itself.
Sensitization Risk Factors
Antibodies +
Antibodies -
p-value
Previous Cardiac
Surgery
23.5%
8.8%
0.19
Pregnancy
20.6%
2.9%
0.05
PRBC
52.1±34.7
35.9±22.3
0.04
FFP
29.4±14.4
23.6±15.2
0.15
Platelets
9.8±9.7
6.8±6.7
0.20
Cryoprecipitate
3.2±2.6
3.1±2.6
0.74
Blood products expressed as mean units ± std dev
.H. Kwon: None. J.Q. Zhang: None. A. Abrahamyan: None.
M
Z.K. Zhang: None. D.W. Gjertson: None. A. Ardehali: None.
J. Kobashigawa: None. R.J. Shemin: None. E.F. Reed: None.
P123β- and γ-Catenin Cooperate to Maintain
Mechanoelectrical Coupling in the Heart
David Swope, Lan Cheng, Jifen Li, Glenn Radice, Thomas Jefferson
Univ, Philadelphia, PA
Arrhythmic right ventricular cardiomyopathy (ARVC) is a
hereditary heart-muscle disease that causes sudden cardiac
death (SCD) in young people. Almost half of ARVC patients
have a mutation in genes encoding cell adhesion proteins
of the desmosome including plakoglobin (also known as
γ-catenin). Plakoglobin (PG) is a unique cell adhesion molecule
as it is located in both desmosomes and adherens junctions
located at the intercalated disc (ID) where it functions to link
cadherins to the cytoskeleton. Redistribution of PG away from
ID is found in virtually all cases of ARVC, regardless of the
specific mutation, suggesting that PG plays a fundamental
role in the pathogenesis of ARVC. To investigate the role of
PG in ARVC, we generated an inducible cardiac-restricted
knockout (CKO) of the PG gene in mice. Despite gap junction
remodelling, PG CKO mice have no apparent conduction
abnormality and survive longer than expected. Importantly,
the PG homolog, β-catenin, showed increased association
with the gap junction protein, connexin43 (Cx43) in PG
CKO hearts. To determine whether β-catenin protects PG
mutant animals from sudden arrhythmic death, we generated
mice lacking both PG and β-catenin specifically in the heart
(i.e. double knockout, DKO). The PG/β-catenin DKO mice
exhibited acute cardiomyopathy, fibrous tissue replacement,
and conduction abnormalities resulting in SCD 3–5 months
(124 day median survival) after deleting the genes. The
PG/β-catenin DKO hearts were susceptible to induced
arrhythmias (8/11 DKO versus 0/9 WT, p < 0.01) consistent
with the SCD phenotype. Furthermore, spontaneous lethal
arrhythmias were captured in PG/β-catenin DKO mice with
telemetric monitoring. In contrast to the PG and β-catenin
single mutants, N-cadherin was significantly reduced at the
ID in the PG/β-catenin DKO mice. Consistent with decreased
desmoglein-2 and plakophilin-2 at the ID, quantitative analysis
of TEM images demonstrated a significant reduction in the
number of desmosomes (7.36 ± 3.68 DKO versus 36.37 ±
4.44 WT per 100-µm ID; n = 10 fields per heart; p < 0.001).
In conclusion, these studies suggest that increasing β-catenin
interaction with Cx43 may enhance gap junction function in
ARVC patients, thus improving electrical conduction in the heart.
D. Swope: None. L. Cheng: None. J. Li: None. G. Radice: None.
P124Withdrawn
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Poster Presentations (continued)
4-deficient mice (CKO) to answer unresolved questions as
to in vivo function of calpains in the heart. Ubiquitous calpain
is consisted of a common regulatory subunit (calpain 4)
and a large catalytic subunit (calpain 1 for μ-calpain and
calpain 2 for m-calpain). In agreement with previous reports,
cardiac-specific deletion of calpain 4 markedly resulted in
a simultaneous decrease in protein levels of calpain 1 and
2, indicating that calpain activity was almost absent in CKO
hearts. CKO showed no cardiac phenotypes under basal
conditions. Then, we subjected CKO and control mice
(CTL) to pressure overload by means of transverse aortic
constriction (TAC). One week after TAC, CKO showed left
ventricle dilatation (LVDd, CKO 3.64 ± 0.2 mm versus CTL
2.66 ± 0.05 mm), and contractile dysfunction (FS, CKO 33.2
± 3.9% versus CTL 47 ± 0.7%). CKO hearts took up Evans
blue, a membrane-impermeant dye, within cardiomyocytes
after TAC, whereas CTL hearts or sham-operated CKO hearts
did not. This indicates plasma membrane was disrupted in
CKO hearts in response to pressure overload. We performed
membrane repair assays on isolated cardiomyocytes from
CKO hearts using a two-photon laser-scanning microscope.
CKO cardiomyocytes continued to take up FM1-43FX dye
for at least 480 sec after disruption of a plasma membrane
by laser irradiation, although CTL cardiomyocytes resealed
within 250 sec. These data indicate that plasma membrane
of cardiomyocytes disrupted by pressure overload failed to
be resealed in CKO hearts. Thus, we conclude that calpains
protect the heart from hemodynamic stresses by promoting
membrane repair.
P125Sunitinib-Induced Cardiomyopathy Is Due to
PDGFR-β Inhibition and Can Be Prevented by
Cotreatment with Thalidomide
Vishnu Chintalgattu, Meredith Rees, Univ of Texas MD Anderson
Cancer Ctr, Houston, TX; Nathan Bryan, Univ of Texas Health Science
Ctr, Houston, TX; Robert Langley, Univ of Texas MD Anderson Cancer
Ctr, Houston, TX; James Culver, Mary Dickinson, Mark L Entman,
Baylor Coll of Med, Houston, TX; Aarif Y Khakoo, Univ of Texas MD
Anderson Cancer Ctr, Houston, TX
. Chintalgattu: None. M. Rees: None. N. Bryan: None. R. Langley:
V
None. J. Culver: None. M. Dickinson: None. M.L. Entman: None.
A.Y. Khakoo: None.
P126Calpain Protects the Heart from Hemodynamic Stress
Manabu Taneike, Shungo Hikoso, Osamu Yamaguchi, Kazuhiko
Nishida, Osaka Univ Graduate Sch of Med, Suita, Japan; Naoki
Mochizuki, Natl Cerebral and Cardiovascular Ctr Res Inst, Suita, Japan;
Issei Komuro, Kinya Otsu, Osaka Univ Graduate Sch of Med, Suita, Japan
Although calpains are well-known Ca2+-dependent intracellular
cysteine proteases, the pathophysiological function of
calpains in the heart remains to be elucidated. Previous
reports have suggested that calpains play detrimental roles
to induce apoptotic or necrotic cell death in pathological
Ca2+-overloaded conditions such as ischemia-reperfusion
injury or myocardial infarction. Recent loss of function studies
have suggested that calpains play important physiological
roles in development, cell migration, organization of actin
cytoskeleton, and plasma membrane repair. In the present
study, we generated and analyzed cardiac-specific calpain
. Taneike: None. S. Hikoso: None. O. Yamaguchi: None. K. Nishida:
M
None. N. Mochizuki: None. I. Komuro: None. K. Otsu: None.
P127Carnitine Palmitoyltransferase 1b Deficiency Leads to
Exacerbated Cardiac Dysfunction and Hypertrophy in
Mice Subjected to Pressure Overload
Lan He, Teayoun Kim, Jian Liu, Peiyong Wang, Univ of Alabama at
Birmingham, Birmingham, AL; Philip A Wood, Burnham Inst for Medical
Res at Lake Nona, Orlando, FL; Qinglin Yang, Univ of Alabama at
Birmingham, Birmingham, AL
Carnitine palmitoyltransferase 1 (CPT1) is a primary ratelimiting enzyme that controls the entry of long-chain fatty
acids into the mitochondrial matrix. CPT1b is the predominant
isoform in the heart essential for myocardial fatty acid oxidation
(FAO). Inhibition of myocardial FAO by specific CPT1 inhibitors
has been proposed to be cardioprotective, but with mixed
results from animal and human studies. To gain more specific
insights, the present study investigates the effect of CPT1b
deficiency in mice subjected to transverse aorta constriction
(TAC)-induced pressure-overload. Because homozygous
knockout of CPT1b causes embryonic lethality, we used the
overtly normal heterozygous CPT1b knockout (CPT1b+/-)
mice and their wild type (WT) littermates for the study. Under a
severe pressure-overload condition, CPT1b+/- hearts showed
substantially increased mortality compared with WT hearts.
Under a milder pressure-overload condition, CPT1b+/- mice
showed more pronounced cardiac hypertrophy than WT mice.
Echocardiographic measurement revealed greater increases of
posterior wall thickness at diastole, left ventricular (LV) internal
dimension at systole and LV mass in CPT1b+/- than in WT
mice. Stroke volume, ejection fraction and fraction shorting
were also further decreased in CPT1b+/- mice. Based on
assessments of heart-weight to body-weight ratio, molecular
markers of cardiac hypertrophy, the cross-sectional area
of cardiomyocytes, fibrosis and cardiomyocyte apoptosis,
cardiac pathological hypertrophy was more pronounce
in CPT1b+/- than in WT mice. Transmission electron
microscope assessment revealed a reduced mitochondrial
volume in CPT1b+/- compared with WT hearts after TAC.
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ABSTRACTS
Introduction: Suntinib malate (SM) is a small molecule
tyrosine kinase inhibitor used for the treatment of metastatic
renal cell carcinoma. However, nearly 20% of SM treated
patients develop cardiomyopathy. We recently reported that
the cardiomyocyte PDGFR-β regulates both cardiac and
coronary microvascular function. In this study, we sought
to define the mechanisms of SM-induced cardiomyopathy.
Methods: C57BL/6 mice were divided into 4 groups:
vehicle control (Ctrl), SM, Ctrl+transverse aortic constriction
(Ctrl-TAC) and SM-TAC. SM was given at 40 mg/kg/d.
An additional group was treated with either SM (40mg/
kg/d) plus vehicle control or SM plus thalidomide (75mg/
kg/d) for 14 days and allowed to recover until day 28. Left
ventricular ejection fraction (LVEF) and coronary flow reserve
(CFR, a measurement of coronary microvascular function)
were assessed by cardiac MRI and ultrasound, respectively.
Pericyte coverage was assessed by immunofluorescent
co-staining of CD31 (vessel) and NG2 (pericyte). Results:
SM induced cardiac and coronary microvascular dysfunction
and impaired cardiac response to stress in a similar manner
to that seen in PDGFR-β knockout mice. These functional
impairments were accompanied by structural vascular
defects and significant loss of microvascular pericyte
coverage. This was recapitulated in aged (1 year) PDGFR-β
knockout mice as well as with the drug CP673,541, which
is a potent, more specific PDGFR inhibitor . Thalidomide is
known to enhance vascular stability through enhancement
of vascular pericyte coverage. Co-treatment with Thal
prevented sunitinib-induced reduction in LVEF (SMLVEF-41%; SM+thalidomide-49%, p<0.01) and preserved
CFR (SM-CFR-2.14; SM+thalidomide 2.95). Thalidomide
also significantly increased microvascular pericyte coverage.
Conclusion: SM-induced cardiac and microvascular
dysfunction is most likely due to PDGFR-β inhibition and can
be prevented by co-treatment with thalidomide. Our findings
not only suggest a novel cardioprotective strategy for cancer
patients at high-risk for sunitinib-induced cardiomyopathy
but also suggest a critical role for pericytes in coronary
microvascular and cardiac function.
57
Poster Presentations (continued)
CPT1b+/- heart sections exhibited dramatic myocardial
lipid accumulation with numerous lipid droplets. Moreover,
CPT1b+/- hearts exhibited substantially elevated triglycerides
and ceramide contents compared with WT hearts. Therefore,
we conclude that CPT1b deficiency is detrimental to the heart
under the pressure-overload condition with exacerbated
cardiac dysfunction and progressive development of
pathological hypertrophy due to lipotoxicity, thus cautious
should be taken in evaluating CPT1b as a therapeutic target
for heart disease.
. He: None. T. Kim: None. J. Liu: None. P. Wang: None. P. Wood:
L
None. Q. Yang: None.
P128Type II Deiodinase Upregulation in Patients with
End-Stage Dilated Cardiomyopathy
ABSTRACTS
Kyle K Henderson, Andrew McElligott, Miensheng Chu, Nilam Patel,
Jennifer T Paul, Loyola Univ Medical Ctr, Maywood, IL
Background: Dilated cardiomyopathy (DCM) is the most
common form of heart disease, with a 50% survival rate
5yrs after diagnosis. Low thyroid hormone levels are an
independent risk factor for heart failure progression. Recent
studies in animal models with heart failure have shown
re-expression of a fetal enzyme, type III deiodinase (D3)
that functions to reduce thyroid hormone concentrations.
Alternatively, an increase in β-adrenergic stimulation during
heart failure may increase expression of type II deiodinase
(D2) that would function to increase thyroid hormone
concentration. We hypothesized that human hearts in
end-stage dilated heart failure would have increased D3/
D2 expression and lower myocardial and serum thyroid
hormone concentrations. Methods and Results: Serum
and left ventricular (LV) tissue from patients with dilated
heart failure undergoing heart transplantation, and organ
donors not meeting the criteria for heart donation were
obtained with full consent and Institutional Review Board
approval. Deiodinase expression was measures with real time
rtPCR and thyroid hormone concentrations measured with
radioimmunoassay. Interestingly, D3 expression and protein
levels were not elevated in DCM patients vs. “non-failing” heart
samples. On the other hand, D2 expression and protein levels
were increased in DCM hearts and tended to have greater
myocardial thyroid hormone concentrations (P =0.10). In spite
of the increase in D2 expression in DCM patients, serum
thyroid hormone concentrations were significantly lower than
serum obtained from healthy volunteers, (P<0.05). Changes
in thyroid hormone receptors and thyroid hormone dependent
downstream signaling changes for Akt, mTOR, and ERK are
currently being investigated. Conclusions: Contrary to our
hypothesis, patients with end-stage DCM have a reduced D3/
D2 expression ratio and tend to have elevated myocardial
thyroid hormone concentrations. Compensatory increases
in D2 expression may enhance myocardial T3 production in
DCM patients; however changes in thyroid hormone receptors
and downstream signaling may negate this effect. Importantly,
serum T3 concentrations were significantly reduced which
likely contributed to patients progression toward end stage
heart failure.
.K. Henderson: None. A. McElligott: None. M. Chu: None. N. Patel:
K
None. J.T. Paul: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P129G Protein-Coupled Receptor Kinase-2 (GRK2) Is a Novel
Regulator of Collagen Synthesis in Adult Human Cardiac
Fibroblasts
Karen M D’Souza, Ricky Malhotra, Jennifer L Philip, Michelle L Staron,
Tiju Theccanat, Shahab A Akhter, Univ of Chicago, Chicago, IL
58
Cardiac fibroblasts (CF) make up 70% of the total cell number
in the heart and play a critical role in regulating normal
myocardial function and in adverse remodeling following
myocardial infarction. Recent studies have shown that
increased intracellular cAMP can inhibit CF transformation
and collagen synthesis in adult rat CF; however, mechanisms
by which cAMP production is regulated in CF have not been
elucidated. The objective of this study was to investigate
the potential role of GRK2 in modulating CF transformation
to myofibroblasts and collagen synthesis in adult human CF
isolated from normal and failing left ventricles. CF isolated
from failing ventricles showed a significant increase in
expression of collagen I, III and VI compared with controls.
α-SMA was increased 2-fold over controls, consistent with
CF transformation to myofibroblasts. Baseline collagen
synthesis was elevated 2-fold in failing CF and was not
inhibited by isoproterenol (ISO)-stimulation in contrast to
normal controls. β-adrenergic receptor (β-AR) signaling was
markedly uncoupled in failing CF as assessed by basal and
ISO-stimulated cAMP production. The primary mechanism
appears to be a 2.5-fold increase in GRK2 activity as GRK2
phosphorylates and uncouples agonist-occupied β-ARs.
Overexpression of GRK2 in normal CF recapitulated a heart
failure phenotype with minimal inhibition of collagen synthesis
following ISO stimulation. In contrast, siRNA-mediated
knockdown of GRK2 expression in normal CF enhanced
cAMP production and led to greater β-agonist-mediated
inhibition of basal and TGFβ-stimulated collagen synthesis
versus control. Inhibition of GRK2 activity by adenoviralmediated βARKct expression or GRK2 knockdown in
failing CF led to a significant decline in collagen and α-SMA
expression. GRK2 inhibition restored β-AR signaling and ISOstimulated inhibition of collagen synthesis and also significantly
decreased collagen synthesis in response to TGFβ stimulation.
In conclusion, GRK2 appears to play a significant role in
regulating CF transformation and collagen synthesis in adult
human CF and increased activity of this kinase may be an
important mechanism of maladaptive ventricular remodeling
as mediated by cardiac fibroblasts.
K.M. D’Souza: None. R. Malhotra: None. J.L. Philip: None.
M.L. Staron: None. T. Theccanat: None. S.A. Akhter: None.
P130Estrogen Receptor β Mediates the Rescue of Cardiac
Function in Advanced Heart Failure by Promoting
Neoangiogenesis and Reducing Fibrosis
Andrea Iorga, Rod Partow-Navid, Humann Matori, Jingyuan Li, Soban
Umar, Mansoureh Eghbali, UCLA, Los Angeles, CA
Estrogen can act via the estrogen receptor alpha (ERa) or
estrogen receptor beta (ERb) to exert its biological effects,
and both of these receptors are present in the heart. We have
previously shown that short-term estrogen (E2) treatment
can rescue pressure overload-induced decompensated
heart failure (HF) in mice, and that this rescue is achieved
mainly through the ERb. Furthermore, E2 has been shown
to regulate angiogenesis in different tissues. Because HF has
been associated with decreased angiogenesis and increased
fibrosis, here we investigated whether the E2-induced rescue
of HF by the selective ERb agonist DPN can regulate cardiac
fibrosis and neoangiogenesis. We used transaortic constriction
to induce HF, and once the ejection fraction (EF) reached
~30%, one group of animals was sacrificed (HF group),
and the other three groups received either 17b-estradiol
via a subcutaneous pellet implant (0.012mg/pellet, n=16),
selective ERa agonist (PPT, 0.625mg/kg/day), or selective
ERb agonist (DPN, 0.625mg/kg/day) for 10 days. Serial
echocardiography was performed to monitor cardiac structure
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Poster Presentations (continued)
and function. As expected, E2 rescued HF by restoring
EF from 33.17±1.12% to 53.05±1.29%. Mice treated with
DPN had a significant EF improvement from 33.17±1.12%
to 45.25±2.1% (n=7), while the EF of PPT-treated mice did
not improve (31.09±2.3%, n=6). Similarly, only the fractional
shortening of DPN-treated mice improved from 15.7±0.58%
in HF to 21.95±1.65% with DPN treatment vs. 14.72±1.24%
with PPT. Next, we examined whether promotion of cardiac
neoangiogenesis and suppression of fibrosis by the selective
ERb agonist are possible mechanisms in the rescue action
of HF by DPN. DPN treatment was able to reverse the
interstitial and perivascular fibrosis observed in HF, while PPT
had no effect. The selective ERb agonist also stimulated
neoangiogenesis, as the capillary density was increased from
0.46±0.04 microvessels/cardiomyocyte in HF to 0.67±0.07
with DPN treatment, whereas PPT treatment had no effect
(0.43±0.03). Our data strongly suggests that upregulation of
cardiac neoangiogenesis and reversal of fibrosis are pivotal
mechanisms in rescuing advanced HF by the estrogen
receptor beta agonist DPN.
. Iorga: None. R. Partow-Navid: None. H. Matori: None. J. Li:
A
None. S. Umar: None. M. Eghbali: None.
P131An Akt-Phosphomimetic Sequence of the Cavb2
C-Terminal Region Protects L-Type Calcium Channels
from Protein Degradation
Francesca Rusconi, Multimedica, Milan, Italy; Magali Cazade, Inst
de Génomique Functionnelle, CNRS UMR5203, INSERM U661,
Univs de Montpellier, Montpellier, France; Fabio Polticelli, Univ Roma
Tre, Rome, Italy; Jean Chemin, Inst de Génomique Functionnelle,
CNRS UMR5203, INSERM U661, Univs de Montpellier, Montpellier,
France; Gianluigi Condorelli, Univ of California, San Diego, San Diego,
CA; Daniele Catalucci, Genetic and Biomedical Res Inst — Natl Res
Council, Milan, Italy
. Rusconi: None. M. Cazade: None. F. Polticelli: None. J. Chemin:
F
None. G. Condorelli: None. D. Catalucci: None.
P132Diabetes-Induced Cardiac Dysfunction Is Rescued
by Endothelial Cationic Amino Acid Transporter 1
Overexpression in Mice
Rebecca H Ritchie, Makhala M Khammy, Marissa A Bowden, Jennifer
Irvine, Karen Andrews, Karina Huynh, Helen Kiriazis, David Kaye, Kylie
Venardos, Baker IDI Heart & Diabetes Inst, Melbourne, Australia
Nitric oxide (NO) bioavailability is markedly impaired in
diabetes, contributing to endothelial and myocardial
dysfunction. Our aim was to determine if endothelial-specific
overexpression of Cationic Amino Acid Transporter 1 (CAT-1,
to enhance L-arginine uptake and NO generation) prevents
diabetes-induced endothelial and cardiac dysfunction in
diabetic mice. Male CAT-1 transgenic and non-transgenic
(Ntg) 6 week old mice were administered streptozotocin (STZ,
55mg/kg ip daily for 5 days). Sham mice received citrate buffer
vehicle. Myocardial function was assessed in anaesthetised
mice in vivo 12 weeks later, via echocardiography. Vascular
reactivity was then assessed in vitro. Compared to their
non-diabetic littermates (n=6), Ntg STZ mice (n=6) exhibited
reduced early-to-late blood flow velocity ratios (E/A,
1.7±0.2 vs 2.4±0.1, p<0.01) and prolonged deceleration
time (DT, 40±1 vs 31±2msec, p<0.002), indicative of
diastolic dysfunction. Decreased E-wave velocity (109±4 vs
76±12msec, p<0.01) and increased A-wave velocity (45±3 vs
55±4msec, p<0.05) were also observed. Systolic dysfunction
was observed in Ntg STZ mice compared to shams, on
reduced fractional shortening (FS, 30±1 vs 39±1%, p<0.002).
In CAT-1 mice, diabetes did not impair E/A ratio (2.1±0.2), FS
(40±3%) or DT (33±1msec, n=8). Thoracic aortic responses
to the endothelium-dependent vasodilator acetylcholine (ACh)
were unchanged by diabetes in Ntg and CAT-1 Tg mice
compared to sham Ntg mice (pEC50 7.0±0.1 vs 7.2±0.1
and 7.1±0.1). There was a non-significant trend for sham
CAT-1 Tg mice to show greater sensitivity to ACh than sham
Ntg mice (pEC50 7.5±0.1 vs 7.1±0.1). Responses to the
endothelium-independent vasodilator sodium nitroprusside
and the vasoconstrictor noradrenaline were unaffected by
diabetes and/or genotype. In conclusion, our results suggest
that endothelial-specific CAT-1 overexpression protects
against cardiac dysfunction via a mechanism independent of
the systemic vasculature. The involvement of the coronary
vasculature remains to be determined. Endothelial CAT-1
up-regulation as a potential therapeutic target for diabetic
cardiomyopathy is an exciting prospect.
.H. Ritchie: None. M.M. Khammy: None. M.A. Bowden: None.
R
J. Irvine: None. K. Andrews: None. K. Huynh: None. H. Kiriazis:
None. D. Kaye: None. K. Venardos: None.
P133 Withdrawn
P134Synoviolin Is a Stress-Inducible Endoplasmic/
Sarcoplasmic Reticulum E3 Ubiquitin Ligase that
Preserves Cardiac Function
ABSTRACTS
Alteration in the density or function of L-Type Calcium
Channels (LTCCs) has been related to cardiovascular
diseases such as heart failure and diabetic cardiomyopathy.
It could therefore be envisioned that increasing LTCC density
may improve cardiac function in heart failure. Recently, we
determined that the Ser-Thr kinase Akt plays a key role
in regulating cardiac inotropism through the modulation
of LTCC density and function. Specifically, we found that
the LTCC pore-forming channel subunit Cava1.2 contains
highly evolutionary conserved PEST sequences (signals
for rapid proteolytic degradation) that are responsible for
direct Cava1.2 protein degradation. Phosphorylation of
the C-terminal coiled coil of the Cavb2 chaperone subunit
enhances LTCC protein stability by preventing PEST-mediated
Cava1.2 degradation. The aim of this study was to further
dissect this Akt-dependent fine-tuning mechanism regulating
LTCC density, searching for potential Akt-phosphomimetic
(APM) molecules that could enhance LTCC density. Using
yeast two-hybrid screening, we found that APM Cavb2
sequences interact with the globular domain of Cavb2 itself
in a solvent-exposed region that we named Tail Interacting
Domain (TID). Biochemical and functional assays as well
as site-specific mutagenesis in TID identified the minimal
aminoacid sequence responsible for the TID-tail interaction.
In addition, through an approach comprising western blot
analyses, fluorescent-based calcium assays, calcium current
(ICaL) measurements, molecular modeling and peptide
arrays, we identified the minimal APMs that efficiently protects
Cava1.2 from protein degradation. Based on our in vitro
results, we suggest that the identified APM sequence/peptide
could be used as a “therapeutic approach” for increasing or
reestablishing impaired cardiac contractility in mouse models
of cardiomyopathy in which the LTCC density is altered, thus
enhancing inotropism.
Shirin Doroudgar, Donna J Thuerauf, Mohsin Khan, Sadia Mohsin,
Mirko Völkers, Natalie Gude, San Diego State Univ, San Diego, CA;
Oliver J Müller, Univ of Heidelberg, Heidelberg, Germany; Mark A
Sussman, Christopher C Glembotski, San Diego State Univ, .
San Diego, CA
We recently reported that synoviolin1 (syvn1) is a novel
endoplasmic reticulum stress response (ERSR) protein that is
up-regulated in the mouse heart by ATF6, a cardioprotective,
nodal transcription factor of the ERSR. Syvn1 is a unique
E3 ubiquitin ligase that retrotranslocates misfolded proteins
from endoplasmic/sarcoplasmic reticulum (ER/SR) to the
cytosol and subsequently polyubiquitinates them, targeting
them for degradation. We now report that syvn1 expression is
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59
Poster Presentations (continued)
induced with tunicamycin, thapsigargin, and dithiothreitol, ER
stressors that activate ATF6. Moreover, adenovirus-mediated
syvn1 overexpression in neonatal rat ventricular cardiac
myocytes (NRVCMs) increases contractility. Consistent
with this finding, syvn1 overexpression increases calcium
transient amplitude as well as diastolic calcium. We also find
that syvn1 overexpression decreases secretion of MANF, a
protective, anti-hypertrophic, ER/SR protein which we find
is conditionally secreted when ER/SR calcium is depleted.
We also report MANF as the first example of a is protein
whose secretion from ventricular myocytes is conditionally
dependent on ER/SR calcium. Furthermore, syvn1 reduces
the growth of NRVCMs treated with the α-adrenergic
agonist, phenylephrine. Moreover, while knockdown of
syvn1 in NRVCMs using syvn1-targeted siRNA increases
cell death, overexpression of syvn1 promotes cell survival.
To investigate the roles of syvn1, in vivo, we examined the
effects of syvn1 overexpression in cardiac myocytes in a
mouse model of trans-aortic banding. Syvn1 overexpression,
in vivo, was achieved by intravenous delivery of recombinant
AAV serotype 9 (AAV9; cardiac specific serotype) with MLC2v
promoter driving syvn1 expression. Baseline cardiac function,
as measured by echocardiography six weeks after gene
delivery, shows no difference between AAV9-control and
AAV9-syvn1 treated mice. Trans-aortic banding decreases
cardiac function in mice injected with AAV9-control. In
contrast, cardiac function is preserved in mice injected with
AAV9-syvn1. The findings in this study suggest that syvn1 is a
novel stress-inducible cardiac E3 ligase with unique functions
in regulating protein secretion, maintaining cell viability, and
preserving cardiac function.
S. Doroudgar: None. D.J. Thuerauf: None. M. Khan: None.
S. Mohsin: None. M. Völkers: None. N. Gude: None. O.J. Müller:
None. M.A. Sussman: None. C.C. Glembotski: None.
This research has received full or partial funding support from the
American Heart Association, Western States Affiliate (California,
Nevada & Utah).
P135Hyperpolarized Carbon-13 Magnetic Resonance Reveals
Early- and Late-Onset Changes to Metabolism in the
Failing Heart
ABSTRACTS
Marie Schroeder, Univ of Oxford, Oxford, United Kingdom; Angus
Z Lau, Sunnybrook Health Sciences Ctr, Toronto, ON, Canada;
Albert P Chen, GE-Healthcare, Toronto, ON, Canada; Jennifer Barry,
Sunnybrook Health Sciences Ctr, Toronto, ON, Canada; Damian J Tyler,
Kieran Clarke, Univ of Oxford, Oxford, United Kingdom; Kim A Connelly,
Graham Wright, Charles H Cunningham, Sunnybrook Health Sciences
Ctr, Toronto, ON, Canada
60
Disordered metabolic substrate utilisation has been implicated
in the pathogenesis of heart failure (HF). Hyperpolarised
(HYP) 13C magnetic resonance, a technique in which the
fate of 13C-labelled metabolites can be followed using
MR imaging or spectroscopy, has enabled non-invasive
assessment of metabolism. The aim of this study was to
monitor carbohydrate metabolism alongside cardiac structure,
function, and energetics, throughout HF progression. HF
was induced in pigs (n=5) by right ventricular pacing at
188 bpm for 5 weeks. Pigs were examined at weekly time
points: cine MRI assessed cardiac structure and function,
HYP 13C2-pyruvate was administered intravenously and
13C MRS was used to assess 13C-glutamate production
via Krebs cycle, 31P MRS assessed myocardial energetics,
and HYP 13C1-pyruvate was administered to enable MRI
of H13CO3- production from pyruvate dehydrogenase
(PDH). At baseline, pigs had a normal left ventricular (LV)
cardiac index (CI) and end diastolic volume (EDVi). The PCr/
ATP was 2.3 ± 0.2. The 13C-glutamate/13C2-pyruvate was
4.3 ± 0.9%, and the H13CO3-/13C1-pyruvate ratio was
1.6 ± 0.2%. After 1–2 weeks of pacing, CI decreased to
3.3 ± 0.5 l/min/m2, PCr/ATP decreased to 1.7 ± 0.1, and
13C-glutamate/13C2-pyruvate decreased to 2.1 ± 0.6%.
With the onset of HF, EDVi increased to 140.3 ± 14.1 ml/
m2 and H13CO3-/13C1-pyruvate decreased to 0.5 ± 0.2%.
In conclusion, we observed an early defect in Krebs’ cycle
that occurred alongside impaired cardiac energetics and
function. Carbohydrate oxidation via PDH was maintained until
the onset of HF. These results encourage use of metabolic
therapies to delay/prevent the onset of heart failure in patients.
. Schroeder: C. Other Research Support; Modest; GE-Healthcare.
M
A.Z. Lau: None. A.P. Chen: A. Employment; Significant; Employee of
GE-Healthcare. J. Barry: None. D.J. Tyler: None. K. Clarke: None.
K.A. Connelly: None. G. Wright: C. Other Research Support; Modest;
GE-Healthcare. C.H. Cunningham: C. Other Research Support;
Modest; GE-Healthcare.
P136E2F2/4 Regulated by Serine 21 Phosphorylation of GSK3α Play Compensatory Roles During Pressure Overload
Takanobu Yamamoto, Yasuhiro Maejima, Peiyong Zhai, Takahisa
Matsuda, Junichi Sadoshima, UMDNJ, Newark, NJ
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine
kinase with two isoforms, alpha and beta, which have
distinct functions in cardiomyocytes (CMs). GSK-3alpha
is phosphorylated at S21 during pressure overload (PO),
and inhibition of S21 phosphorylation in GSK-3alpha S21A
knock-in (alpha-KI) mice promotes hypertrophy and heart
failure in response to PO, accompanied by decreases in
the total number of CMs in the heart. Since GSK-3alpha
downregulates cyclinD1 in the nucleus, GSK-3alpha may
negatively regulate E2F-mediated transcription. Reporter
gene assays showed that the transcriptional activity of
E2F was increased by GSK-3alpha knockdown (1.75 fold,
p<0.05). To evaluate the role of E2F isoforms in regulating
cardiac hypertrophy and function during PO, E2F1-/-,
E2F2-/+, E2F4-/+, and wild type (WT) mice were subjected
to transverse aortic constriction (TAC). Left ventricular (LV)
weight/ tibial length (LVW/TL) was significantly greater and
LV ejection fraction (LVEF) was significantly decreased in
both E2F2-/+ and E2F4-/+ after 2 weeks of TAC (LVW/
TL: E2F2-/+=7.1±0.3, E2F4-/+=7.0±0.4, WT=5.9±0.3,
p<0.05 vs. WT; LVEF: E2F2-/+=53±1%, E2F4-/+=61±2%,
WT=75±1%, p<0.05 vs. WT). Thus, downregulation of
either E2F2 or E2F4 induced a phenotype similar to that
of alpha-KI in response to TAC. To examine the causative
role of E2F2/E2F4 downregulation in mediating the cardiac
phenotype in alpha-KI mice, adenovirus (Ad) harboring either
E2F2 or E2F4 was injected into alpha-KI hearts. Rescue
with E2F2 or E2F4 attenuated cardiac hypertrophy (LVW/
TL: alpha-KI+E2F2=7.1±0.4, alpha-KI+E2F4=7.3±0.3,
alpha-KI+LacZ=8.7±0.4, p<0.05 vs. alpha-KI+LacZ) and
improved LV dysfunction (LVEF: alpha-KI+E2F2=66±3%,
alpha-KI+E2F4=60±2%, alpha-KI+LacZ=39±2%, p<0.05
vs. alpha-KI+LacZ) in alpha-KI mice under PO conditions.
Injection of either Ad-E2F2 or Ad-E2F4, but not of Ad-LacZ,
significantly increased the number of Ki67-positive myocytes
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Poster Presentations (continued)
in the alpha-KI mice (alpha-KI+LacZ =0.7±0.3%, alphaKI+E2F2=10.4±2.3%, alpha-KI+E2F4=9.2±1.5%, p<0.05
vs. alpha-KI+LacZ). These results suggest that maintaining
the activity of E2F2 and E2F4 through S21 phosphorylation
of GSK-3alpha plays an essential role in preserving cardiac
function during PO.
myocardial ischemia-reperfusion injury. It is not known
whether PTEN regulates cytokine production and cardiac
remodeling. Here, we have investigated the hypothesis
that PTEN mediates post-MI remodeling by inhibiting
IL-10. Methods and Results: MI was induced in
wildtype (WT) mice and Pten heterozygous (HET) mice.
In a separate experiment, Pten adenoviruses or empty
viruses were injected into the peri-infarct area of WT mice.
Left ventricular (LV) anatomical and functional changes
were assessed by echocardiography. Mononuclear cell
infiltration was measured by histology. TNF-α, IL-10,
matrix metalloproteinase (MMP)-2 and MMP-9 protein
levels were analyzed in the peri-infarct area. At the end of
the experiments, LV end-diastolic diameter (LVEDD) was
decreased and fractional shortening (FS) was increased
in HET mice compared with WT mice (LVEDD: 4.13±0.22
vs. 5.42±0.26 mm, p<0.01; FS: 21.9±2.6 vs. 11.5±1.1
%, p<0.001). Moreover, heart weight and infarct size were
decreased in HET mice compared with WT mice. However,
FS was decreased and LV rupture (LVR) was increased
in Pten adenovirus-treated mice compared with empty
vector-treated mice [FS: 8.2±1.1 vs. 21.5±3.2 %, p <
0.01; LVR: 100% (16/16) vs. 33% (3/9)]. Mononuclear cell
infiltration was attenuated in HET mice and worsened in
Pten adenovirus-treated mice. IL-10 protein levels were
upregulated and TNF-α, MMP-2, and MMP-9 protein levels
were downregulated in HET mice. The opposite effects were
found in Pten over-expressing hearts. Conclusions: PTEN
downregulation increases IL-10 production and inhibits
inflammatory responses and attenuates cardiac remodeling;
conversely, PTEN over-expression inhibits IL-10 expression
and increases cardiac injury. Therefore, our studies
demonstrate that PTEN mediates post-MI remodeling, at
least in part, by regulating IL-10 production.
T. Yamamoto: None. Y. Maejima: None. P. Zhai: None. T. Matsuda:
None. J. Sadoshima: None.
P137Metabolomic Analysis of the Early and Late
Hypertrophic Heart
Alan C Brooks, Brian E Sansbury, Zhengzhi Xie, Robert E Brainard,
Lewis J Watson, Kenneth R Brittian, Sumanth D Prabhu, Steven P Jones,
Aruni Bhatnagar, Bradford G Hill, Univ of Louisville, Louisville, KY
A.C. Brooks: None. B.E. Sansbury: None. Z. Xie: None. R.E. Brainard:
None. L.J. Watson: None. K.R. Brittian: None. S.D. Prabhu: None.
S.P. Jones: None. A. Bhatnagar: None. B.G. Hill: None.
P138PTEN Mediates Post-Myocardial Infarction Remodeling
Nirmal Parajuli, Johns Hopkins Univ, Baltimore, MD; Yuan Yuan,
Fourth Military Medical Univ, Xian, China; Djahida Bedja, Zheqing P Cai,
Johns Hopkins Univ, Baltimore, MD
Background: Following myocardial infarction (MI), innate
immunity is activated, leading to cytokine production
and initiation of inflammation. Interleukin (IL)-10 has
been reported to attenuate post-MI remodeling by
inhibiting expression of tumor necrosis factor (TNF)-α.
The phosphatase and tensin homologue deleted on
chromosome ten (PTEN) plays an important role in
N. Parajuli: None. Y. Yuan: None. D. Bedja: None. Z.P. Cai: None.
P139Heart Failure Is Initiated By and Progresses Because of
Normal Responses of Energy Metabolism to Stress
John E Foker, James M Berry, Brian A Harvey, Univ of Minnesota,
Minneapolis, MN; Nicholas T Befera, Duke Univ, Durham, NC; Kevin J
Tveter, West Virginia Univ, Morgantown, WV; Richard W Bianco, Univ .
of Minnesota, Minneapolis, MN
Objectives: The mechanism underlying heart failure (HF)
after an index cardiac event is unknown but should have
several characteristics. HF is a modern problem, so it will
likely be a general response and be able to encompass
the variety of initiating cardiac lesions as well as significant
stress (e.g. exertion). The consequences should lead to
the many alterations found in HF. [ATP] falls when energy
demand outstrips supply (e.g. ischemia) but do not
recover promptly with reperfusion because AMP is quickly
catabolized and unavailable for recharging. [ATP] recovery
is slow and limited by the availability of ribose-5-P. Ribose
is not used as fuel and is channeled into nucleotide (ATP)
synthesis allowing the effects of [ATP] recovery on function
to be studied. Our hypothesis was that HF results from
these responses of energy metabolism to stress which
lower [ATP] and affect the numerous myocardial reactions
whose activity depends on [ATP]. The ability of ribose to
quickly increase [ATP] allows these effects to be studied.
Methods: The effects of ribose infusion on two models of
myocardial stress were studied. (1) An intact canine model
of global ischemia which allowed serial myocardial biopsies
and detailed function analysis. (2) A rat myocardial infarction
model which allowed ECHO analysis of the remote
myocardium. Results:
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ABSTRACTS
The metabolic adaptations to acute myocardial pressure
overload are characterized by alterations in metabolism that
drive the hypertrophic response and that balance workload
with energy demand. Under conditions of chronic pressure
overload, it is known that substrate utilization becomes
less flexible and that the heart shifts energy preference
from fatty acids to glucose. Nevertheless, the metabolic
changes that underlie the progression of compensated
hypertrophy to heart failure are incompletely understood and
attempts to correct the known metabolic defects to delay
decompensation have been largely unsuccessful. To identify
key changes in metabolic phenotype that could underlie
progression to heart failure, we measured metabolites in a
transverse aortic constriction (TAC) mouse model using an
unbiased metabolomic approach. Hearts were harvested
1 d, 1 wk and 8 wks after sham or TAC operation, and
metabolites were extracted from the hearts and analyzed via
GC/MS and LC/MS/MS. The signal intensities of 288 named
metabolites were re-scaled to median values. Welch’s t-test
and two-way ANOVA were used to identify metabolites that
changed significantly with pressure overload and progression
to heart failure. Echocardiographic measurements showed
a significant decrease in ejection fraction after 1 d (65±2%
vs. 49±5%) and 8 wks (61±2% vs. 34±8%) of TAC; 1 wk
of TAC showed a compensated phenotype characterized
by a largely preserved ejection fraction. One day after TAC,
only 1.7% of the metabolites changed significantly; however,
nearly all amino acids measured were increased by 1 wk.
By 8 wks, amino acids returned to near sham levels and a
significant and robust decrease in phospholipid, carnitine,
inositol, sterol, and fatty acid metabolites occurred. These
findings demonstrate that the temporal changes in metabolic
phenotype are more complex than previously thought. The
preservation of pathways involved in lipid and amino acid
metabolism may be important for maintaining myocardial
energetics and preventing pump failure under conditions of
chronic pressure overload.
61
Poster Presentations (continued)
Global ischemia (20’)
[ATP] recovery control w/
ribose infusion
9.9 + 1.4 days
2.8 + 0.6 days
Diastolic compliance
recovery control w/ ribose
infusion
9.4 + 1.1 days
2.4 + 0.9 days
Myocardial infarction
Pre MI
2 weeks
post MI
LV systolic diameter (cm)
control w/ ribose infusion
0.40 + 0.03 0.39
+ 0.06
0.73 + 0.10
0.47 + 0.22
Remote LV wall thickness
(cm) control w/ ribose
infusion
0.13 + 0.03 0.13
+ 0.02
0.10 + 0.01
0.18 + 0.12
LV ejection fraction control
w/ ribose infusion
74 + 5.9% .
76 + 2.3%
22 + 6.3%
52 + 5.0%
onclusions: 1) When energy demand outstripped supply
C
(either from ischemia or increased work) myocardial [ATP] falls
and recovery was slow. 2) Ribose is the limiting precursor for
[ATP] recovery and infusion allowed the relationship between
[ATP] and function to be studied. 3) In both myocardial
ischemia recovery and increased load (from MI) ribose
enhanced [ATP] and improved function. 4) The response of
myocardial energy metabolism response to stress results in
lower [ATP] which will decrease the activity of many function
related reactions. 5) HF could begin and progress from these
reactions of energy metabolism to cardiac stress.
J.E. Foker: None. J.M. Berry: None. B.A. Harvey: None. N.T. Befera:
None. K.J. Tveter: None. R.W. Bianco: None.
P140Not published at presenter’s request.
P141Gender Differences in the Development of Severe
Pulmonary Hypertension and Right Ventricular
Dysfunction in Apolipoprotein E-Deficient Mice Are
Eliminated with Increasing Age
ABSTRACTS
Rod Partow-Navid, Soban Umar, Humann Matori, Andrea Iorga,
Alan M Fogelman, Mohamad Navab, Mansoureh Eghbali, UCLA, Los
Angeles, CA
62
Apolipoprotein E (ApoE) is a multifunctional protein and its
deficiency leads to the development of atherosclerosis in mice.
Patients with pulmonary hypertension (PH) have reduced
expression of ApoE in lung tissue. ApoE is known to inhibit
endothelial and smooth muscle cell proliferation and has
anti-inflammatory and anti-platelet aggregation properties.
Young ApoE deficient mice have been shown to develop
high fat diet-induced PH in a gender specific manner. Estrous
cyclicity peaks at 7–8 months and declines by 9 months of
age in mice. Here we investigated the effects of monocrotaline
(MCT) on young and middle-aged ApoE deficient mice.
Middle-Aged (MA) (11–12 month old) male (n=4) and female
(n=4) and young (7–8 month old) male (n=5) and female (n=5)
ApoE deficient mice were injected with a single intraperitoneal
dose of MCT (60 mg/kg). Mice were closely monitored for
~4 weeks with serial echocardiography for cardiopulmonary
hemodynamic assessment. Direct cardiac catheterisation was
performed terminally to record peak systolic right ventricular
pressure (RVP). RV, LV, IVS and lung tissue was dissected and
weighed. Trichrome staining and histochemical analyses were
performed. At ~4 weeks after MCT, MA male and female and
young male mice developed severe PH (RVP: MA male=64±5
mmHg, MA female=71±4 mmHg, young male=60±5 mmHg,
p=n.s between all the groups) whereas young females
developed significantly less severe PH (RVP: 37±5 mmHg,
p<0.05 vs. MA male and female, and young male). MA male
and female and young male mice developed severe RV
dysfunction (RV ejection fraction (RVEF): MA male=31±2%,
MA female=28±4%, young male=36±1%, p=n.s between all
the groups) whereas young females showed significantly better
RV function (RVEF: 43±2%, P<0.05 vs. MA male and female,
and young male). MA male and female mice also developed
more severe RV hypertrophy (RV/LV+Septum, MA male=0.49,
MA female=0.53, young female=0.39). MA male and female
mice also manifested increased peripheral pulmonary artery
muscularization and pulmonary fibrosis. Interestingly, the
gender differences witnessed between young ApoE deficient
male and female mice in the development of severe PH and RV
dysfunction are abolished as the mice increase in age.
R. Partow-Navid: None. S. Umar: None. H. Matori: None. A. Iorga:
None. A.M. Fogelman: None. M. Navab: None. M. Eghbali: None.
P142The Actin-Binding Protein Vinculin Interacts with Zonula
Occludens-1 and Controls Endoplasmatic Reticular
Stress-Induced Ubiquitin/Proteasome Pathway Activation
in Cardiac Myocytes
Alice E Zemljic-Harpf, Joseph Godoy, VA Healthcare, Univ of
California, San Diego, La Jolla, CA; Elizabeth K Asfaw, VA Healthcare,
Veterans Medical Res Fndn, La Jolla, CA; Andrea A Domenighetti, Univ
of California, San Diego, La Jolla, CA; Robert S Ross, VA Healthcare,
Univ of California, San Diego, La Jolla, CA
In cardiac-myocytes (CMC) Vinculin (Vcl) links actin filaments
to integrin and cadherin based cellular junctions. Zonula
occludens -1 (ZO-1) binds connexin (Cx) 43, cadherin and
actin in CMC. Vcl and ZO-1 anchor the sarcomere to the
sarcolemma. Patients harboring mutations in the Vinculin
gene develop cardiomyopathy. CMC specific inactivation of
the Vcl gene (cVclKO) caused mal-distribution of Cx 43 and
predisposed cVclKO mice to sudden death by ventricular
tachycardia during preserved cardiac function. Mice that
survived developed heart failure. We hypothesized that Vcl
disruption affects gap junctional integrity through an interaction
with ZO-1. Results: Co-immunoprecipitation studies showed
that endogenous Vcl from wild-type CMC lysates bound Cx43
as well as ZO-1. Immunomicroscopy of Vcl, Cx43 and ZO-1 in
wild-type heart, as well as in isolated adult CMC showed that
Vcl, Cx43 and ZO-1 co-localize at the intercalated disc (ICD).
cVclKO hearts showed that ZO-1 expression was absent from
ICDs in Vcl null CMC. Yeast two-hybrid protein interaction
screening revealed that the 3rd PDZ/SH3 domain of ZO-1
interacted with the Vcl head domain. Adenoviral-mediated Cre
expression in Vclfl/fl neonatal mouse CMC reduced Vcl mRNA
to 27% ± 0.002 (p<0.005), and Vcl protein expression to 47%,
± 0.044, p<0.01, when compared to LacZ expressing controls
(VclWT). With this we found: 1) decreased Cx43 (to 17% ±
0.07), ZO-1 (60% ± 0.03), talin (43% ± 0.24), β1D integrin
(21% ± 0.09) and RhoA protein expression despite unchanged
transcript levels (all p<0.0001), 2) a 6 fold increased Akt
activation (S473/total Akt), abolished FOXO1 activation, 3)
increased protein expression of the ER chaperone calnexin,
activated Bad and JNK and 4) restored Cx43, ZO-1, talin and
β1D integrin protein after proteasomal inhibition. A 70% Vcl
reduction resulted in cardiomyocyte death. Conclusion: This
is the first report of direct interaction of Vcl with ZO-1. Vcl
reduction decreased Cx43, ZO-1, β1D, talin and RhoA protein
expression, which were rescued by proteasomal inhibition.
This indicates that Vcl plays a critical role in stabilizing cellmatrix and cell-cell contact sites by controlling ER stress
induced proteasomal degradation. Vinculin is crucial to
preserve cardiac myocyte integrity.
A.E. Zemljic-Harpf: None. J. Godoy: None. E.K. Asfaw: None.
A.A. Domenighetti: None. R.S. Ross: None.
This research has received full or partial funding support from the
American Heart Association, Western States Affiliate (California,
Nevada & Utah).
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Poster Presentations (continued)
P143Activation of GPR30 Attenuates Diastolic Dysfunction and
LV Remodeling in Oophorectomized mRen2.Lewis Rats
Hao Wang, Jewell A Jessup, Sarah Lindsey, Marina S Lin, Clarissa
Chagas, Leanne Groban, Wake Forest Univ Sch of Med, WinstonSalem, NC
The incidence of diastolic dysfunction increases in women
after menopause but the mechanisms involved are not
completely understood. GPR30 is a novel estrogen receptor
expressed in various tissues including the heart. Studies have
shown that activation of GPR30 by its agonist G1, improves
contractile function and reduces infarct size in isolated rat
and mouse hearts subjected to ischemia/reperfusion injury
and attenuates diastolic dysfunction in a salt-sensitive rat
model of hypertension. We hypothesized that the activation of
GPR30 by G1 protects against the development of diastolic
dysfunction and cardiac hypertrophy in oophorectomized
mRen2.Lewis rats, an established model that emulates the
cardiac phenotype of the postmenopausal woman. Surgical
bilateral oophorectomy (OVX) was performed in female mRen2.
Lewis rats at 4 weeks of age, and G1 (50 or 100 µg/kg/day)
was given subcutaneously via minipump starting at 13 weeks
of age for 2 weeks. Both doses of G1 significantly improved
lusitropic function and structure, independent of changes in
blood pressure. Compared to vehicle-treated OVX rats, G1
reduced the tissue Doppler-derived index of left ventricular
filling pressure (E/e’), left ventricle mass, wall thickness, and the
biomarkers of hypertrophy, ANF and BNP mRNA levels. Using
cultured H9c2 cardiomyocytes, in vitro studies further showed
that 1) G1 inhibited angiotensin (ANG) II-induced hypertrophy,
evidenced by reductions in cell size, and ANF and BNP
gene expression; 2) the antagonist of GPR30, G15, inhibited
estrogen’s protective effects on ANG II-induced hypertrophy;
and 3) G1 induced phosphorylation of Erk and Akt. These
data demonstrate the protective role of GPR30 in a model of
diastolic dysfunction and cardiac hypertrophy, and provide
insight into the underlying mechanisms.
H. Wang: None. J.A. Jessup: None. S. Lindsey: None. M.S. Lin:
None. C. Chagas: None. L. Groban: None.
P144ER Stress-Mediated Reduction of SERCA2a Protein Is
Not Involved in Increase in Lethal Heart Failure After
Myocardial Infarction in Obese Type 2 Diabetes
Tatsuya Sato, Akifumi Takada, Takayuki Miki, Masaya Tanno, Atsushi
Kuno, Hidemichi Kouzu, Takahito Itoh, Daisuke Sunaga, Tetsuji Miura,
Sapporo Medical Univ Sch of Med, Sapporo, Japan
T. Sato: None. A. Takada: None. T. Miki: C. Other Research Support;
Modest; Otsuka Pharmaceutical Co.. M. Tanno: None. A. Kuno: None.
H. Kouzu: None. T. Itoh: None. D. Sunaga: None. T. Miura: C. Other
Research Support; Modest; Otsuka Pharmaceutical Co..
P145Cathelicidin Antimicrobial Peptide Is Upregulated After
Myocarditis and Inhibits Fibroblast Migration via P2x7
Receptor Signaling
Hiroyuki Nakayama, Kazuki Matsui, Tomomi Yamashita, Haruyo
Kawaguchi, Yasushi Fujio, Osaka Univ, Osaka, Japan
Rationale: Experimental autoimmune myocarditis in BALB/c
mice is a model of inflammatory heart disease. It is well known
that the process of this model exhibits the acute inflammation
phase followed by the healing phase. However, the precise
mechanisms how the inflammation terminates and the healing
proceeds remain to be elucidated. Objective:To identify the
molecule which functions in the healing phase of EAM and
investigate its pathophysiological roles on cardiac cells.
Methods and Results: To induce EAM, BALB/c mice were
immunized twice with α-myosin heavy chain peptide. The
severity of inflammation peaked at 21days after immunization
and mitigated at 28days. To analyze the alterations of gene
expression profile during healing phase, we performed
microarray analyses using mRNA from hearts of 21day and
28day after immunization. Among up-regulated genes, we
focused on cathelicidin antimicrobial peptide (CAMP) which has
been reported to be involved in anti-inflammatory response and
epithelial tissue repair. Histochemical staining using anti-CAMP
antibody revealed that CAMP expresses in inflammatory cells
in EAM hearts. Treatment with LL37, a human homologue of
CAMP, mediated activation of ERK, JNK and p38 in fibroblasts
from neonatal rat hearts, whereas it failed to activate those MAP
kinases in rat neonatal cardiomyocytes, suggesting CAMP is
engaged in healing process by regulating function of fibroblasts.
In addition, LL-37 treatment mediated morphological changes
of fibroblasts and decreased migration distances assessed by
wound healing assay as well (283±43 mm in treated cells vs
183±54 mm in cells treated with 10 mg/ml of LL37, p<0.01).
The expression of focal adhesion kinase is decreased in LL37
treated cardiac fibroblasts compared with non-treated cells.
Moreover, blockade of P2X7 receptor which is known as a
receptor for LL37 restored the migration distances after LL37
treatment, suggesting that LL37 suppresses cardiac fibroblast
motility via P2X7 receptor signaling. Conclusion: Our results
indicate that CAMP is up-regulated after myocarditis and
inhibits fibroblasts migration via P2X7 receptor signaling. Those
results suggest that CAMP could be a useful tool to inhibit
fibrotic changes in inflammatory heart disease.
ABSTRACTS
Rationale: Our previous studies showed that increased
endoplasmic reticulum (ER) stress in the myocardium impairs
cytoprotective signaling in obese type 2 diabetes mellitus
(T2DM). Objective: We examined roles of ER stress in
alterations of ventricular function and mortality after myocardial
infarction by T2DM. Methods and Results: SERCA2a protein
level was significantly lower by 45% and Ser16- and Thr17phosphorylated phospholamban levels tended to be lower
in a rat model of T2DM (OLETF) than those in non-diabetic
control rats (LETO). Treatment with an ER stress modulator,
4-phenylbutyric acid (4-PBA) or tauroursodeoxycholic acid
(TUDCA), restored SERCA2a protein in OLETF to the level of
that in LETO. Although left ventricular (LV) ejection fractions
were comparable in OLETF and LETO, LV end-systolic
elastance (1685±86 vs. 1953±68 mmHg/ml) and LVdP/
dtmax (5886±337 vs. 7389±457 mmHg/s) were lower
and tau was larger (12.1±0.9 vs. 8.9±0.8 msec) in OLETF.
These impairments of LV function in OLETF were restored by
treatment with 4-PBA (LV end-systolic elastance: 1813±58
mmHg/ml, LVdP/dtmax: 7053±546 mmHg/s and tau: 8.5±0.5
msec). Elevation of LV systolic pressure and dP/dtmax levels
by dobutamine infusion was reduced, and inhibitory effects of
verapamil on the dobutamine-induced positive inotropism were
blunted in OLETF. In patch-clamp experiments using isolated
cardiomyocytes, increase in L-type Ca2+ channel current after
isoproterenol treatment was markedly attenuated in OLETF as
compared with the change in LETO. Mortality rate within 48
hrs after myocardial infarction was significantly higher in OLETF
than in LETO (64.0% vs. 7.7%). Telemetric recording indicated
that rapid progression of heart failure but not arrhythmia was
responsible for the high mortality rate in OLETF. In contrast to
the improvement of baseline ventricular dysfunction, neither
4-PBA nor TUDCA significantly reduced mortality rate after
infarction in OLETF. Conclusions: ER stress plays a major
role in reduction of SERCA2a by T2DM. Blunted response of
cardiac L-type Ca2+ channels to adrenoceptor activation, rather
than reduced SERCA2a, may contribute to increase in lethal
heart failure after myocardial infarction in T2DM.
H. Nakayama: None. K. Matsui: None. T. Yamashita: None.
H. Kawaguchi: None. Y. Fujio: None.
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63
Poster Presentations (continued)
P146Fas-ligand Induces Cardiomyopathy via the Activation
of the Erk1/2 Pathway
Anne-Cecile Huby, Enkhsaikhan Purevjav, Jeffrey A Towbin,
The Heart Inst, Cincinnati Children’s Hosp Medical Ctr, Cincinnati, OH
Fas receptor and its ligand, Fas-L, are the most well
characterized co-stimulatory molecules playing an essential
role in the induction of programmed cell death or apoptosis.
Increase of circulating Fas-L in serum and in cardiomyocytes
are described in different models of cardiac diseases such as
myocarditis, pressure overload, AND myocardial infarction/
ischemia. In addition, associated activation of inflammatory
pathwayshas been observed. However, no studies have yet
revealed mechanisms underlying the pathogenecity of FasL
in the heart. Transgenic mice (C57/Bl6) with cardiac-specific
over-expression of Fas-L were investigated. We characterized
cardiac function by echocardiography. Cellular and molecular
studies were performed on heart tissue using genomic,
histologic, immunological, and protein analysis techniques.
Fas-L transgenic mice had a significant rate of mortality
(p<0.0001) compared to WT due to heart failure. Histological
studies of Fas-L hearts revealed predominant perivascular
fibrosis associated with inflammation. Expression studies of
secondary messengers of the activated Fas/FasL system
demonstrated up-regulation of an active CASP-3 confirming
induction of apoptosis in myocardium. Down-regulation of
FLIP, NIK and NF-κB was evident. Interestingly, RIP was
up-regulated leading to the activation of ERK1/2 (4.27±0.158
vs 10.086±1.591 in WT and FasL mice hearts, respectively,
p=0.022). Analysis of inflammatory mediators revealed increase
in secretion of interleukines (IL-17, IL-6, IL-1β and TNF-α)
and diffuse infiltration of CD3 positive cells. Studies of fibrotic
pathways showed an increased expression of periostin and
osteopontin, but not activation of TGFβ1, showing that periostin
and osteopontin can be activated independently of TGFβ1 via
the ERK1/2 pathway, Our findings suggest that Fas-L induces
cardiac failure, via an activation of the ERK1/2 pathway, which
is an important player involved in the hypertrophic response in
the heart. Targeting the ERK1/2 pathway in cardiac diseases
associated with Fas-L expression may therefore be able to
prevent progression to cardiomyopathy.
A. Huby: None. E. Purevjav: None. J.A. Towbin: None.
P147Antiangiogenic Effect of Doxorubicin on Human Cardiac
Microvascular Endothelial Cells Does Not Result from
Reduced Vascular Endothelial Growth Factor Production
ABSTRACTS
Eugene A Konorev, Zuyue Sun, Univ of Hawaii Hilo Coll of Pharmacy,
Hilo, HI
64
Clinical use of the potent anticancer agent doxorubicin (DOX)
is limited by a dose-dependent cardiomyopathy. Although
cardiac muscle has been thought to be a primary target of
the cardiotoxic effect of doxorubicin, our studies indicate that
adult cardiomyocytes may be resistant to the direct effects
of this agent. These studies have prompted us to identify
alternative targets for doxorubicin in adult heart that may
contribute to the development of cardiomyopathy. Additionally,
recent literature report (Lee et al. PNAS 2009;106:2353–2358)
indicated that DOX inhibited tumor angiogenesis due to the
reduced production of vascular endothelial growth factor
(VEGF) by tumor cells. We set out therefore to test the
hypothesis that DOX inhibits angiogenesis by human cardiac
microvascular endothelial cells (HCMVEC) via inhibition of
VEGF production. We detected reduced proliferation/viability
of HCMVEC cultures treated with DOX. DOX increased
caspase-3-like activity and development of apoptosis in
HCMVEC, and inhibited the formation of vascular tubule
formation in a co-culture of HCMVEC with human cardiac
fibroblasts (HCF) with an IC50 of 8 nM. HCF cultures released
7-fold greater amounts of VEGF into the culture media as
compared with HCMVEC under the normoxic conditions
while 1% hypoxia and 100 μM CoCl2 further increased the
production of VEGF by HCF. DOX moderately inhibited
hypoxia- and CoCl2-induced VEGF release by these cells at
concentrations above 500 nM whereas inhibition of vascular
tubule formation by DOX in a co-culture system occurred
at much lower concentrations and was not associated with
the reduced production of VEGF. We conclude that DOX
is a very potent antiangiogenic agent in the human cardiac
microvascular endothelial cell system. However, this effect
of DOX is not caused by the reduced production of VEGF.
Inhibition of vascular network formation in the heart is likely to
contribute to the development of doxorubicin cardiomyopathy
in treated patients.
E.A. Konorev: None. Z. Sun: None.
P148Urotensin II Impairs Cardiac Functions and Proliferation
of Cardiac Side Population Cells in Mice During Chronic
Pressure Overload.
Yunzeng Zou, Hui Gong, Hong Ma, Guoping Zhang, Zhidan Chen,
Guoliang Jiang, Chunjie Yang, Yuan Yan, Jie Yuan, Lei Li, Jian Wu,
Junbo Ge, Fudan Univ, Shanghai, China
Urotensin II (UII), a potent vasoactive “somatostatin-like”
peptide, has been known to play a role in cardiovascular
diseases including cardiac hypertrophy and heart failure.
But its effect on cardiac side population cells (CSPs), one
of somatic stem cells in the heart potentially participating in
cardiac protection after injury, is unclear. The present study
was therefore conducted to examine influences of UII on the
differentiation, proliferation and function of CSPs. CSPs were
isolated from neonatal rat hearts by fluorescence-activated
cell sorting (FACS) and cultured in the absence or at the
presence of UII. The expressions of alpha-cardiac myosin
heavy chain, alpha-smooth muscle actin and Von Willebrand
factor at mRNAs and protein levels were analyzed by reverse
transcriptional PCR (RT-PCR) and immunofluoresence to
evaluate the differentiation of CSPs into cardiomyocytes,
smooth muscle cells and endothelial cells, respectively. The
proliferation of CSPs was assessed by Luminescent Cell
Viability Assay. The influence of UII on the proliferation of CSPs
in vivo was also evaluated by FACS. Our results revealed that
UII did inhibit the proliferation of CSPs through up-regulation
of phosphorylated c-Jun N-terminal protein kinase (JNK),
although it didn’t affect the differentiation of cultured CSPs
into cardiaomyocytes, smooth muscle cells and endothelial
cells. In vivo experiments also showed that injection of UII
reduced the number of CSPs and impaired cardiac functions
compared with vehicle injection in mice subjected to a chronic
pressure overload, and an UII antagonist urantide induced a
preserved cardiac function with an increased number of CSPs.
These data indicate that UII reduces the number of CSPs by
inhibiting the proliferation of CSPs possibly through increase
of JNK phosphorylation, and blockage of UII may be a useful
strategy for cardiac protection in the hypertensive.
Y. Zou: None. H. Gong: None. H. Ma: None. G. Zhang: None.
Z. Chen: None. G. Jiang: None. C. Yang: None. Y. Yan: None.
J. Yuan: None. L. Li: None. J. Wu: None. J. Ge: None.
P149Mozobil Increases Myocardial Fibrosis via the Recruitment
of Fibrocytes in the Angiotensin II-Treated Heart
Alec G Falkenham, Tim Lee, Jean Francois Legare, Dalhousie Univ,
Halifax, NS, Canada
Background: Fibroblast progenitor cells (fibrocytes) are
important to the development of myocardial fibrosis and
suggested to migrate to the chemokine CXCL12. CXCL12
has one primary receptor, CXCR4, to which the inhibitor
Mozobil competitively binds. Mozobil has been shown to inhibit
recruitment of fibrocytes to the lungs following bleomycin injury,
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Poster Presentations (continued)
but its affects on progenitor cells in the heart remains unclear.
We hypothesized that if CXCL12 is necessary for fibrocyte
recruitment, Mozobil would inhibit fibrocyte infiltration into the
myocardium and consequently, decrease fibrosis. Methods:
C57/Bl6 mice were treated with saline (control), AngII, or
AngII+Mozobil via osmotic mini-pump. Mice were sacrificed
at day 3 and tissues were collected for RNA, protein and
histology. Heart sections were stained with H&E and Sirius
Red and analyzed for cellular infiltrate and collagen deposition,
respectively. Infiltrate was measured using standardized grid
counts on a single heart section. The percent area affected
was the number of grids containing cellular infiltrate over the
total grids containing heart tissue (~375 grids per section).
Fibrocytes were cultured from mouse peripheral blood
following mobilization with Mozobil. Fibrocytes were stained for
collagen-1, CD133 and CXCR4. Results: Cultured fibrocytes
were positive for markers CD133 and collagen-1, as well as
CXCR4. AngII infusion resulted in significant cellular infiltration,
affecting 30.0 ± 4.0% (n=8) of the myocardium, relative
to saline (0.3% ± 0.4; n=8). In contrast, animals receiving
AngII+Mozobil had 78.9±3.1% (n=5) of the myocardium
affected (p<0.05). Increased infiltrate was also associated with
significantly increased collagen deposition: Control (4.3% ±
0.1), AngII (11.9% ± 1.9), and AngII+Mozobil (29.0% ± 4.8).
Furthermore, the infiltrate was positive for the progenitor
marker CD133. Conclusion: Our findings confirm isolated
fibrocytes express CXCR4. Unexpectedly however, our findings
suggest CXCL12 is not required for the migration of fibrocytes
to the myocardium, based on the failure of CXCR4 blockade to
limit migration. Our findings suggest that increased migration
into the heart may be through the increased mobilization of
progenitor cells from the bone marrow.
A.G. Falkenham: None. T. Lee: None. J. Legare: None.
P150Estrogen Protects Against and Reverses Adverse
Ventricular Remodeling in Pulmonary HypertensionInduced Right Ventricular Failure
Rangarajan D Nadadur, Soban Umar, Andrea Iorga, Humann Matori,
Rod Partow-Navid, Mansoureh Eghbali, UCLA, Los Angeles, CA
R.D. Nadadur: None. S. Umar: None. A. Iorga: None. H. Matori:
None. R. Partow-Navid: None. M. Eghbali: None.
P151Differential Effects of Concurrent and Sequential
Combined Lapatinib and Doxorubicin Treatment on
Cardiac Function in Mice
David Goukassian, Sharath Sasi, Juyong Lee, Steward St. Elizabeth’s
Medical Ctr and Tufts Univ Sch of Med, Boston, MA; James R Stone,
Massachusetts General Hosp and Harvard Medical Sch, Boston, MA;
Daniela Budiu, Christopher Lawson, Michael Maysky, Lynn Hlatky,
Joseph Carrozza, James P Morgan, Giannoula Klement, Xinhua Yan,
Steward St. Elizabeth’s Medical Ctr and Tufts Univ Sch of Med, Boston, MA
Background: Clinical studies showed that Trastuzumab,
a monoclonal antibody that blocks the HER2 receptor,
exacerbated chemotherapy drug doxorubicin (DOX)-induced
heart failure. Lapatinib, a small molecule inhibitor of HER2 and
EGF receptor (EGFR), is currently studied in clinical trials for
cancer therapy, in combination with DOX, concurrently and
sequentially. Therefore, it is important to assess the cardiac
effect of the combination treatment using lapatinib and DOX.
Methods: Two mouse models were used. In the first model,
mice were treated with a single dose of DOX (20 mg/kg, i.p.),
concurrently with lapatinib (100 mg/kg, oral gavage, daily). In
the second model, mice were treated with DOX (2 mg/kg, i.p.
twice a week) for seven weeks, followed by lapatinib (100 mg/
kg, oral gavage, daily) for three weeks. Survival was analyzed
by the Kaplan-Meier method. Cardiac function was monitored
by hemodynamic measurements. Results: When mice were
treated concurrently with lapatinib and DOX, ten-day survival
was significantly lower in DOX+lapatinib vs. DOX mice (13
vs. 0 %, n=7–8/group, P<0.001). Five to seven days after
the treatment, cardiac contractile function was significantly
lower in DOX+lapatinib vs. control mice [left ventricular
systolic pressure (LVSP): 83 ± 2 vs. 93 ± 2 mmHg, P<0.05;
dP/dtmin: 4020 ± 360 vs. 7112 ± 580 mmHg/sec, P<0.05;
cardiac output (CO): 1236 ± 266 vs. 2734 ± 209 unit/min,
P<0.05, n=5/group]; however, these indices were maintained
in DOX mice (LVSP: 99 ±4 mmHg; dP/dtmin: 7787 ± 965
mmHg/sec; CO: 2315 ± 366 unit/min, P=NS vs. control,
n=5–6/ group). On the other hand, when mice were treated
sequentially with DOX and lapatinib, seventy-day survival was
higher in DOX+lapatinib vs. DOX mice (63 vs. 43 %, n=7–8/
group). Cardiac output was decreased in DOX vs. control mice
(1733±135 vs. 3438±268 unit/min, P<0.05, n=4–5/group).
However, cardiac output was less depressed in DOX+lapatinib
mice (2817±559 unit/min, P=NS vs. control, n=4/group).
Conclusions: These results showed that concurrent and
sequential combined lapatinib and DOX treatment produced
opposite effects on survival and cardiac function in mice. This
study suggests that the cardiac safety of lapatinib and DOX
combination therapy depends on the specific treatment regimen.
ABSTRACTS
Pulmonary hypertension (PH) leads to right-ventricular failure
(RVF). RVF is characterized by adverse RV remodeling
including hypertrophy and changes in the cardiac Extracellular
Matrix (ECM) such as fibrosis and re-expression of cardiac
fetal genes. Among the potentially re-expressed genes are
the novel ECM interacting proteins Osteopontin (OPN) and
Osteocalcin (OCN). Gender differences found in experimental
PH are attributed to protective effects of Estrogen (E2). We
hypothesize that gender differences observed in experimental
PH are partially due to the effects of E2 on the cardiac ECM,
and that exogenous E2 may be able to reverse adverse
RV remodeling. Male and female rats received single
monocrotaline (MCT, 60mg/kg) injection. Some rats were
given E2 (42.5μg/kg/day) from day 21–30. Saline treated
rats were controls. Cardiopulmonary hemodynamics were
serially monitored and RV pressures (RVP) were recorded
terminally. RV fibrosis was assessed by trichrome staining.
Gene expression was determined by RT PCR and plasma OPN
by ELISA. All rats developed PH by day 21 and RVF by day
30. Male rats developed more severe PH-induced RVF than
females (RVP=70 vs. 41.5±5 mmHg; RV/(LV+IVS)= 0.69±0.07
vs. 0.47±0.04; RVEF = 30.4±1.8 vs. 42.8±2% resp., all
p<0.05). Males also revealed more severe RV fibrosis and
greater re-expression of OPN (4.5 fold vs. females, p<0.05)
and OCN (2-fold vs. females, p<0.05). Plasma OPN was also
elevated in RVF males (1.00±0.11 in control to 1.47±0.18
pg/ml, p<0.05) but not RVF females (0.848±0.18 in control
to 0.859±0.294 pg/ml, p=ns). Since females experienced
less severe RV remodeling than males, MCT injected males
were treated with exogenous E2 from day 21–30. Some E2
treated male rats were sacrificed at day 30, and some were
kept an additional 12 days after E2-withdrawal (E2-W group).
E2 reversed PH-induced RVF (RVP=38mmHg; RV/(LV+IVS)
= 0.28±0.03; RVEF=61.5±0.8%, all p<0.05 vs. male RVF)
and fibrosis. OPN and OCN were fully restored following E2
withdrawal by day 42. E2 therapy also restored circulating OPN
levels (p<0.05 vs. RVF) showing that OPN has potential value
as a plasma marker for PH-induced RV failure. These results
suggest that E2 protects against adverse RV remodeling in
females, and reverses adverse RV remodeling in males.
. Goukassian: None. S. Sasi: None. J. Lee: None. J.R. Stone: None.
D
D. Budiu: None. C. Lawson: None. M. Maysky: None. L. Hlatky: None.
J. Carrozza: None. J.P. Morgan: None. G. Klement: None. X. Yan: None.
This research has received full or partial funding support from the
American Heart Association, Founders Affiliate (Connecticut,
Maine, Massachusetts, New Hampshire, New Jersey, New York,
Rhode Island, Vermont).
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
65
Poster Presentations (continued)
P152Cardiac mTOR Preserves Cardiac Function and Inhibits
the Inflammatory Response After Transient Ischemia
Toshinori Aoyagi, Brendan T Inouye, Takashi Matsui, Ctr for
Cardiovascular Res, John A Burns Sch of Med, Univ of Hawaii,
Honolulu, HI
Recent reports strongly suggested that the mammalian target
of rapamycin (mTOR) is necessary for cardiomyocyte survival
in pathological cardiac hypertrophy. Using cardiac-specific
transgenic mice overexpressing mTOR (mTOR-Tg), we reported
that cardiac mTOR is sufficient to protect the heart against
heart failure in pathological hypertrophy, accompanied by a
decrease in production of proinflammatory cytokines. However,
the role of cardiac mTOR in ischemic cardiac injury remains
undefined. To assess the effects of cardiac mTOR in cardiac
ischemia, we studied the response of hearts from mTOR-Tg
mice to global ischemia-reperfusion injury (IRI) (20 min ischemia,
40 min reperfusion) in the ex vivo Langendorff perfusion model.
mTOR-Tg mice demonstrated improved functional recovery
compared with littermate control (WT) mice [percent recovery
of LV developed pressure (LVDP); 44.6±7.3 vs. 24.7±5.3 %,
mTOR-Tg vs. WT, n=9 each, p<0.05]. Biochemical analyses
demonstrated that the level of mTOR protein decreased
under IRI stress in WT while the level of mTOR protein in
post-IRI mTOR-Tg mice was comparable to non-ischemic
control mice. Both mTORC1 (p70S6K) and mTORC2 (Akt)
activation in mTOR-Tg mice were increased by IRI, while these
were not changed in WT mice after IRI. Quantitative RT-PCR
demonstrated that expression of proinflammatory cytokines in
mTOR-Tg mice after IRI was significantly lower than WT mice
(IL-6, 7.1±1.5 vs. 14.6±1.6, p<0.005; TNF-alpha, 2.1±0.4 vs.
5.1±0.8, p<0.01; post-IRI mTOR-Tg mice vs. WT mice, fold
change over non-ischemic control WT mice, n=9 each). To
further characterize the inflammatory response, we measured
cytokine in the effluent samples by ELISA. We observed that
secretion of IL-6 in post-IRI mTOR-Tg mice was significantly
lower than that in WT mice (50.1±11.5 vs. 95.6±23.8 pg/ml,
post-IRI mTOR mice vs. post-IRI WT mice, n=9 each, p<0.05).
IL-6 is known as a strong prognostic predictor in patients with
heart failure. Taken together, these findings suggest that cardiac
mTOR is sufficient to substantially preserve cardiac function and
prevent the inflammatory response, which may have relevance
for settings in heart failure.
T. Aoyagi: None. B.T. Inouye: None. T. Matsui: None.
ABSTRACTS
P153Role of Nonreceptor Tyrosine Kinases in Cardiac Fibrosis
in a Mouse Model of Pressure Overload Hypertrophy
66
Sundaravadivel Balasubramanian, Harinath Kasiganesan, Lakeya
Quinones, Yuhua Zhang, Amy Bradshaw, Dhandapani Kuppuswamy,
MUSC, Charleston, SC
During prolonged hypertrophic insult to the myocardium, while
the function of cardiomyocytes needs to be protected, the
hyperactivation of cardiac fibroblasts has to be curbed to prevent
fibrosis. Previously, we showed that integrin-mediated nonreceptor tyrosine kinase (NRTK) activation is required for normal
functioning of both cardiac fibroblasts and cardiomyocytes.
We hypothesized that inhibition of NRTKs in cardiac fibroblasts
without affecting cardiomyocytes would be beneficial to the
stressed myocardium. Our initial studies using kinase inactive
forms of Src, Pyk2 and FAK expressed adenovirally in isolated
primary cardiac fibroblasts showed that the pro-fibrotic signaling
events as studied by fibronectin and collagen deposition are
downregulated. Our in vivo studies in mouse transverse aortic
constriction (TAC) model suggest that dasatinib, a multikinase
NRTK inhibitor administration via a peritoneally implanted miniosmotic pump is able to preserve ventricular geometry and
function and reduce the accumulation of fibrotic extracellular
matrix (ECM) proteins upon 4 wk pressure overload. Data
obtained from cell culture experiments with kinase inactive
NRTKs and dasatinib suggest that NRTK inhibition is able to
reduce the proliferation, migration and mitogenic signaling in
cardiac fibroblasts without affecting the cell survival pathways
in cardiomyocytes. These data indicate that NRTKs play a
significant pro-fibrotic role in cardiac fibroblasts and curbing the
activity of NRTKs could be a potential therapeutic approach to
treat fibrosis in hypertrophic heart diseases.
S. Balasubramanian: None. H. Kasiganesan: None. L. Quinones:
None. Y. Zhang: None. A. Bradshaw: None. D. Kuppuswamy: None.
P154Focal Adhesion Kinase Plays a Critical Role in Upregulating
Fibrogenesis in Load-Induced Cardiac Hypertrophy
Ana Paula Dalla Costa, State Univ of Campinas, Campinas, Brazil;
Carolina F Clemente, Brazilian Natl Lab for Biosciences, Campinas,
Brazil; Thais H Theizen, José Roberto Souza, Leandro Cardoso, State
Univ of Campinas, Campinas, Brazil; Kleber G Franchini, Brazilian Natl
Lab for Biosciences, Campinas, Brazil
Myocardial fibrosis is maladaptive, accelerating the evolution
of diseased hearts to failure. The pathogenesis of myocardial
fibrosis is critically dependent on complex processes of
activation (i.e. enhanced proliferation, production and secretion
of soluble factors, collagen and matrix metalloproteinases)
and terminal differentiation of cardiac fibroblasts into
myofibroblasts, resultant from the mobilization of numerous
signaling molecules by physical and humoral stimuli. Noting that
Focal Adhesion Kinase (FAK) is activated in areas of ongoing
myocardial fibrosis, we sought to examine whether it is a
critical mediator of fibrogenesis in load-induced hypertrophic
hearts. Isolated fibroblasts from hypertrophic hearts of mice
subjected to transverse aortic constriction (TAC; 1 to 8 weeks)
were highly activated as recognized by markers that indicate
enhanced proliferation (nuclear Ki67), production of collagen
and matrix metalloproteinase-2 (MMP-2) and differentiation
into myofibroblasts (expression of α-smooth muscle actin —
α-SMA). In these cells, FAK was upregulated, as also were
its dowstream pathways Src/ERK1/2 and PI3K/AKT/mTOR.
Depletion of FAK (~80%) after treatment with small interfering
RNA (siRNA-FAK) markedly attenuated cardiac hypertrophy
and fibrosis, and significantly reduced the number of activated
fibroblasts harvested from overloaded hearts. Restoration of
FAK function by overexpressing a full-length FAK construct in
these cells, selectively enhanced the activity of the downstream
PI3K/AKT/mTOR and rescued the activated phenotype of
fibroblasts. Transfection with an inactive FAK mutant (Tyr397
substituted by phenylalanine) did not rescue the activated
phenotype of fibroblasts harvested from overloaded hearts
depleted of FAK. However, cells harvested from overloaded
hearts depleted of FAK and treated with the mTOR activating
aminoacid leucine showed typical phenotype of activated
fibroblasts. These findings uncover a role for FAK in regulating
the signaling cascade PI3K/AKT/mTOR in cardiac fibroblasts,
which seems to be critical for the pathogenesis of myocardial
fibrosis in hypertrophic hearts. Targeting this pathway may
provide a novel strategy for treating hypertrophic heart diseases.
A.C. Dalla Costa: None. C.F.M. Clemente: None. T.H. Theizen: None.
J.M. Souza: None. L. Cardoso: None. K.G. Franchini: None.
P155HSF-1 Deletion Induces MDR1 Gene in the Heart and
Protects from Doxorubicin-Induced Cardiotoxicity
Karthikeyan Krishnamurthy, Lawrence Druhan, Govindasamy
Ilangovan, The Ohio State Univ, Columbus, OH
Krishnamurthy Karthikeyan1; Lawrence Druhan2 and
Govindasamy Ilangovan1 Davis Heart & Lung Research
Institute, Department of Internal Medicine1 Department of
Anesthesiology2, The Ohio State University, Columbus, OH
Heat shock factor 1 (HSF-1) has been found to be a frontline
responder of different stresses in eukaryotic cells. Deletion
of HSF-1 has been reported to develop defects in mice. In
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Poster Presentations (continued)
the present work we report that deletion of HSF-1 induced
multidrug resistance 1 (MDR1) gene and P-glycoprotein (P-gp)
expression. Both RT-PCR and western blotting, confirmed the
higher level of MDR1 gene expression and P-gp protein in HSF1 knock out mouse hearts. P-gp is well known ATP binding
cassette, which extrudes intracellular drugs upon binding ATP.
Hence the phenotype of the P-gp pump in HSF-1-/- mice was
studied in the form of Doxorubicin (Dox) extrusion in the heart.
Cardiomyocytes isolated from HSF-1-/- and HSF-1+/- mice
retained very less intracellular Dox, compared to wild type
counterparts. Similarly, both HSF-1+/- and HSF-1-/- mice were
less susceptible for Dox-induced cardiotoxicity, as seen from
reduced cardiac dysfunction in these group against Dox (as
confirmed by cardiac MRI and echocardiography). Further
confirmatory studies revealed that deletion of HSF-1 enhances
NF-B, which subsequently induces MDR1 gene. These results
reveal that inactivation of HSF-1 in the heart will be a potential
approach to prevent Dox-induced cardiotoxicity.
failure. To explore the role of miR-22 in the mouse heart
physiology, we generated miR-22 null (KO) mice. Although,
miR-22 KO mice showed normal cardiac structure and
function at baseline, these mice are sensitized to maladaptive
remodeling (cardiac dilation) and decompensation in response
to pressure overload by transverse aortic constrictions (TAC)
stimulation. Genome-wide molecular analysis of KO hearts
revealed attenuated expression of numerous CarG-dependent
genes encoding proteins that reside at the sarcomeric Z-disc
(including Myh7, Acta1, Mlp, Melusin, MyoZ2) indicating that
miR-22 is required for optimum muscle gene expression.
Alterations in sarcomeric gene expression is especially
interesting as this suggests a primary role of miR-22 in
controlling cardiac contractility and adaptation to stress.
Targetomics analysis revealed that mechanistically this effect
could be modulated in part by miR-22 target PURB (Purine
Rich element binding protein B), a transcriptional/translational
repressor. In conclusion we define a critical role of miR-22
in cardiac adaptation to hemodynamic stress. Furthermore,
these data provides a previously unseen essential molecular
mechanism that underlies homeostatic control of sarcomeric
protein expression in the heart.
K. Krishnamurthy: None. L. Druhan: None. G. Ilangovan: None.
This research has received full or partial funding support from the
American Heart Association, Great Rivers Affiliate (Delaware, Kentucky,
Ohio, Pennsylvania & West Virginia).
P156IRE1α Activity Is Abbreviated in Myocytes
DeAnna L Steiger, Yibin Wang, David Geffen Sch of Med,
Los Angeles, CA
D.L. Steiger: None. Y. Wang: None.
P157Not published at presenter’s request.
P158MicroRNA-22 Modulates Cardiac Gene Expression and
Controls Compensation to Hemodynamic Stress in Mice
Priyatansh Gurha, Baylor Coll of Med, Houston, TX; Robert Kelm Jr,
Univ of Vermont, Coll of Med, Houston, TX; Mark Entman, George
Tafet, Section of Cardiovascular Res, Baylor Coll of Med and The
Methodist DeBakey Heart and Vascular Ctr, Houston, TX; Alan Bradley,
Wellcome Trust Sanger Inst, Cambridge, United Kingdom; Antony
Rodriguez, Baylor Coll of Med, Houston, TX
This research has received full or partial funding support from the
American Heart Association, National Center.
P159Th17 Cells Are Indispensable for Cardiac Inflammation in
Autoimmune Myocarditis
Tomomi Yamashita, Hiroyuki Nakayama, Yasushi Fujio, Osaka Univ,
Suita, Japan
Background: Chronic inflammation is closely associated with
heart failure; however, the molecular and cellular mechanisms
of inflammatory reactions remain to be fully elucidated.
Interleukin(IL)-17 is a pro-inflammatory cytokine that is
produced by Th17 cells. Th17 cells are induced by IL-6 and
transforming growth factor(TGF-β), through up-regulation of
its specific transcriptional factor, retinoic acid receptor-related
orphan nuclear receptor(ROR)γt. Th17 cells are reported to
participate in pathogenesis of chronic inflammatory disease.
In this study, we examined the functional roles of Th17
signaling in cardiac inflammation using murine experimental
autoimmune myocarditis(EAM) model. Methods and Results:
EAM was induced in wild-type(WT) and/or RORγt-eGFP
knock-in Balb/c mice by immunization twice with α-myosin
heavy chain peptide. Three weeks after the first immunization,
the mRNA expression of RORγt was up-regulated and
correlated with the disease severity in heart. Additionally, in
immunostaining and flow cytometry analysis, we observed
a significant number of Th17 cells among inflammatory cells
infiltrating myocardium from WT and RORγt-eGFP knock-in
heterozygous mice. These data showed that Th17 cells were
remarkably recruited in EAM heart. Moreover, depression of
Th17 differentiation suppressed disease severity completely
with inhibition of RORγt and IL-17 transcriptional activity,
whereas IL-17 neutralizing antibody to lesser extent. Finally,
RORγt-eGFP knock-in homozygous mice were protected
from cardiac inflammation. Conclusion: Our results indicated
that Th17 activation through RORγt pathway is a critical
mechanism for the development of myocarditis. However,
IL-17 is not crucial for induction of EAM, suggesting that other
Th17 producing pro-inflammatory cytokines are involved. It
could be proposed that Th17 cells are novel therapeutic target
against chronic inflammation in heart.
ABSTRACTS
ackground: A unifying feature of Ischemia/reperfusion
B
injury, myocardial infarction and heart failure is disruption of
endo(sarco)plasmic reticulum homeostasis and activation
of the intracellular signaling pathway known as “ER Stress”
or “Unfolded Protein Response” (UPR). UPR signaling is
activated through three ER Stress sensor proteins, including
IRE1α. IRE1α can, through direct interaction, induce adaptive
or apoptotic signaling programs. Although ER stress signaling
is known to be active following various cardiac injuries, it
is unknown whether IRE1α contributes to an adaptive or
apoptotic program. We sought to characterize the functional
impact of IRE1-mediated UPR signaling in cardiomyocytes.
Hypothesis: IRE1α-mediated UPR signaling contributes to
cardiac hypertrophy or cell death in response to pathological
stresses. Method: NRVM were treated with adenovirusmediated IRE1α-myc overexpression. Protein and RNA
were harvested two and five days following treatment and
IRE1α activation, activity and downstream signaling was
monitored by PCR, qPCR, and immunoblot. Results: Ectopic
expression led to robust phosphor-IRE1α Ser724 and IRE1α
RNase activity toward Xbp-1. BiP and EDEM expression was
induced, indicative of adaptive UPR signaling. Apoptotic UPR
signaling was not induced (CHOP expression). Surprisingly,
IRE1α activity and downstream signaling was transient.
After five days, both IRE1α RNase activity and downstream
signaling were extinguished, despite continued robust
IRE1α protein expression and activation (phospho-Ser724).
Conclusion: NRVM possess uncharacterized intrinsic
mechanisms to protect against unwarranted IRE1α signaling.
P. Gurha: None. R. Kelm Jr: None. M. Entman: None. G. Tafet:
None. A. Bradley: None. A. Rodriguez: None.
T. Yamashita: None. H. Nakayama: None. Y. Fujio: None.
Recent evidence suggests that miRNAs play an important
role in cardiac morphogenesis and pathophyiology of heart
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
67
Poster Presentations (continued)
secreted Hi-FGF-2 can be blunted by limited proteolysis of
its N-terminal extension. Experimental: Heparin-sepharosebound Hi-FGF-2 present in rat tissue extracts converted to an
approximately 18 kDa protein (*Lo-FGF-2) through truncation
of its N-terminal extension, in the absence of protease
inhibitors. Serine protease inhibitors such as PMSF fully
prevented this conversion. Similarly, purified recombinant rat
Hi-FGF-2 was converted to *Lo-FGF-2 by thrombin, a serine
protease, and the effect was prevented by hirudin, a thrombin
specific inhibitor. Conditioned media from unstimulated
or angiotensin II-stimulated rat cardiac non-myocytes
(fibroblasts) were used to treat neonatal cardiomyocytes
in culture, in the absence or presence of thrombin.
Conditioned medium from stimulated (but not unstimulated)
cells contained Hi-FGF-2, which promoted cardiomyocyte
hypertrophy (increase in cell surface area), and the effect
was due to secreted FGF-2 because it was prevented by
neutralizing anti-FGF-2 antibodies. Thrombin itself had no
effect on cell size but prevented the pro-hypertrophic effect
of the conditioned media; while converting the secreted
Hi-FGF-2 to *Lo-FGF-2. Conclusions: The truncation of
the N-terminal extension of secreted Hi-FGF-2 by serine
protease(s) can provide a strategy to prevent undesirable
paracrine effects of Hi-FGF-2 (such as cardiac hypertrophy),
while maintaining potentially beneficial (cytoprotective,
angiogenic) effects associated with Lo-FGF-2.
P160N-Type Ca2+ Channel Blockade Prevents Sudden Death in
Mice with Heart Failure
Yuko Yamada, Koichiro Kuwahara, Hideyuki Kinoshita, Yoshihiro
Kuwabara, Yasuaki Nakagawa, Junko Shibata, Takeya Minami, Shinji
Yasuno, Satoshi Usami, Toshio Nishikimi, Kenji Ueshima, Yasuo Mori,
Kazuwa Nakao, Kyoto Univ, Kyoto, Japan
ABSTRACTS
Background: Increased sympathetic nerve activity is involved
in the increased arrhythmogenicity seen in patients with
chronic heart failure. N-type Ca2+ channel (NCC) modulates
sympathetic nerve activity by regulating calcium entry at
sympathetic nerve terminal, which triggers the release of the
neurotransmitter noradrenalin. The ability of NCC blockade
to prevent lethal arrhythmias associated with heart failure
has never been tested, however. Methods and Results: We
compared effects of cilnidipine (Cil), dual N-and L-type Ca2+
channel blocker, with those of nitrendipine (Nit), a selective
L-type Ca2+ channel blocker, in a cardiac-specific, dominantnegative form of neuron-restrictive silencer factor transgenic
mice (dnNRSF-Tg), a mouse model of dilated cardiomyopathy
leading to sudden arrhythmic death. Hemodynamic and
echocardiographic analyses showed no significant difference
among the control, Cil, and Nit groups. In contrast, Cil
dramatically improved the survival rate (p<0.001) and reduced
ventricular arrhythmia (p<0.05) among dnNRSF-Tg mice,
compared with control vehicle or Nit (n=22 for WT, n=20 for Tg
without drugs, n=22 for Tg with Cil, n=23 for Tg with Nit). Cil
improved the imbalance in autonomic nerve activity assessed
by heart rate valiability, and also reduced the increased urinary
excretion of noradrenalin seen in dnNRSF-Tg, whereas Nit
did not. Conclusions: NCC blockade ameliorated disturbed
autonomic nerve function and improved survival in a mouse
model of dilated cardiomyopathy and sudden death. Our
findings suggest NCC blockade is a potentially useful
approach to preventing sudden cardiac death in patients with
heart failure.
Y. Yamada: None. K. Kuwahara: None. H. Kinoshita: None.
Y. Kuwabara: None. Y. Nakagawa: None. J. Shibata: None.
T. Minami: None. S. Yasuno: None. S. Usami: None. T. Nishikimi:
None. K. Ueshima: None. Y. Mori: None. K. Nakao: None.
P161Conversion of Secreted High-Molecular-Weight FGF-2
to an 18 kDa Low-Molecular-Weight FGF-2 by Limited
Proteolysis Suppresses the Paracrine Induction of
Cardiomyocyte Hypertrophy
Jon-Jon R Santiago, Brian P Bestvater, Robert R Fandrich, Elissavet
Kardami, Inst of Cardiovascular Sciences, Winnipeg, MB, Canada
Background: High molecular weight, 23 kDa rat FGF-2 (HiFGF-2) is composed of an N-terminal extension as well as the
18 kDa core sequence present in low molecular weight (Lo-)
FGF-2. Hi- and Lo-FGF-2 have distinct biological activities:
unlike Lo-FGF-2, Hi-FGF-2, which is secreted by cardiac nonmyocytes, promotes cardiomyocytes hypertrophy in vivo and
in vitro. We tested if the paracrine pro-hypertrophic effects of
68
.R. Santiago: None. B.P. Bestvater: None. R.R. Fandrich: None.
J
E. Kardami: None.
P162Granulocyte Colony-Stimulating Factor Administration
Reduced the Inducibility of Ventricular Tachyarrhythmias
in Cardiac Hypertrophied Rats Through Phosphorylated
Connexin-43
Nobutake Shimojo, Takeshi Machino, Donzhu Xu, Taizo Kimura, Rumi
Koshitsuka, Yuri Kameda, Satoshi Sakai, Kazutaka Aonuma, Univ of
Tsukuba, Tsukuba, Japan
Introduction: Cardiac hypertrophy is an independent risk factor
for sudden cardiac death from ventricular tachyarrhythmias
(VT). Granulocyte colony-stimulating factor (G-CSF) has
recently been reported to suppress VT after myocardial
infarction by modulating the function of gap junctions between
cardiomyocytes via maintaining Connexin-43 (Cx43). We
hypothesized that the G-CSF could also regress an enhanced
vulnerability to VT in the cardiac hypertrophy without ischemic
fibrosis through regulating Cx43. Methods: Dahl salt-sensitive
rats were maintained for a 6 week-period on a high-salt diet
as left ventricular hypertrophy models (LVHs) and a low-salt
diet as controls (CONs). After 3-time subcutaneous injection
(50 μg/kg) of G-CSF and vehicle, the inducibility of VT was
evaluated by rapid ventricular burst pacing. The electrical
pulses for the induction of VT were square waves with 6 ms
width at 6 V and delivered at 25 Hz for 30 sec. White blood cell
was counted to confirm a response to the G-CSF treatment.
Expression levels of phosphorylated and total Cx43 in the rat
ventricles were analyzed by immunoblotting. Results: The LVHs
showed apparent cardiac hypertrophy without pathological
fibrotic changes. The G-CSF reduced the inducibility of VT
compared to the vehicle in the LVHs (11 % vs. 63 %, p=0.04).
Furthermore, the G-CSF eliminated the inducibility of VT in the
CONs, although a difference in the inducibility of VT between
with and without the G-CSF treatment did not reach statistical
significance in the CONs (0 % vs. 33 %, p=0.60). White blood
cell count in one microliter of blood was elevated by the G-CSF
treatment in both LVHs (15083±4397 vs. 6976±1308, p<0.01)
and CONs (19370±1174 vs. 7700±2335, p<0.01). The G-CSF
increased phosphorylated Cx43 levels compared to the vehicle
in both LVHs (1.4-fold vs. 1.2-fold, p<0.01) and CONs (1.4-fold
vs. 1.0-fold, p=0.04), whereas the G-CSF did not affect total
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Poster Presentations (continued)
Cx43 levels in all groups. Conclusion: We demonstrated that
the G-CSF administration ameliorated the electrophysiological
stability in the rat model of cardiac hypertrophy by
modulating the function of gap junctions through accelerating
phosphorylation of Cx43.
N. Shimojo: None. T. Machino: None. D. Xu: None. T. Kimura:
None. R. Koshitsuka: None. Y. Kameda: None. S. Sakai: None.
K. Aonuma: None.
P163Rescue of Preexisting Severe Pulmonary Hypertension
and Right Ventricular Failure by Intralipid
Soban Umar, Humann Matori, Reza Foroughi, Parisa Partownavid,
Mansoureh Eghbali, UCLA, Los Angeles, CA
S. Umar: None. H. Matori: None. R. Foroughi: None.
P. Partownavid: None. M. Eghbali: None.
P164N-acetylglucosamine Conjugated to Nanoparticles
Enhances Myocyte Uptake and Improves Delivery of a
Small Molecule P38 Inhibitor for Postinfarct Healing
Warren D Gray, Emory Univ and Georgia Inst of Technology, Atlanta,
GA; Pao Lin Che, Milton Brown, Emory Univ, Atlanta, GA; Xinghai Ning,
Niren Murthy, Georgia Inst of Technology, Atlanta, GA; Michael E Davis,
Emory Univ and Georgia Inst of Technology, Atlanta, GA
An estimated 985,000 new myocardial infarctions (MI)
will occur in the U.S. in 2011. While many will survive the
initial insult, the early damage will eventually lead to heart
failure for which the only definitive cure is transplantation.
Cardiomyocyte (CM) apoptosis is a large contributor to
cardiac dysfunction, and although potential therapeutic
molecules exist to inhibit apoptotic pathways, drug delivery
W.D. Gray: None. P. Che: None. M. Brown: None. X. Ning: None.
N. Murthy: None. M.E. Davis: None.
P165 Withdrawn
P166Not published at presenter’s request.
P167CXCR4 Antagonism Is Equally Effective as Sirolimus
in Reducing Neointima Formation After Arterial Injury
Without Impairing Reendothelialization
Karim Hamesch, Pallavi Subramanian, Xiaofeng Li, Anna Thiemann,
Kathrin Heyll, Inst for Molecular Cardiovascular Res, Aachen, Germany;
Klaus Dembowsky, Eric Chevalier, Polyphor Ltd, Allschwil, Switzerland;
Christian Weber, Inst for Cardiovascular Prevention, Munich, Germany;
Andreas Schober, Inst for Molecular Cardiovascular Res, Aachen, Germany
The chemokine receptor CXCR4 is crucial in neointima
formation which is a problem after stent implantation.
Drug-eluting stents reduce the restenosis risk but delayed
re-endothelialization promotes late stent thrombosis. We
compared CXCR4 antagonism with sirolimus treatment in
preventing neointima formation after arterial injury in mice.
Apolipoprotein E-/- mice after carotid wire injury were given
a CXCR4 antagonist (POL5551 (P), a Protein Epitope
Mimetic (PEM)) or sirolimus (n=6–8/group). The drugs were
administered continuously (P: 2 and 20 mg/kg/d; sirolimus:
1.25 mg/kg/d; osmotic pumps) or intermittently (P: 20 mg/kg;
IP, once daily) for 28 days. The neointimal area, smooth muscle
cell (SMC) content, macrophage content, and endothelial
coverage were quantified by planimetry from injured arteries
stained with Elastica van Giesson, or after immunostaining for
α-smooth muscle actin, Mac-2, and von Willebrand Factor,
respectively. Peripheral Sca-1+/Lin- smooth muscle progenitor
cells (SPCs) were measured by flow cytometry. Treatment
with sirolimus as well as with a continuous (C) or intermittent
(I), high (H) or low (L) dose (D) of POL5551 (P) reduced the
neointimal area compared to corresponding vehicle (Sirolimus:
69%, CHD-P: 70%, CLD-P: 63%, IHD-P: 53%). The relative
neointimal content of macrophages increased by sirolimus
application (70%). In contrast, the macrophage content
decreased by CHD-P and IHD-P application (57% and 37%,
respectively). However, CLD-P application did not alter the
macrophage content. Sirolimus, CHD-P, and IHD-P highly
diminished the neointimal SMC content (78%, 85%, and 67%,
respectively), while CLD-P treatment did not. SPC increase in
the circulation 1 day post injury was prevented by sirolimus,
CHD-P, and IHD-P, but not by CLD-P. The endothelial coverage
was not reduced by P, but significantly by sirolimus (14%).
In conclusion, CXCR4 antagonism by POL5551 is equally
effective as sirolimus in reducing neointima formation after
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
Pulmonary hypertension (PH) is characterized by pulmonary
vascular remodeling leading to right ventricular (RV) hypertrophy
and failure (RVF). Intralipid (ILP) is the first safe lipid emulsion
for human use. Several constituents of ILP, like γ-linolenic acid
and soy-phytoestrogens are protective for lungs and heart.
Previously we showed that ILP prevents the development of
PH. Here we test a more clinically relevant hypothesis if ILP can
rescue pre-existing severe PH and RV dysfunction. To induce
PH, male rats received single monocrotaline (MCT, 60mg/kg)
injection. At day 21, when severe PH had been well-established
(PH group, n=7), rats were given ILP (1mL of 20% ILP/day,
i.p., n=7) from day 21–30. Other MCT rats were left untreated
to develop RVF (RVF group, n=9) by day 30. Saline treated
rats were control (CTRL, n=5). Serial echocardiography was
done to monitor cardiopulmonary hemodynamics. Cardiac
catheterisation was performed just before sacrifice to record
RV pressure (RVP). CD31 staining for RV capillary density and
smooth muscle actin staining for pulmonary arteriolar medial
hypertrophy were performed. Rats developed severe PH and
RV dysfunction 21 days after MCT in PH group [RVP=67±1
vs. 31±1mmHg in CTRL, RV-ejection fraction (RVEF)=39%
vs. 65±1%; RV/(LV+IVS)=0.64±0.05 vs. 0.24±0.02; medial
hypertrophy=2.8 fold vs. CTRL; all p<0.05]. Interestingly, we
found that even 10-day ILP therapy not only prevented the
progression of PH to RVF but also rescued pre-existing PH and
RV dysfunction [RVP=44±1 mmHg vs. 70±2 mmHg in RVF;
RVEF=53±1 vs. 30±1%; RV/(LV+IVS)=0.39±0.02 vs. 0.66±0.08;
medial hypertrophy=3 fold less vs. RVF; all p<0.05]. ILP therapy
resulted in 100% survival compared to only 25% survival in
RVF. Next we investigated if ILP rescue was associated with
stimulation of RV neoangiogenesis. We found that RV capillary
density was decreased in PH (0.7±0.07 vs. 1±0.07 capillaries
per myocyte in CTRL; p<0.05) and worsened in RVF (0.52±0.07
capillaries per myocyte; p<0.05 vs. CTRL). ILP therapy resulted
in stimulation of neoangiogenesis (capillary density=0.98±0.01
capillaries per myocyte; p<0.05 vs. PH and RVF). ILP therapy
rescues severe pre-existing PH and retards the development of
RV failure possibly via stimulation of RV neoangiogenesis.
methods are lacking. This damage is largely regional and
thus localized delivery of therapeutics holds great potential.
However, CMs are relatively non-phagocytic, which precludes
existing delivery schemes using polymeric particles that
target phagocytic cells. Recently, the carbohydrate N-acetylglucosamine (GlcNAc) was discovered to be bound and
internalized by CMs, providing a potential mechanism for
drug delivery. Here we demonstrate efficacy of a drug delivery
system comprising a drug-loaded biodegradable polyketal
nanoparticle that is surface-decorated with GlcNAc. Inclusion
of the sugar enhanced uptake by CMs in vitro as measured
by intracellular activated fluorescence. When delivered in
vivo following ischemia-reperfusion injury, GlcNAc-decorated
particles loaded with the p38 inhibitor SB239063 reduced
infarct size and improved acute cardiac function. This was in
contrast to our published data demonstrating no acute effect
of non-sugar decorated, p38 inhibitor-loaded particles. These
data suggest a novel therapeutic option to enhance uptake of
drug-loaded nanoparticles to CMs, and perhaps to reduce the
large amount of CM cell death following myocardial injury.
69
Poster Presentations (continued)
arterial injury in mice. It might be more beneficial than sirolimus
because it does not impair re-endothelialization and leads
to a more stable lesional phenotype. These results indicate
that POL5551 might be a promising alternative for restenosis
prevention after stent implantation.
. Hamesch: None. P. Subramanian: None. X. Li: None. A. Thiemann:
K
None. K. Heyll: None. K. Dembowsky: A. Employment; Significant;
Polyphor Ltd. E. Chevalier: A. Employment; Significant; Polyphor Ltd.
C. Weber: None. A. Schober: B. Research Grant; Significant;
Polyphor Ltd.
P168Activated Protein C Inhibits the Development of
Myocardial Fibrosis
ABSTRACTS
Mryanda J Sopel, Tanya L Myers, Timothy D Lee, Robert S Liwski,
Jean-Francois Legare, Dalhousie Univ, Halifax, NS, Canada
Background: Activated Protein C (aPC) is a naturally
produced circulating anticoagulant with anti-inflammatory and
cytoprotective properties proposed to have beneficial effects
on the myocardium. Myocardial fibrosis is a pathophysiologic
process evident in many cardiovascular diseases and is
believed to directly contribute to eventual organ failure.
Using a well-described model of myocardial fibrosis after
Angiotensin II (AngII) infusion our aim was to investigate the
novel therapeutic function of aPC in the development of
fibrosis. Methods: C57Bl/6 mice were continuously infused
with AngII (2.0 μg/kg/min), AngII and aPC (0.4 μg/kg/min), or
saline for a 3d period. Hearts were harvested and processed
for analysis. Cellular infiltration and collagen deposition were
analyzed using histologic staining. Cellular apoptosis was
assessed using a TUNEL assay. Quantitative RT-PCR was
used to assess transcript levels of molecular mediators.
Results: Infusion of AngII for 3d resulted in multifocal areas
of myocardial cellular infiltration associated with significant
collagen deposition compared to saline control animals
(p≤0.01). The addition of aPC with AngII infusion inhibited this
fibrotic response. Co-administration with aPC also inhibited
the upregulation of the pro-fibrotic cytokine CTGF expression
seen in AngII infused animals (p≤0.01). Apoptosis was
also significantly inhibited when aPC was co-administered
based on a decrease of in TUNEL positive cells and an
increase expression ration of BCL/BAX suggesting an antiapoptotic effect (p≤0.01), Furthermore, aPC also inhibited
the up-regulation of the cell adhesion molecule p-selectin
seen in AngII infused animals (p≤0.01). Conclusion: The
co-administration of aPC in a model of myocardial fibrosis
was able to abrogate completely the fibrotic response. The
mechanism of action of aPC appears to be a decreased
cellular adhesion molecule expression in the myocardium
leading to decreased cellular infiltration and enhanced cellular
survival. This data suggests that aPC has the potential as a
therapeutic agent in cardiovascular diseases.
M.J. Sopel: None. T.L. Myers: None. T.D.G. Lee: None. R.S. Liwski:
None. J. Legare: None.
P169A Novel Mitochondrial Permeability Transition Pore
Inhibitor, Bendavia, Reduces Microvascular Obstruction
(No-Reflow) due to Myocardial Ischemia/Reperfusion
Injury in the Rabbit
Sharon L Hale, Wangde Dai, Robert A Kloner, Good Samaritan Hosp,
Los Angeles, CA
Background: The no-reflow phenomenon is an important
detrimental component of reperfusion injury with clinical
implications, as it is an independent predictor of outcome after
myocardial infarction. Whether the mitochondrial permeability
transition pore (MPTP) is involved in vascular integrity and
no-reflow post reperfusion is unknown. Therefore, we tested
Bendavia, a compound that is known to inhibit the MPTP,
for its effects on no-reflow. Methods: Anesthetized rabbits
70
were subjected to 30 min coronary artery occlusion and 3 hrs
reperfusion. Treated animals (n=49) received Bendavia (0.05
mg/kg/hr) after the onset of ischemia, 1, 10 or 20 min before
reperfusion. Control animals (n=15) received saline. Risk zone
was measured by Unisperse blue staining, anatomic no-reflow
area using thioflavin S and necrosis by triphenyltetrazolium
chloride. Results: The ischemic insult was similar in both
groups (31% of left ventricle (LV) in Bendavia and 30% in
control). Bendavia treatment resulted in a non-significant 11%
reduction in necrosis, expressed as a percentage of the risk
zone. However for any given risk zone size, no-reflow was
significantly reduced by Bendavia, as there was a significant
group effect on the relationship between the extent of the noreflow zone and that of the risk zone (ANCOVA, p = 0.0085).
Overall, the no-reflow zone, expressed as a % of the risk
zone was 22 ± 2% in treated hearts vs. 28 ± 3% in controls
(p=0.07). In hearts with less severe risk zones (below the
mean value of 31% of LV), Bendavia treatment significantly
reduced the no-reflow zone by 35% (17±2 vs. 26±4% of risk
zone, p <0.05); in hearts with large risk zone, Bendavia nonsignificantly reduced no-reflow by 17%. Bendavia treatment
had no effect on hemodynamic parameters or regional
myocardial blood flow during the ischemic period. Conclusion:
These data suggest that the MPTP may play a role in the
pathophysiology of no-reflow after myocardial ischemia/
reperfusion. Bendavia reduced the extent of the no-reflow
zone for any given risk zone size, suggesting that it helped
prevent microvascular obstruction.
S.L. Hale: C. Other Research Support; Modest; Stealth Peptides.
W. Dai: None. R.A. Kloner: C. Other Research Support; Modest;
Stealth Peptides.
P170Nicorandil Inhibits Gαq-Induced Chronic Heart Failure
in Transgenic Mice
Masamichi Hirose, Iwate Medical Univ, Shiwa-gun, Iwate, Japan;
Yasuchika Takeishi, Fukushima Medical Univ, Fukushima, Japan;
Hisashi Shimojo, Toshihide Kashihara, Tsutomu Nakada, Shinshu
Univ, Matsumoto, Japan; Ulrike Mende, Brown Univ, Providence, RI;
Mitsuhiko Yamada, Shinshu Univ, Matsumoto, Japan
Introduction: Beneficial effects of nicorandil on the treatment
of hypertensive heart failure (HF) and ischemic heart disease
have been suggested. However, whether nicorandil has
inhibitory effects on HF and ventricular arrhythmias caused
by the activation of G protein alpha q (Gαq) -coupled
receptor (GPCR) signaling pathway still remains unknown.
We examined effects of chronic and acute administration of
nicorandil on the development of HF and ventricular action
potential (VAP) in transgenic mice with transient cardiac
expression of activated Gαq (Gαq-TG), respectively. Method
and Results: Nicorandil (6 mg/kg/day) or vehicle was
chronically administered in Gαq-TG mice for 24 weeks from
8 weeks of age, and then ventricular function, and electrical
and structural changes were investigated in the hearts.
Chronic nicorandil administration improved the reduction
of left ventricular fractional shortening (p < 0.001) in GαqTG hearts. During 10 min of electrocardiogram recording,
premature ventricular contractions (more than 20 beats/min)
were observed in 7 of 10 vehicle-treated Gαq-TG but in none
of 10 nicorandil-treated Gαq-TG hearts (p < 0.01). QT interval
was significantly shorter in nicorandil-treated Gαq-TG than in
vehicle-treated Gαq-TG hearts (p < 0.05). Chronic nicorandil
administration improved the increased ventricular interstitial
fibrosis (p < 0.05) but not cardiac hypertrophy in Gαq-TG left
ventricles. Real time RT-PCR revealed that mRNA expression
levels of s sulfonylurea receptor 2B (SUR-2B) were decreased
in vehicle-treatd Gαq-TG but not in nicorandil-treated Gαq-TG.
In addition, chronic nicorandil increased endotherial nitric oxide
syntheses gene expression in Gαq-TG hearts (p < 0.05). Acute
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Poster Presentations (continued)
Serial 7-day Holter ECGs were used to detect AF recurrences.
Results: At baseline, HSP70 was detectable in 14 patients
(21 %), but there was no correlation between clinical or
echocardiographic variables and the presence or the level of
HSP70. Patients with paroxysmal AF (n=39) showed lower
anti-HSP70 antibodies (median 43, IQR 28–62 µg/ml) than
patients with persistent AF (n=28; 53, 41–85 µg/ml, p=.035).
Using multivariable regression analysis, AF type was the only
variable associated with anti-HSP70 antibodies (Beta=.342,
p=.008). At 6 months, HSP70 was present in 27 patients (41
%, p<.001 vs. baseline) with an overall increase (median 0,
IQR 0–0 vs. 0, 0–0.09 ng/ml, p=.029). Similarly, there was an
increase of anti-HSP70 antibodies (48, 36–72 vs. 57, 43–87
µg/ml, p<.001). AF recurrence rates were higher in patients
with HSP70 increase >0.025 ng/ml (32 vs. 11 %, p=.038)
or anti-HSP70 antibodies increase >2.5 µg/ml (26 vs. 4 %,
p=.033). Conclusion: HSP70 and anti-HSP70 antibodies may
be involved in the progression of AF and AF recurrence after
catheter ablation.
nicorandil administration (1 microM) significantly shortened the
prolonged VAP duration and reduced the number of PVCs in
vehicle treated Gαq-TG hearts. Conclusions: These findings
suggest that nicorandil inhibits ventricular electrical and
structural remodeling and arrhythmias through the shortening
of VAP duration and the increased expression of SUR-2B and
eNOS in a mouse model of HF.
M. Hirose: None. Y. Takeishi: None. H. Shimojo: None. T. Kashihara:
None. T. Nakada: None. U. Mende: None. M. Yamada: None.
P171Low Concentrations of Aspirin Activate Cytoprotective
Signaling Pathway Genes in Human Endothelial Cells: A
Gene Expression Profiling Study
Reena V Kartha, Belinda W Cheung, Subbaya Subramanian, Henning
Schröder, Univ of Minnesota, Minneapolis, MN
R.V. Kartha: None. B.W.Y. Cheung: None. S. Subramanian: None.
H. Schröder: None.
P172Circulating Heat Shock Protein 70 and Antiheat Shock
Protein 70 Antibodies in Atrial Fibrillation: Relation with
Atrial Fibrillation Type and Response to Catheter Ablation
Jelena Kornej, Claudia Reinhardt, Jedrzej Kosiuk, Arash Arya, Gerhard
Hindricks, Volker Adams, Daniela Husser, Andreas Bollmann, Heart Ctr
Leipzig, Leipzig, Germany
Background: HSP and anti-HSP antibodies have been
associated with AF development and progression. This study
investigated the possible association between circulating
heat shock protein 70 (HSP70) and anti-HSP70 antibodies as
well their changes and rhythm outcome after atrial fibrillation
(AF) catheter ablation. Methods: In 67 patients with AF
(59±11 years, 66 % male, 66 % lone AF) undergoing catheter
ablation, circulating HSP70 and anti-HSP70 antibodies levels
were quantified before and 6 months after catheter ablation.
. Kornej: None. C. Reinhardt: None. J. Kosiuk: None. A. Arya:
J
None. G. Hindricks: None. V. Adams: None. D. Husser: None.
A. Bollmann: None.
P173Targeting α-1-Adrenergic Receptors for Heart
Failure Therapy
Megan D Montgomery, Bat-Erdene Myagmar, Paul C Simpson,
UCSF/SFVAMC, San Francisco, CA
Alpha-1 adrenergic receptors (α1-ARs) have beneficial effects
in the heart and cardiac myocytes. In pilot studies in vivo, a
sub-hypertensive dose of the α1A subtype-selective agonist
A61603 has cardioprotective signaling and prevents and
rescues cardiomyopathy in mouse models. These studies
give a possible explanation for adverse cardiac effects of
α1-antagonists in clinical trials and are a rationale to test
α1-agonists in heart failure. Our objective was to establish
models to screen α1-agonists for beneficial cardiac signaling.
We tested the hypothesis that different α1-AR agonists have
distinct beneficial cardiac effects at very low doses. To test this,
we studied α1-AR-mediated cardioprotective and adaptive
hypertrophic signaling in isolated neonatal rat ventricular
myocytes (NRVM) and adult mouse ventricular myocytes
(AMVM). We compared A61603 to all clinically approved α1agonists: epinephrine (EPI), norepinephrine (NE), phenylephrine
(PE), dobutamine (DOB), methoxamine (MET), and midodrine
(MID) at 5 doses, all in the presence of the beta-blocker
propranolol (200 nM). Cardioprotective signaling was measured
by α1-AR-ERK survival pathway activation and cell viability by
MTT assay after doxorubicin (20 μM)-induced toxicity. Cellular
adaptive hypertrophy was measured by protein synthesis in
NRVM and fetal gene protein expression in AMVM. In NRVM,
A61603 was the most potent (EC50 = 4 nM) and efficacious
agonist for ERK activation. The rank-order potency was:
A61603 > NE > EPI > PE > MET > DOB > MID. For protein
synthesis, A61603 had high efficacy and potency (EC50 = 12
nM). The rank-order potency was: A61603 > NE > DOB > PE
> EPI > MET > MID. In AMVM, A61603 was potent for ERK
(EC50 = 58 nM) and increased cell viability 48% (1.48 vs. 1.00
control) with doxorubicin toxicity. A61603 (200 nM) increased
the number of myocytes expressing beta-myosin heavy chain
and atrial natriuretic factor by immunocytochemistry. We
conclude that myocyte culture models are predictive of α1agonist efficacy and potency in adaptive and protective cardiac
effects. A61603, an imidazoline with high affinity for the α1AAR, is more potent and efficacious in myocyte models than are
clinically available α1-agonists, and is a promising candidate for
therapeutic testing in vivo.
ABSTRACTS
Low-dose aspirin is recommended for individuals with risk
for heart disease and stroke in order to prevent thrombotic
occlusive events. In addition to its well-established platelet
inhibitory effects, increased expression of the antioxidant heme
oxygenase-1 is considered to contribute to cardiovascular
protection by aspirin. However little is known about the
genome-wide changes in gene expression mediated by aspirin.
In order to identify other molecular pathways and targets
affected by aspirin, we investigated the global effects of aspirin
treatment (3 μM and 100 μM) in vitro in human endothelial cells
(Ea.hy 926) using HT-12 bead arrays (Illumina). The filtered
genes were further analyzed using Ingenuity Pathway Analysis
(IPA) software. We determined the gene expression profiles
of endothelial cells treated with aspirin (3 μM and 100 μM)
along with vehicle controls. Gene microarray analysis revealed
significant upregulation of 134 genes (p <0.005 and >2 fold
change) and downregulation of over 100 genes (p <0.005 and
≤0.5 fold change). Interestingly, the pattern of gene expression
was similar between the two analyzed concentrations of 3 μM
and 100 μM. The majority of upregulated genes were involved in
gene expression, transcriptional regulation, cell-to-cell signalling
and interaction and encoded transcription factors such as
MAP kinases, phosphoinositide-3-kinases and cell cycle
regulators (p<0.005). Several components of heterotrimeric
G-proteins and G-protein-coupled receptors were also found
to be upregulated which can potentially trigger phospholipase
C signaling, suggesting an increase in overall intracellular
second messenger signaling. In summary, our gene expression
analysis suggests that aspirin at low and therapeutically relevant
concentrations (3 μM) is effective in triggering cytoprotective
signaling pathways some of which play a role in the regulation
of heme oxygenase-1. Moreover, our study shows that lowdose aspirin results in altered expression profiles of a variety of
genes. Future studies will have to address the biological and
clinical relevance of these novel aspirin targets.
M.D. Montgomery: None. B. Myagmar: None. P.C. Simpson: B.
Research Grant; Significant; NIH and Department of Veterans Affairs.
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
71
Poster Presentations (continued)
P174Dynamics of the Renin-Angiotensin-Aldosterone System:
Implications of Angiotensin-Peptide Fingerprinting on the
Clinical Use and Development of Antihypertensive Drugs
Marko Poglitsch, Manfred Schuster, Hans Loibner, APEIRON Biologics
AG, Vienna, Austria
ackground: The Renin-Angiotensin-Aldosterone-System
B
(RAAS) is an extensively studied peptide hormone system
which is critically involved in the regulation of blood pressure
and other important physiologic functions. Angiotensin-(1–10)
which is liberated from angiotensinogen by kidney secreted
Renin, is sequentially metabolized to different N- and
C-terminal truncated fragments with partially described
biologic activities. Beside Angiotensin II (Ang-(1–8)), various
other angiotensin peptides including Ang-(1–7), Ang-(1–9),
Ang-(2–8) and Ang-(3–8) are known to possess physiologic
activity while Ang-(1–7) and more recently Ang-(1–9) were
described to antagonize pathologic effects of Ang-(1–8) via
vasodilation and antifibrotic action respectively. The role of
Ang-(1–8) in hypertension converts enzymes participating in
Ang-(1–8) generation to favorable targets for antihypertensive
drugs. Methods: Human blood was collected from healthy
volunteers followed by ex-vivo incubation at 37°C in the
presence of different inhibitors of angiotensin metabolism.
Angiotensin concentrations were determined employing a
novel sensitive LC-MS/MS based method, which allows the
simultaneous quantification of a broad panel of angiotensin
peptides in human plasma. Results: The investigation of
different angiotensin metabolism modifiers revealed agent
specific patterns of angiotensin peptide levels reflecting the
specificity of these inhibitors. Spiking experiments allowed
the determination of turnover rates for distinct angiotensin
conversion reactions surrounded by their physiological matrix.
CONCLUSION: The measurement of blood concentrations
of angiotensin peptides provides important information about
the substrate specificities and activities of enzymes involved
in their metabolism. This allows the identification of potential
causes for pathologic conditions, facilitating a patient specific
therapeutic modulation of the RAAS. The overall assessment
of angiotensin peptide concentrations (Angiotensin-PeptideFingerprinting) represents a powerful tool to gain a more
detailed understanding of blood pressure regulation, enabling
the pharmacologic characterization of RAAS modifying agents
in use and development.
ABSTRACTS
M. Poglitsch: None. M. Schuster: None. H. Loibner: None.
72
P175Selective Regional Injections of Alginate Hydrogel into
Cardiac Veins Improve Left Ventricular Function and
Reverse Chamber Remodeling in Dogs with Chronic
Heart Failure
Hani N Sabbah, Mengjun Wang, Michael S Sabbah, Sharad Rastogi,
Ramesh C Gupta, Henry Ford Hosp, Detroit, MI; Sam Helgerson,
Lonestar Heart, Inc, Laguna Hills, CA
Background: Progressive LV enlargement and dysfunction
are characteristics of heart failure (HF) and are associated
with increased mortality and morbidity. We tested the
hypothesis that selective regional injections of alginate
hydrogel implants (AHI, Algisyl-LVR, Lonestar Heart, Inc.)
into isolated segments of epicardial veins of the failing LV will
increase LV wall thickness and lead to reduced LV size, lower
LV wall stress and improved LV systolic and diastolic function.
Methods: 12 dogs with coronary microembolization-induced
HF (LV ejection fraction, EF<30%) were studied. During an
open-chest procedure, 1.5 to 2.0 cm segments of the 2nd
diagonal branch and the 2nd marginal branch of the cardiac
epicardial veins were isolated by ligation with silk sutures. In
6 dogs, the isolated segments were each injected with 1.0
ml of AHI and in 6 dogs with normal saline (controls). LV ED
volume (EDV), ES volume (ESV), EF, stroke volume (SV), ED
wall stress (EDWS), deceleration time of early mitral inflow
velocity (DT), and severity of functional mitral regurgitation
(MR) were measured before therapy (Pre) and at 6 months
after AHI or saline delivery (Post). The treatment effect (Δ) was
calculated as the difference between Pre and Post. Results:
In preliminary we showed that an intravenous injection of 1.0
ml AHI increased regional LV end-diastolic (ED) and endsystolic (ES) wall thickness by 5.8 ± 0.7 % and 9.0 ± 1.0 %,
respectively. Compared to saline controls, treatment of HF
dogs with AHI prevented the increase in EDV and significantly
decreased ESV, EDWS and severity of functional MR (Table).
In addition, AHI significantly increased LV EF, SV and DT
indicating improved LV systolic and diastolic function (Table).
Treatment with Alginate implants also showed a safe and nonarrhythmogenic chronic profile. Conclusion: LV reconstruction
using selective regional injections of AHI into epicardial cardiac
veins represents a safe novel approach for the treatment of
advanced chronic HF.
Hemodynamic and Ventriculographic Results
Control (n=6)
AHI (n=6)
P-Value
ΔEDV (ml)
2.5 ± 1.0
-1.1 ± 0.4
0.114
ΔESV (ml)
4.3 ± 1.7
-3.9 ± 0.4
0.001
ΔEF (%)
-3.8 ± 1.5
5.0 ± 0.4
0.001
ΔSV (ml)
-1.8 ± 0.8
0.11 ± 0.04
0.001
ΔEDWS (gm/
cm2)
9.4 ± 6.5
-23.1 ± 4.6
0.006
ΔDT (msec)
-4.7 ± 0.4
27.0 ± 4.4
0.001
ΔMR (%)
1.7 ± 0.8
-3.6 ±1.3
0.004
.N. Sabbah: G. Consultant/Advisory Board; Modest; Lonestar Heart,
H
Inc.. M. Wang: None. M.S. Sabbah: None. S. Rastogi: None.
R.C. Gupta: None. S. Helgerson: A. Employment; Significant;
Lonestar Heart, Inc..
P176Not published at presenter’s request.
P177Traditional Chinese Medication Qiliqiangxin Inhibits
Cardiac Hypertrophy, Remodeling, and Dysfunction
During Chronic Pressure Overload in Mice
Yunzeng Zou, Hui Gong, Li Lin, Ning Zhou, Lei Li, Yanyan Liang,
Jian Wu, Jingmin Zhou, Junbo Ge, Zhongshan Hosp, Fudan Univ,
Shanghai, China
Qiliqiangxin (QL), a traditional Chinese medicine, has been
used in the treatment of chronic heart failure. However,
whether QL can prevent cardiac hypertrophy and remodeling
in the hypertensive is unknown. We here compared
the effects of QL with Losartan on the development of
cardiac hypertrophy in a mice model of pressure overload.
Constriction of transverse aorta (TAC) or sham operation
was imposed to C57B/L6 mice and QL (0.6mg/Kg/day),
Losartan (13.4mg/Kg/day) or vehicle was then administrated
to them. Cardiac hypertrophy, remodeling, functions and
fibrosis were evaluated by echocardiography, catheterization,
histology, and examination of specific gene expression and
ERK phosphorylation. Local apoptosis, autophagy, TNF-α/
IGF-1, angiotensin II type 1 receptor (AT1-R), and especially
the proliferation of cardiomyocytes and phosphorylation of
ErbB2 and ErbB4 were examined in vivo to elucidate the
mechanisms. Two weeks later, TAC resulted in a significant
cardiac hypertrophy in vehicle group, which was significantly
suppressed in either QL or Losartan group. At the end of 4
weeks, QL treatment effectively abrogated TAC-induced the
development of myocardial remodeling, dysfunction, fibrosis,
and the increases in apoptosis, autophagy, TNF-α to IGF-1
ratio and AT1-R expression, which were comparable to
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Poster Presentations (continued)
Losartan treatment. However, QL, but not Losartan, enhanced
proliferation of cardiomyocytes at 4 weeks after TAC, which
was paralleled with dowregulation of C/EBPβ, upregulation of
CITED4, and increases in ErbB2 and ErbB4 phosphorylation.
Thus, QL inhibits myocardial inflammation and cardiomyocyte
death, and promotes cardiomyocyte proliferation, leading to
an ameliorated cardiac remodeling and function in a mice
model of pressure overload. The possible mechanisms may
involve inhibition of AT1-R and activation of ErbB receptors.
Y. Zou: None. H. Gong: None. L. Lin: None. N. Zhou: None. L. Li:
None. Y. Liang: None. J. Wu: None. J. Zhou: None. J. Ge: None.
P178Not published at presenter’s request.
P179Human Peripheral Blood B Cells Improve Cardiac
Function in Aged Rats After Myocardial Infarction
Traci T Goodchild, Michael B Sweet, Jai Pal Singh, Saint Joseph’s
Translational Res Inst, Atlanta, GA; Mark C Poznansky, Massachusetts
General Hosp, Harvard Medical Sch, Boston, MA; Nicolas A Chronos,
Saint Joseph’s Translational Res Inst, Atlanta, GA
T.T. Goodchild: None. M.B. Sweet: None. J. Singh: None.
M.C. Poznansky: None. N.A.F. Chronos: None.
P180Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives
Atrophic Remodeling of the Heart
Kedryn K Baskin, Rebecca Salazar, Univ of Texas Health Science
Ctr, Houston, TX; Wenhao Chen, Univ of Toledo, Toledo, OH; Heinrich
Taegtmeyer, Univ of Texas Health Science Ctr, Houston, TX
The heart adapts to changes in load by remodeling
both metabolically and structurally. During this process,
cardiomyocytes break down unnecessary or damaged
K.K. Baskin: None. R. Salazar: None. W. Chen: None.
H. Taegtmeyer: None.
This research has received full or partial funding support from the
American Heart Association, South Central Affiliate (Arkansas,
New Mexico, Oklahoma & Texas).
P181Withdrawn
P182 Withdrawn
P183Cardiac Deletion of CaMKII Delta and Gamma
Protects Against Heart Failure Despite Activation
of Calcineurin Signaling
Michael M Kreusser, Lorenz H Lehmann, Stanislav Keranov, Univ of
Heidelberg, Heidelberg, Germany; Josef-Hermann Gröne, Deutsches
Krebsforschungszentrum, Heidelberg, Germany; Hugo A Katus, Univ
of Heidelberg, Heidelberg, Germany; Eric N Olson, Univ of Texas
Southwestern Medical Ctr at Dallas, Dallas, TX; Johannes Backs, Univ
of Heidelberg, Heidelberg, Germany
CaMKII delta and gamma are the major CaMKII genes
expressed in the heart, and both are up-regulated in response
to pressure overload. Recently, we have demonstrated that
CaMKII delta single knockout mice are protected against
cardiac hypertrophy and remodeling. However, the role of
CaMKII gamma and potential redundant functions of CaMKII
delta and gamma are still elusive. The aim of the present study
was to evaluate the function of CaMKII delta and gamma by a
cardiomyocyte-specific double knockout mouse model(delta/
gamma-CKO). Strikingly, whereas delta and gamma single
knockout mice displayed only slightly reduced levels of
cardiac phospholamban (PLN) phosphorylation at the CaMKII
phosphorylation site threonin 17, in delta/gamma-CKO mice
there was almost no residual PLN-threonin-17 phosphorylation
detectable. Surprisingly and in contrast to delta and gamma
single knockout mice, delta/gamma-CKO mice did develop
cardiac hypertrophy after transverse aortic constriction (TAC).
Despite cardiac hypertrophy we observed markedly reduced
cardiac fibrosis and apoptosis. Microarray analysis revealed
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ABSTRACTS
Background: Aging is associated with increased mortality
following acute myocardial infarction (AMI). Early cell therapy
clinical trials for AMI have shown a modest improvement
in cardiac function. Previous evidence suggest that B cells
may be protective in tissue injury. Our initial studies showed
that intramyocardial delivery of bone marrow derived B cells
improves cardiac function in young rats. Aim: In this study,
we determined the effect of B cell therapy in aged animals
subjected to AMI using a clinically relevant protocol. Methods:
Human PB B cells were isolated from buffy coats using CD19+
selection. Young (8–10wks) and aged (10–12mos) athymic rats
had baseline echocardiography then coronary artery ligation
(d1). At d7, rats had repeat echocardiography and received
1x107 B cells or vehicle intravenously. At d9 and d21, rats
underwent follow-up echocardiography. Explanted hearts were
measured for infarct size and B cell localization was determined
in infarcted aged rat hearts after d8 in a subset of animals by
IHC. Results: Fractional ventricular diameter shortening (FS)
decreased at d7 compared to baseline in young (27.9 ±0.9
vs. 55.6±0.6%, P<0.001) and aged (31.6±0.9 vs. 53.4±0.5%,
P<0.001) rats. At d9, FS was higher in B cell compared to
control treated aged rats (44.9±2.6 vs. 29.3±3.0%, P<0.001).
FS was minimally changed in B cell compared to control
treated young rats (37.3±3.3 vs. 31.2±3.5%, P=NS). At d21,
FS was higher in aged rats receiving B cells compared to
controls (38.6±2.2 vs. 30.0±2.6%, P<0.001) and trended
higher in B cell treated young rats compared to control
(34.1±1.8 vs. 28.0±2.6%, P=0.058). FS was higher in aged
rats receiving B cells stored for 1.5hrs at room temperature
compared to freshly prepared B cells (35.1±3.2% vs.
27.1±2.4%, P=0.035). Infarct size was smaller in B cell treated
aged rats compared to controls (12.9±2.3 vs. 22.4±2.4% LV,
P=0.010); no difference in infarct size was detected in young
rats. B cells were detected in the infarct of aged rats at d8.
Conclusion: These data support that intravenous delivery
of peripheral blood B cells is efficacious in improving cardiac
function and limiting infarct size in aged rats, an experimental
model more relevant to clinical population.
proteins and use the resulting amino acids for the synthesis
of new proteins and/or energy provision. Protein degradation
via the ubiquitin proteasome system is controlled by ubiquitin
ligases, which determine the specific proteins to be degraded.
Atrogin-1, a muscle specific ubiquitin ligase, is required for
skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits
the development of cardiac hypertrophy. We now tested the
hypothesis that Atrogin-1 is required for atrophic remodeling
of the unloaded heart. Hearts from wild type (WT) and
Atrogin-1 -/- mice (8–10 weeks old, n=8–12) were subjected
to mechanical unloading by heterotopic transplantation. In WT
hearts, seven days of unloading significantly reduced heart
weight and myocyte cross-sectional area, while hearts lacking
Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase
in heart weight, 2-fold increase in myocyte area). Conventional
markers of atrophic remodeling, such as the reactivation of
the fetal gene program (ANF, MHC isoform switch), were
detected in both WT and Atrogin-1 -/- transplanted hearts.
Proteasome activity and markers of autophagy were increased
after unloading, although not significantly different between
WT and Atrogin-1 -/- hearts. Pathways regulating protein
synthesis were enhanced in the absence of Atrogin-1; there
was an increase in activated Akt and its downstream pathway
including mTOR, 4E-BP1, and p70 S6 kinase. Additionally,
two known targets of Atrogin-1 involved in hypertrophy and
protein synthesis, calcineurin and eukaryotic initiation factor
3f, were upregulated in unloaded Atrogin-1 deficient hearts.
Consequently, “unloaded” cardiomyocytes lacking Atrogin-1
in vitro exhibit increased basal rates of protein synthesis.
The results suggest that Atrogin-1 not only enhances protein
degradation, but also keeps protein synthesis in check. Thus
Atrogin-1 has a duel role in regulating cardiac mass.
73
Poster Presentations (continued)
a distinct different gene expression profile pointing to an
activation of calcineurin in delta/gamma-CKO mice after TAC.
Phosphorylation of calcineurin at serine 197, which leads to an
inactivation of its enzymatic activity, was almost abolished in
delta/gamma-CKO mice. To test the therapeutical implications
of a complete myocardial CaMKII knockout, an tamoxifeninducible knockout system was established. Knockout of
CaMKII delta and gamma was induced by administration of
tamoxifen three weeks after TAC surgery. Whereas control
mice did develop overt heart failure and cardiac remodeling
16 weeks after TAC, delta/gamma-iCKO mice recovered from
cardiac dysfunction. Taken together, our mouse genetic studies
demonstrate that CaMKII delta and gamma are promising drug
targets to restore cardiac function after pathological stress.
These data also unmask a cross talk of CaMKII to endogenous
calcineurin signaling, which results in adaptive cardiac
hypertrophy and not pathological remodeling.
M.M. Kreusser: None. L.H. Lehmann: None. S. Keranov: None.
J. Gröne: None. H.A. Katus: None. E.N. Olson: None. J. Backs: None.
P184Rescue of Reductive Stress by Nrf2 Deficiency in Protein
Aggregation Cardiomyopathy Mice
ABSTRACTS
Rajasekaran Namakkal Soorappan, Gayatri D Khanderao, Corey J
Miller, Curtis Olson, Vasanthi Rajasekaran, Univ of Utah Health Care, Salt
Lake City, UT; Sankaranarayanan Kannan, M D Anderson Cancer Ctr,
Houston, TX; Sheldon E Litwin, Medical Coll of Georgia, Augusta, GA;
Christopher J Davidson, Univ of Utah Health Care, Salt Lake City, UT
Background: We recently discovered that the overexpression
of human mutant αB-crystallin (hR120GCryAB) in mouse
resulted in protein aggregation cardiomyopathy (PAC) and
reductive stress (RS) in the heart tissue (Rajasekaran et.al
Cell, 2007). Further, we identified that sustained activation of
Nrf2 (nuclear erythroid 2 related factor 2)/ARE signaling as a
causal mechanism for RS in the MPAC mice. We hypothesize
that abolishing Nrf2 could prevent RS in R120GTG mice and
thereby rescue them from cardiomyopathy and heart failure.
Methods: To elucidate loss-of-function mechanisms for Nrf2
on RS and MPAC, we produced (1) NTG/WT, (2) R120GTG/
WT, (3) R120GTG/Nrf2+/- and (4) R120GTG/Nrf2-/- mice by
intercrossing R120GTG X Nrf2-KO. Primarily, we determined the
rate of survival and performed echocardiography to understand
the degree of pathogenesis and cardiac function over time
(n=6-10). Next, we analyzed the redox state and protein/
mRNA expression for major antioxidants including enzymes that
are involved in glutathione metabolism in the heart. Results:
Disruption of Nrf2 prolonged the survival of MPAC mice along
with no signs of cardiac dysfunction (by ECHO). The R120GTG
mice with Nrf2 deficiency (either Nrf2+/- or Nrf2-/-; n=4) had
significantly decreased glutathione (but equal to NTG) levels
when compared with R120GTG/WT cohorts. Such a decline
in GSH resulted in quenching of “reducing power” to prevent
“reductive stress” and facilitated redox homeostasis in the
myocardium. Interestingly, the intercross mice showed no
cardiac hypertrophy even at 12 months while the R120GTG
exhibited 90% mortality due to PAC/heart failure. Further, protein
and mRNA levels for major antioxidants were found to be normal
in the R120GTG/Nrf2+/- while there was significant down
regulation of these parameters in the R120GTG/Nrf2-/- mice
when compared to NTG/WT suggesting that Nrf2-independent
pathways are inadequate to maintain the antioxidant capacity
under stressed conditions. Conclusions: These results
demonstrate a critical role for Nrf2 underlying antioxidant
potential in the myocardium. Abrogating RS via Nrf2 might be
highly relevant to cure the protein aggregation diseases that are
coupled with elevated intracellular reducing potential.
R. Namakkal Soorappan: None. G. Khanderao: None. C.J. Miller:
None. C. Olson: None. V. Rajasekaran: None. S. Kannan: None.
S.E. Litwin: None. C.J. Davidson: None.
74
This research has received full or partial funding support from the
American Heart Association, Western States Affiliate (California,
Nevada & Utah).
P185Cardiac Characterization of Phlpp1 Knockout Mice:
A Novel Phosphatase to Terminate Akt Signaling
Nicole H Purcell, Katherine Huang, Xiaoxue Zhang, Univ of California,
San Diego, La Jolla, CA; Natalie Gude, Mark Sussman, San Diego
State Univ, San Diego, CA; Asa B Gustafsson, Shigeki Miyamoto, .
Joan Heller Brown, Univ of California, San Diego, La Jolla, CA
The serine/threonine kinase Akt, also known as protein
kinase B (PKB), regulates a wide variety of cellular processes
including metabolism, cell growth, survival, protein synthesis,
and gene transcription. Akt is activated by upstream kinases
through sequential phosphorylation on its activation loop
(Thr308) followed by phosphorylation on the hydrophobic
motif (Ser473). Studies using overexpression of Akt in vitro
and in vivo have implicated the kinase in either attenuating
or increasing cardiac hypertrophy, depending on signal
intensity and localization of Akt in the cell. Recently the
protein phosphatase PHLPP (PH domain leucine-rich repeat
protein phosphatase) was shown to dephosphorylate Akt
on its hydrophobic motif (Ser473), thereby decreasing
kinase activity. We hypothesized that PHLPP could serve
as an important regulator of Akt signaling in the heart and
that loss of PHLPP would accentuate Akt activation under
physiological conditions. We generated phlpp1 null mice to
investigate the cardiac phenotype induced by modulating the
kinetics of Akt activation in vivo. Myocytes were isolated from
PHLPP1 knock-out and wild-type mice to demonstrate loss of
PHLPP1 protein and increased Akt phosphorylation following
stimulation with the cytokine LIF. Histological examination of
hearts revealed that PHLPP1 KO mice have an increased
capillary to myocyte ratio compared to age matched wild-type
mice at baseline. Increased capillary networks were also
detected in the PHLPP1 KO mice relative to wild-type mice
using corrosion casting. To determine the role of PHLPP
removal on cardiac hypertrophy, PHLPP1 null mice and wildtype mice were subjected to two weeks pressure overload
by transverse aortic constriction (TAC). Cardiac hypertrophy
was unexpectedly attenuated in PHLPP1 null mice (26%
increase in HW/BW ratio vs. 48% in wild-type mice). In
contrast, PHLPP1 null mice display an accentuated response
to physiological hypertrophy induced by swimming compared
to wild-type controls (46% vs. 38% respectively). Our data
suggests that there is enhanced Akt activation following
PHLPP removal and this attenuates pathological hypertrophy
due to changes in capillary density.
N.H. Purcell: None. K. Huang: None. X. Zhang: None. N. Gude:
None. M. Sussman: None. A.B. Gustafsson: None. S. Miyamoto:
None. J. Heller Brown: None.
P186IL-10 Mitigates Adverse Cardiac Remodeling and
Improves Left Ventricle Function in Pressure OverloadInduced Hypertrophy
Suresh K Verma, Prasanna Krishnamurthy, Neha Senger, Melissa Thal,
Eneda Hoxha, Erin Lambers, Tina Thorne, Gangjian Qin, Douglas W
Losordo, Raj Kishore, Feinberg Cardiovascular Res Inst, Chicago, IL
Background: Hypertrophy associated heart failure remain the
leading cause of death worldwide. Inflammatory cytokines
play critical role in adverse cardiac remodeling and heart
failure. Therefore, approaches geared towards inhibiting
cardiac inflammation may provide therapeutic benefits. Thus,
we tested the hypothesis that anti-inflammatory cytokine,
IL-10, therapy might inhibit pressure overload-induced cardiac
remodeling. Methods and Results: Cardiac hypertrophy
was induced in Wild-type (WT) and IL-10-knockout (KO) mice
by isoproterenol (ISO; 45mg/kg b.wt./day, 14 days) infusion
through mini-osmotic pumps. Control groups mice received
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Poster Presentations (continued)
saline. ISO-induced increase in heart weight/tibia length
ratio in WT was further increased in KO mice. ISO-mediated
cardiac dysfunction (%EF and %FS) in WT mice were
exaggerated in KO mice. KO mice displayed higher expression
of fetal genes (ANP and BNP) and inflammatory cytokines
(TNFα and IL-1β). ISO-treated KO mice had notably higher
fibrosis and apoptosis than the WT. Interestingly, systemic
recombinant IL-10 administration markedly attenuated effects
of ISO and improved left ventricle function, reversed fetal
gene expression, fibrosis and apoptosis both in WT and in
KO mice. To further understand the mechanism of IL-10
protection, NRCM and H9C2 cells were treated with ISO
(10µM) and/or IL-10 (20ng/mL), in vitro. ISO significantly
increased the mRNA expression of ANP and BNP as well
as of inflammatory cytokines which was markedly reduced
by IL-10. ISO treatment also induced the activation of p38,
ERK1/2 MAP kinases and of NFkB while it inhibited STAT3 phosphorylation. Interestingly, co-treatment with IL-10
suppressed p38 and ERK1/2 phosphorylation while enhancing
STAT-3 phosphorylation. Interestingly, ISO-induced nuclear
translocation of NFkB was mimicked by STAT3 specific
inhibitor cucurbitacin, suggesting STAT3 as the downstream
target of IL-10 effects. Conclusion: Our studies suggest
that IL-10 treatment not only inhibits the progression but
also reverses the pressure overload-induced adverse cardiac
remodeling. Ongoing investigations will further provide a better
understanding on the mechanistic and therapeutic aspects of
IL-10 on hypertrophic remodeling and heart failure.
.K. Verma: None. P. Krishnamurthy: None. N. Senger: None.
S
M. Thal: None. E. Hoxha: None. E. Lambers: None. T. Thorne: None.
G. Qin: None. D.W. Losordo: None. R. Kishore: None.
P187Casein Kinase 2/Histone Deacetylase 2/Krüppel-like
Factor 4 Is a Novel Axis of Development of Cardiac
Hypertrophy
Gwang Hyeon Eom, Chonnam Natl Univ Medical Sch, Gwangju,
Korea, Republic of; Young Kuk Cho, Chonnam Natl Univ Hosp,
Gwangju, Korea, Republic of; Jeong-Hyeon Ko, Sera Shin, Nakwon
Choe, Yoojung Kim, Hosouk Joung, Hae Jin Kee, Hyun Kook,
Chonnam Natl Univ Medical Sch, Gwangju, Korea, Republic of
G. Eom: None. Y. Cho: None. J. Ko: None. S. Shin: None. N. Choe:
None. Y. Kim: None. H. Joung: None. H. Kee: None. H. Kook: None.
P188Suppression of MK2 Signaling Protects Against Pressure
Overload-Induced Cardiac Dysfunction
Maya Khairallah, Dharmendra Dingar, Montreal Heart Inst, Montreal,
QC, Canada; Ramzi J Khairallah, Univ of Maryland — Baltimore,
Baltimore, MD; George Vaniotis, Yanfen Shi, Montreal Heart Inst,
Montreal, QC, Canada; Matthias Gaestel, Hannover Medical Sch,
Hannover, Germany; William C Stanley, Univ of Maryland — Baltimore,
Baltimore, MD; Jean Claude Tardif, Bruce G Allen, Montreal Heart Inst,
Montreal, QC, Canada
p38 mitogen-activated protein kinases (MAPKs) regulate a
broad range of cellular activities but have been implicated
in the pathogenesis of cardiovascular diseases. The
mechanisms whereby p38 exerts divergent effects are
unknown. Current p38 inhibitors both produce serious side
effects and loose efficacy with chronic use. Hence, a better
understanding of the downstream targets of p38, i.e. MAPKactivated protein kinases (MKs), is essential. We hypothesized
that inhibition of MK2 activity (MK2-/-) during chronic pressure
overload is cardioprotective. Twelve-week-old MK2-/- and
littermate control (MK2+/+) mice were subjected to transverse
aortic constriction (2 wks, n=8). MK2-/- mice showed a
~20% reduced increase in heart weight-to-tibia length ratio
(Fig A) despite similar increases in mean blood pressure and
ANP expression. Compared to controls, banded MK2-/hearts also showed preserved left ventricular: (i) fractional
shortening, (ii) ejection fraction (preserved at 72.5 ± 2.2%
after banding for MK2-/- but decreased from 67.9 ± 3.6%
to 58.8 ± 3.4% in MK2+/+, p<0.05, Fig B), and (iii) diastolic
function evidenced by the ratio of transmitral and myocardial
early diastolic velocities (E/Em preserved after banding
in MK2-/- but increased by 78.8 ± 11.1% in MK2+/+,
p<0.01). MK2 deficiency did not reduce interstitial fibrosis.
For equivalent respiration rates, mitochondria from MK2-/hearts showed a significant decrease in Ca2+-sensitivity for
mitochondrial permeability transition pore opening (p<0.05,
Fig C). Overall, these results suggest that MK2 mediates part
of the detrimental remodeling evoked by chronic pressure
overload-induced activation of p38.
M. Khairallah: None. D. Dingar: None. R.J. Khairallah: None.
G. Vaniotis: None. Y. Shi: None. M. Gaestel: None. W.C. Stanley:
None. J.C. Tardif: None. B.G. Allen: None.
ABSTRACTS
ackground: Cardiac hypertrophy is characterized by
B
transcriptional reprogramming of fetal gene expression,
and histone modifiers are tightly linked to the regulation
of those genes. We previously reported that activation of
histone deacetylase (HDAC) 2, one of the class I HDACs,
mediates hypertrophy. Here we suggest that disinhibiting of
kruppel-like factor 4 (Klf4) by casein kinase-2α1 (CK2α1)dependent phosphorylation of HDAC2 S394 develop the
cardiac hypertrophy. Methods and Results: Hypertrophic
stimuli phosphorylated Hdac2 S394, which was necessary
for its enzymatic activation and thereby for the development
of hypertrophic phenotypes. Transgenic mice overexpressing
Hdac2-wild type exhibited cardiac hypertrophy, whereas
those expressing phosphorylation-resistant Hdac2 S394A
did not. Compared with that in age-matched normal human
hearts, phosphorylation of Hdac2 S394 was dramatically
increased in hypertrophic cardiomyopathy patients.
Hypertrophy-induced phosphorylation of Hdac2 S394 and
its enzymatic activity were completely blocked either by
CK2-blockers or by CK2a1 siRNA. Hypertrophic stimuli led
CK2α1 to be activated, and its chemical inhibitors blocked
hypertrophy in both phenylephrine-treated cardiomyocytes
and in isoproterenol-administered mice. However, by utilizing
KLF4-binding element-disrupted Nppa promoter, treatment
with either TBB or TBCA failed to reduce the mutant promoter
activity. These results emphasized that CK2α1-induced
hypertrophic events are dependent on both Hdac2 and KLF4.
CK2α1-transgenic mice developed hypertrophy, which was
attenuated by administration of trichostatin A, an HDAC
inhibitor. Overexpression of CK2α1 caused hypertrophy in
cardiomyocytes, whereas its chemical inhibitors as well as
Hdac2 S394A blunted it. Hypertrophy in CK2α1-transgenic
mice was exaggerated by crossing these mice with Hdac2transgenic mice. By contrast, however, it was blocked when
CK2α1-transgenic mice were crossed with Hdac2 S394Atransgenic mice. Conclusions: We have demonstrated a
novel mechanism in the development of cardiac hypertrophy
by which CK2 activates HDAC2 via phosphorylating HDAC2
S394 and consequence down-regulation of KLF4.
P189RhoA Functions as an Antihypertrophic Switch in the
Mouse Heart
Davy Vanhoutte, Jop Van Berlo, Allen J York, Yi Zheng, Jeffery D
Molkentin, Cincinnati Children’s Hosp Medical Ctr, Cincinnati, OH
Background: Small GTPase RhoA has been previously
implicated as an important signaling effector within the
cardiomyocyte. However, recent studies have challenged
the hypothesized role of RhoA as an effector of cardiac
hypertrophy. Therefore, this study examined the in vivo role of
RhoA in the development of pathological cardiac hypertrophy.
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
75
Poster Presentations (continued)
Methods and Results: Endogenous RhoA protein expression
and activity levels (GTP-bound) in wild-type hearts were
significantly increased after pressure overload induced
by transverse aortic constriction (TAC). To investigate the
necessity of RhoA within the adult heart, RhoA-LoxP-targeted
(RhoAflx/flx) mice were crossed with transgenic mice expressing
Cre recombinase under the control of the endogenous
cardiomyocyte-specific β-myosin heavy chain (β-MHC)
promoter to generate RhoAβMHC-cre mice. Deletion of RhoA with
β-MHC-Cre produced viable adults with > 85% loss of RhoA
protein in the heart, without altering the basic architecture and
function of the heart compared to control hearts, at both 2
and 8 months of age. However, subjecting RhoAβMHC-cre hearts
to 2 weeks of TAC resulted in marked increase in cardiac
hypertrophy (HW/BW (mg/g): 9.5 ± 0.3 for RhoAβMHC-cre versus
7.7 ± 0.4 for RhoAflx/flx; and cardiomyocyte size (mm2): 407
± 21 for RhoAβMHC-cre versus 262 ± 8 for RhoAflx/flx; n ≥ 8 per
group; p<0.01) and a significantly increased fibrotic response.
Moreover, RhoAβMHC-cre hearts transitioned more quickly into
heart failure whereas control mice maintained proper cardiac
function (fractional shortening (%): 23.3 ± 1.2 for RhoAβMHC-cre
versus 29.3 ± 1.2 for RhoAflx/flx; n ≥ 8 per group; p<0.01; 12
weeks after TAC). The latter was further associated with a
significant increase in lung weight normalized to body weight
and re-expression of the cardiac fetal gene program. In
addition, these mice also displayed greater cardiac hypertrophy
in response to 2 weeks of angiotensinII/phenylephrine infusion.
Conclusion: These data identify RhoA as an antihypertrophic
molecular switch in the mouse heart.
D. Vanhoutte: None. J. Van Berlo: None. A.J. York: None. Y. Zheng:
None. J.D. Molkentin: None.
P190Role of mTOR Signaling in LEOPARD-SyndromeAssociated Hypertrophic Cardiomyopathy
ABSTRACTS
Maike Krenz, Christine Schramm, Deborah Fine, Michelle Edwards,
Ashley Reeb, Univ of Missouri-Columbia, Columbia, MO
76
The identification of mutations in PTPN11 (encoding
the protein tyrosine phosphatase Shp2) in families with
congenital heart disease has facilitated mechanistic studies
of various cardiovascular defects. However, the roles of
normal and mutant Shp2 in the developing heart are still
poorly understood. In particular, how Shp2 loss-of-function
(LOF) mutations cause LEOPARD Syndrome, which is
characterized by congenital heart defects such as pulmonary
valve stenosis and hypertrophic cardiomyopathy (HCM),
remains unclear. We employed both in vitro and in vivo
models to investigate signaling mechanisms downstream of
Q510E-Shp2, a particularly aggressive mutation leading to
early-onset HCM. In cultured rat neonatal cardiomyocytes,
Q510E-Shp2 expression significantly increased cell size. This
increase was accompanied by hyperactivation of signaling
through mammalian target of rapamycin (mTOR). mTOR
inhibition with rapamycin reversed the pro-hypertrophic
effects of Q510E-Shp2 in culture. Mice with cardiomyocytespecific overexpression of Q510E-Shp2 starting before birth
also showed up-regulation of signaling through Akt/mTOR/
p70S6K and developed HCM in the early neonatal period.
Q510E-Shp2 expression increased cardiomyocyte sizes and
septum thickness, and caused cardiomyocyte disarray and
fibrosis. Echocardiographically, hearts were hypocontractile
and displayed increased wall thicknesses. Importantly,
rapamycin administration rescued the Q510E-Shp2-induced
phenotype in vivo. Our studies establish a role for mTOR
signaling in HCM caused by the LOF mutation Q510E-Shp2.
Pharmacological inhibition of mTOR was sufficient to rescue
the HCM phenotype in both models. These findings have
important implications not only for the development of novel
therapeutic approaches for LEOPARD Syndrome patients, but
also for closely related forms of congenital heart disease such
as Noonan Syndrome.
M. Krenz: B. Research Grant; Significant; AHA Scientist Development
Grant. C. Schramm: None. D. Fine: None. M. Edwards: None.
A. Reeb: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P191Calpain-Generated Free PKCα Catalytic Domains
Induce HDAC5 Nuclear Export and Regulate Cardiac
Gene Transcription
Yan Zhang, Scot Matkovich, Abhinav Diwan, Min-Young Kang,
Gerald W Dorn II, Washington Univ in St Louis, St Louis, MO
Receptor-mediated activation of protein kinase (PK) C is a
central pathway regulating cell growth, homeostasis, and
programmed death. Recently, we showed that calpainmediated proteolytic processing of PKC in ischemic
myocardium activates PKC signaling in a receptor-independent
manner by releasing a persistent and constitutively active free
catalytic C-terminal fragment, PKCα-CT. This unregulated
kinase provokes cardiomyopathy, but the mechanisms
remain unclear. We examined hypothesis that PKCα-CT has
transcriptional activity. Using immunoblot analysis and confocal
microscopy, we found that PKCα-CT localized in part to nuclei
and spontaneously induced cytosolic relocalization HDAC5 of
the transcriptional regulator. Co- expression of calpain 1 with full
length PKCα can generate PKCα-CT and produced the same
HDAC5 cytosolic relocalization, whereas full length PKCα alone
had no such effect. HDAC5 cytosolic relocalization induced
by PKCα-CT was abolished by the protein kinase inhibitor
GO6976, but not by PKD inhibitor CID 755673. The in vivo
relevance of these findings was examined in transgenic mice
expressing PKCα and PKCα-CT. To assess the consequence
on gene expression, we performed global transcriptome
profiling by Affymetrix microarrays and mRNA sequencing.
The two techniques substantially agreed. Compared to control
hearts, 621 mRNAs were regulated at least 1.3 fold in PKCα-CT
hearts (P< 0.001), only 59 in full-length PKCα hearts. MEF2dependent inflammatory pathway genes which are putative
HDAC targets were upregulated in PKCα-CT heart: 15 MEF2
target mRNAs were upregulated in PKCα-CT hearts (p<0.001),
only one in PKCα hearts. These results reveal that PKCα-CT is
a potent regulator of pathological cardiac gene expression by
localizing to nuclei and directly promoting nuclei-cytoplasmic
shuttling of HDAC5. Receptor-independent effect of PKCαCT and HDAC phosphorylation in ischemic hearts has broad
ramifications for understanding and preventing the pathological
transcriptional stress response.
Y. Zhang: None. S. Matkovich: None. A. Diwan: None. M. Kang:
None. G. Dorn II: None.
P192MicroRNA-199b Targets the Nuclear Kinase
Dyrk1a in an Auto-Amplification Loop Promoting
Calcineurin/NFAT Signaling
Kanita Salic, Paula A da Costa Martins, Cardiovascular Res
Inst Maastricht, Hubrecht Inst and Interuniversity Cardiology Inst
Netherlands, Royal Netherlands Acad of Sciences, Maastricht,
Utrecht, Netherlands; Monika M Gladka, Cardiovascular Res Inst
Maastricht, Maastricht, Netherlands; Stefanos Leptidis, Hamid el
Azzouzi, Cardiovascular Res Inst Maastricht, Hubrecht Inst and
Interuniversity Cardiology Inst Netherlands, Royal Netherlands Acad
of Sciences, Maastricht, Utrecht, Netherlands; Arne Hansen, Inst of
Experimental and Clinical Pharmacology and Toxicology, Univ Medical
Ctr Hamburg-Eppendorf, Hamburg, Germany; Gianluigi Condorelli,
Inst of Biomedical Technologies, Consiglio Nazionale delle Ricerche,
Milan, Italy; Maria L Arbones, Ctr for Genomic Regulation and Ctr
de Investigación Biomédica en Red de Enfermedades Raras, Ctr de
Regulació Genòmica, Barcelona, Spain; Thomas Eschenhagen, Inst of
Experimental and Clinical Pharmacology and Toxicology, Univ Medical
Ctr Hamburg-Eppendorf, Hamburg, Germany; Leon J De Windt,
Cardiovascular Res Inst Maastricht, Maastricht, The Netherlands
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Poster Presentations (continued)
icroRNAs (miRs) are a class of single-stranded, non-coding
M
RNAs of ~22 nucleotides in length, and growing evidence
indicates that miRs are implicated in myocardial disease
processes. A key pathway involved in heart failure consists of
the phosphatase calcineurin and its downstream transcription
factor Nuclear Factor of Activated T-cells (NFAT). We performed
microRNA profiling in hearts from calcineurin transgenic mice
and demonstrated that microRNA-199b (miR-199b) is a direct
calcineurin/NFAT target gene. MiR-199b increases in expression
in mouse and human heart failure, and targets the nuclear
NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation
regulated kinase 1a (Dyrk1a), constituting a pathogenic feed
forward mechanism with impact on calcineurin-responsive gene
expression. Interestingly, cardiac miR-199b levels inversely
correlated with cardiac Dyrk1a expression in biopsies of failing
human hearts secondary to ischemic heart disease or nonischemic dilated cardiomyopathy. Furthermore, mutant mice
overexpressing miR-199b or haploinsufficient for Dyrk1a are
sensitized to calcineurin/NFAT signaling or pressure overload
and exhibit stress-induced cardiomegaly by reduced Dyrk1a
expression. From a therapeutic point of view, in vivo inhibition
of miR-199b by a specific antagomir normalized Dyrk1a
expression, reduced nuclear NFAT activity, and caused marked
inhibition and even reversal of pre-established hypertrophy
and fibrosis in mouse models of heart failure. Our results reveal
that microRNAs impact cardiac cellular signaling and gene
expression, and implicate miR-199b as a therapeutic disease
target in heart failure.
. Salic: None. P. da Costa Martins: F. Ownership Interest; Modest;
K
Modest. M. Gladka: None. S. Leptidis: None. H. el Azzouzi: None.
A. Hansen: None. G. Condorelli: None. M. Arbones: None.
T. Eschenhagen: None. L. De Windt: F. Ownership Interest; Modest
P193Nuclear Localization of the α1B-Adrenergic Receptor
Subtype Is Required for Hypertrophic Signaling in
Cardiac Myocytes
Steven C Wu, Andrew L Cypher, Chastity L Healy, Casey D Wright,
Yuan Huang, Timothy D O’Connell, Sanford Res/USD, Sioux Falls, SD
.C. Wu: None. A.L. Cypher: None. C.L. Healy: None. C.D. Wright:
S
None. Y. Huang: None. T.D. O’Connell: None.
P194CCAAT/Enhancer-Binding Protein Beta Negatively Regulates
Hemodynamic Stress-Induced Cardiac Hypertrophy
Jota Oyabu, Osamu Yamaguchi, Takafumi Oka, Shungo Hikoso,
Kazuhiko Nishida, Issei Komuro, Kinya Otsu, Osaka Univ Graduate Sch
of Med, Suita-City, Osaka, Japan
The CCAAT/enhancer-binding proteins (C/EBP) beta is a
member of family of leucine-zipper transcription factors that
regulate gene expression to control cellular proliferation,
differentiation, inflammation and metabolism. Previous reports
showed that the protein and mRNA levels of C/EBP beta were
increased in rat ischemia-reperfused or human failing hearts.
Recent study indicates an important role of C/EBP beta in
physiological cardiac hypertrophic responses. In the present
study, we generated cardiac-specific C/EBP beta-deficient
mice (CKO) to elucidate its in vivo function in pathological
cardiac hypertrophy. We crossed floxed C/EBP beta mice
with mice expressing Cre recombinase in cardiac-specific
manner. Echocardiographic and physiological analyses
revealed that CKO showed no cardiac phenotypes under
basal conditions at 10 weeks old, indicating that the C/EBP
beta is not essential for mouse heart development. Then, we
subjected CKO and control mice (CTL) to pressure overload
by means of transverse aortic constriction (TAC). In wildtype mouse hearts, the expression level of C/EBP beta was
increased after TAC. One week after TAC, CKO showed left
ventricle (LV) hypertrophy (LV/body weight, CKO 4.64 ± 0.08
mg/g versus CTL 4.14 ± 0.06 mg/g, p<0.01, LV mass index,
CKO 106.1 ± 1.7 versus CTL 98.5 ± 1.8, p<0.05), without
showing contractile dysfunction. Cross-sectional area of
cardiomyocytes was increased in CKO after TAC (CKO 373.6
± 2.1 μm2 versus CTL 300.5 ± 3.6 μm2, p<0.01). The increase
in the atrial natriuretic factor (ANF) or alpha skeletal actin
mRNA expression, molecular markers for cardiac remodeling,
was observed in CKO hearts. Furthermore, overexpression of
C/EBP beta in isolated neonatal rat cardiomyocytes inhibited
an increase in 3H-leucine uptake induced by phenylephrine
stimulation. C/EBP beta is expressed as three distinct protein
isoforms, namely FL, LAP, and LIP, which are encoded by a single
gene, but transcribed from different initiation sites. Luciferase
reporter gene assay showed that overexpression of LAP
isoform attenuated the transcriptional activity of ANF induced
by phenylephrine, but not LIP. Thus, we conclude that C/EBP
beta attenuates the pathological cardiac hypertrophy induced by
hemodynamic stresses.
J. Oyabu: None. O. Yamaguchi: None. T. Oka: None. S. Hikoso:
None. K. Nishida: None. I. Komuro: None. K. Otsu: None.
P195Nuclear Translocation of GRK5 Following Hypertrophic
Stimuli Is a Calmodulin-Dependent Process
Jessica I Gold, Jeffrey S Martini, J Kurt Chuprun, Walter J Koch,
Thomas Jefferson Univ, Philadelphia, PA
ABSTRACTS
We previously demonstrated that α1-adrenergic receptors
(α1-ARs) in the heart are required for physiologic hypertrophy
during development and prevent a maladaptive response
to pathologic stress. We have also shown that the major
subtypes, α1A and α1B, both localize to the nucleus in adult
cardiac myocytes. Importantly, we have defined a nuclear
α1A-ERK survival and an α1A-PKCδ-cTnI inotropic signaling
pathway that both originate at the nucleus and are transduced
to cytosolic targets, suggesting that the α1A is required for
cardiac myocyte survival and contractility. However, less
is known about the molecular function of the α1B. In the
current study, we examined the role of the α1B-subtype in
hypertrophic signaling. First, we identified a bi-partite nuclear
localization sequence (NLS) in the carboxy-terminal tail of the
receptor. Mutation of the NLS (α1B-NLSmut) disrupted its
localization to the nucleus when expressed in adult cardiac
myocytes. We then compared hypertrophic signaling of the
wild-type α1B- to the mutated receptor by reconstitution in
cardiac myocytes lacking endogenous α1B (α1BKO) receptors.
Activation of the wild-type receptor by the α1-agonist
phenylephrine in α1BKO myocytes restored hypertrophic
signaling, as we observed increased phosphorylation of protein
kinase C (PKC) isoforms δ and ε, and histone deacetylases
(HDAC) 4 and 5. We also observed increased expression of
the hypertrophic gene marker, atrial natriuretic factor (ANF).
Expression of the α1B-NLSmut failed to activate hypertrophic
signaling in α1BKO cardiac myocytes despite phenylephrine
stimulation. Together, our data show that nuclear localization
of the α1B-subtype is required for hypertrophic signaling
and overall further suggest that α1-adrenergic receptors are
functional only at the nucleus in cardiac myocytes.
Maladaptive ventricular hypertrophy often precedes the
pathological endpoint of Heart Failure (HF). G protein
Coupled Receptors (GPCRs), such as the β-adrenergic
receptor, play a crucial role in the development of cardiac
hypertrophy. GPCR Kinase 5 (GRK5) is a primary GRK
expressed in cardiomyocytes. Traditionally, it phosphorylates
and desensitizes agonist-bound GPCRs. Recent, exciting
data from our lab shows that GRK5 translocates into the
nucleus and phosphorylates Histone Deacetylase 5 (HDAC5)
in response to hypertrophic stimuli. This novel kinase activity
of GRK5 causes nuclear export of HDAC5, enhancing
transcription of hypertrophic genes regulated by myocyte
elongation factor 2 (MEF2). We hypothesize that that nuclear
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
77
Poster Presentations (continued)
GRK5 activity plays a critical role in cardiomyocytes, facilitating
pathological hypertrophy and HF, and that hypertrophic GPCR
signaling induces GRK5 nuclear translocation. To test this
hypothesis, we utilized neonatal rat ventricular cardiomyocytes
(NRVMs) to determine GRK5 localization following the
hypertrophic stimulus of phenylephrine (PE) treatment. To
determine the mechanism causing nuclear translocation, we
analyzed the effects targeted inhibition of key post-GPCR
hypertrophic signaling pathways on GRK5 cellular localization.
Treatment of NRVMs with PE for 1 hour increases nuclear
GRK5 accumulation by 100%. Chronic infusion of PE also
lead to a 50% increase in nuclear GRK5 in the hearts of mice
overexpressing cardiac-specific GRK5. Calmodulin inhibition
(CaM) in NRVMs via calmidizolium chloride decreases nuclear
GRK5 accumulation, while inhibition of PKC and PKD shows
no significant effect. Using a luciferase assay, we have
shown that overexpression of CaM increases MEF2 activity
3-fold and causes increased nuclear accumulation of GRK5.
Further, overexpression of a GRK5 mutant that lacks CaM
binding activity, results in a lack of PE- and CaM-induced
nuclear translocation. We have now found that hypertrophic
stimuli increases myocyte GRK5 nuclear translocation via a
CaM-dependent process. Defining the role of CaM signaling
in pathological GRK5 nuclear localization may offer a new
therapeutic target for maladaptive hypertrophy and HF.
J.I. Gold: None. J.S. Martini: None. J. Chuprun: None. W.J. Koch: None.
This research has received full or partial funding support from the
American Heart Association, Great Rivers Affiliate (Delaware, Kentucky,
Ohio, Pennsylvania & West Virginia).
P196The Impact of MicroRNA195 on the Lkb1/AMPK Signaling
Axis and Hypertrophic Cardiomyopathy
ABSTRACTS
Hao Chen, Laurel Mckee, Gustavo Untiveros, Jessica Perez, John
Konhilas, Univ of Arizona, Tucson, AZ
78
Objectives: MicroRNAs (miRs) have been identified as chief
post-transcriptional regulators of cardiac disease progression.
In addition, a critical role of the adenosine monophosphateactivated kinase (AMPK) pathway in the development of
myocardial hypertrophy has been revealed. Yet, regulation
of the AMPK pathway by miRs in the heart has not been
addressed. We hypothesized that components of the AMPK
pathway are targeted by miRs and alter AMPK signaling
in a mouse model of hypertrophic cardiomyopathy (HCM).
Methods and Results: Using real-time PCR, a candidate miR
screen that included 22 miRs implicated in pathological cardiac
disease and/or metabolic dysregulation was performed on
hearts from 60-, 120-, and 240-day-old transgenic HCM male
mice harboring an R403Q mutation in the myosin heavy gene.
Among early (60 day) elevated miRs were miR-195 and -451.
Both miR-195 and -451 have conserved target sites in the 3’
UTR of CAB39 (MO25), a central component of the MO25/
STRAD/LKB1 complex that acts as an upstream kinase for
AMPK and its subsequent activation. We further confirmed
the elevation of miR-195 and -451 by Northern blotting. Next,
we demonstrated specific expression and a similar distribution
pattern of miR-195 and -451 in cardiomyocytes of R403Q
HCM hearts by in situ hybridization. To determine whether
the conserved sites in MO25 3’ UTR acted as functional
targets, either the miR-195 or miR-451 target sequence
was cloned into a luciferase expression vector. MiR-195
but not miR-451 suppressed luciferase activity compared
to the missense sequence control vector in C2C12 cells. In
addition, over-expression of miR195 in C2C12 cells knocked
down MO25 expression levels and downstream AMPK
signaling (phosphorylation of Acetyl CoA carboxylase). Finally,
parallel changes were measured in 60 day R403Q HCM
male hearts that included reduced MO25 expression and
lowered phosphorlation of AMPK and Acetyl CoA carboxylase.
Conclusion: Our findings indicate that miR-195 targets the
LKB1/AMPK signaling axis and suggest a functional role for
miR-195 elevation in R403Q HCM disease progression.
H. Chen: None. L. Mckee: None. G. Untiveros: None. J. Perez:
None. J. Konhilas: None.
P197Reversible Acetylation of GSK3β Regulates the
Development of Cardiac Hypertrophy
Ravi Sundaresan Nagalingam, Vinodkumar B Pillai, Sadhana Samant,
Don Wolfgeher, Univ of Chicago, Chicago, IL; Madhu Gupta, Univ of
Illinois at Chicago, Chicago, IL; Mahesh P Gupta, Univ of Chicago,
Chicago, IL
Glycogen synthase kinase 3β (GSK3β) is a critical regulator
of diverse cellular functions involved in the maintenance of
cellular structure, function and survival. In the heart, GSK3β
has been shown to play a key role in antagonizing the
development of cardiac hypertrophy. It has been shown that
GSK3β activity is inhibited by phosphorylation at Ser9 during
the development of cardiac hypertrophy. However, recent
studies indicated that blocking the Ser9 phosphorylation
alone is not sufficient to protect GSK3β activity, suggesting
that alternative mechanisms might be involved in regulation
of GSK3β activity. In the present study, we tested the
hypothesis whether GSK3β is acetylated in established
hypertrophy, which leads to inhibition of GSK3β activity, and
thereby promoting progression and maintenance of cardiac
hypertrophy. We examined acetylation, phosphorylation
and catalytic activity of GSK3β in pressure overload model
of cardiac hypertrophy at different stages of hypertrophy
development. The results showed that GSK3β catalytic
activity was significantly down-regulated throughout the
course of hypertrophy development. However, the inhibitory
phosphorylation of GSK3β was observed only at initial
stages, but not at later stages of pressure overload (4
weeks). Interestingly, we observed a significantly increased
acetylation of GSK3β, which corresponded to the reduced
activity at this later stage of hypertrophy. We then determined
the impact of GSK3β acetylation on its catalytic activity,
by measuring the ability of GSK3β to phosphorylate the
substrate glycogen synthase. The results showed that
acetylation decreased the catalytic activity of the GSK3β by
almost 3 fold. Next, we performed mass spectrometry to find
out the target lysine residues of GSK3β. Proteomic analysis
revealed that multiple lysine residues in different regions of
GSK3β were acetylated. By using site directed mutagenesis,
we found that acetylation inhibits the catalytic activity of
GSK3β by suppressing its substrate binding ability, which
is independent of inhibitory-Ser 9 phosphorylation. These
studies for the first time show a phosphorylation independent
mechanism regulating GSK3β activity during progression and
maintenance of cardiac hypertrophy.
. Nagalingam: None. V. Pillai: None. S. Samant: None.
R
D. Wolfgeher: None. M. Gupta: None. M. Gupta: None.
This research has received full or partial funding support from the
American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa,
Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota,
South Dakota & Wisconsin).
P198Aberrant Activation of γ2-AMPK Causes Cardiac
Hypertrophy Independent of Glycogen Storage
Maengjo Kim, Univ of Washington, Seattle, WA; Roger Hunter, Kei
Sakamoto, Univ of Dundee, Dundee, United Kingdom; Christine E
Seidman, Jonathan G Seidman, Harvard Medical Sch, Boston, MA;
Rong Tian, Univ of Washington, Seattle, WA
AMP-activated protein kinase (AMPK) is an energy sensor
and a key regulator of cell metabolism, hence a promising
drug target. The cardiac functions of AMPK have not been
understood. Point mutations in the regulatory γ2-subunit
(encoded by PRKAG2 gene) have been shown to cause a
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Poster Presentations (continued)
unique form of cardiomyopathy in humans characterized
by cardiac hypertrophy, arrhythmias and glycogen storage.
We have previously shown that PRKAG2 mutation caused
aberrant activation of AMPK in the absence of energy deficit
and subsequently triggered re-routing of excessive glucose
into glycogen pool. In this study, we addressed two questions:
1) whether cardiac hypertrophy in PRKAG2 cardiomyopathy
was secondary of glycogen storage; 2) which hypertrophic
signaling pathways are involved. We sought to reduce
glycogen storage in transgenic mice expressing a mutant
PRKAG2 (N488I) in the heart (TGγ2N488I) by crossing them
to knock-in mice harboring a mutation in the muscle form of
glycogen synthase (GYS1KI) that greatly reduced GYS activity
in response to glucose-6-phosphate. Compared to TGγ2N488I,
TGγ2N488I-GYS1-KI (double mutant) hearts showed lower GYS
activity (0.7 ± 0.07 vs. 6.9 ± 0.49 nmol/min/mg, p<0.0001)
and reduced glycogen content (35 ± 4.5 vs. 169 ± 40 umol/g,
p<0.0001). Nonetheless, cardiac hypertrophy remained in
the double mutant. The heart weight to body weight ratios
were 6.8 ± 0.7 mg/g for TGγ2N488I, 6.7 ± 0.5 mg/g for the
double mutant compared to 4.0 ± 0.2 mg/g in the wild type.
Furthermore, we have observed significant changes in FOXO
(forkhead-O transcription factor) and mTOR (mammalian
target of rapamycin) pathways in the TGγ2N488I hearts.
Increased phosphorylation of FOXO3a (Ser321, Ser253) and
FoxO1a (Ser256) led to nuclear exclusion and degradation
of FOXO proteins. Increased mTOR activity was evidenced
by enhanced phosphorylation of Ser2448 as well as its
downstream targets S6 and 4E-BP. Taken together these data
indicate that aberrant γ2-AMPK activation causes cardiac
hypertrophy independent of excessive glycogen accumulation.
We found that increased mTOR activity and decreased FOXO
signaling contributes to cardiac hypertrophy in TGγ2N488I mice,
suggesting novel mechanisms underlying cardiac hypertrophy
caused by abnormal γ2-AMPK activity.
M. Kim: None. R. Hunter: None. K. Sakamoto: None. C.E. Seidman:
None. J.G. Seidman: None. R. Tian: None.
This research has received full or partial funding support from the
American Heart Association, Pacific Mountain Affilliate
P199Placental Growth Factor Regulates Cardiac Adaptation
and Hypertrophy Through a Paracrine Mechanism
Federica Accornero, Jop Van Berlo, Matthew D Benard, CCHMC,
Cincinnati, OH; Peter Carmeliet, Vesalius Res Ctr, Leuven, Belgium;
Jeffery D Molkentin, CCHMC, Cincinnati, OH
F. Accornero: None. J. Van Berlo: None. M.D. Benard: None.
P. Carmeliet: None. J.D. Molkentin: None.
P200Transcriptional Regulation of tcap Conveys an Inhibitory
Mechanotransductive Response to
Modulate Cardiomyocyte Size
Jingchun Yang, Yonghe Ding, Xiaojing Sun, Xiaolei Xu, Mayo Clinic,
Rochester, MN
To sense and to respond to mechanical stimuli is a
fundamental process of cardiomyocytes during both heart
development and diseases. However, how mechanical stimuli
control cardiomyocyte size remains elusive. Here, we report a
novel inhibitory mechanotransductive response suggested by
loss-of-functions studies of α-Actinin2 (Actn2), a predominant
sarcomeric Z-disc protein. During zebrafish cardiogenesis,
depletion of Actn2 results in a defective Z-disc, while other
sarcomeric substructures remain normal. As a consequence,
ventricle chamber size is severely reduced. Interestingly, this
chamber size reduction can be rescued by the cessation of
heart contraction, suggesting the involvement of an inhibitory
mechanotransductive reponse. At the molecular level, both
tcapa and tcapb, two zebrafish tcap homologues that are
transcriptionally responsive to stretch, are transcriptionally
activated in actn2 knockdown embryos while activation of
these gene results in smaller cardiomyocytes. Depletion of
either gene rescues the reduced cardiomyocyte size in actn2
knockdown embryos. Consistent to embryonic studies, tcaps
are transcriptionally activated during pathogenesis of anemiainduced cardiomyopathy, while over expression of tcapa via
transgenics exerts a cardioprotective effect. Together, we
conclude that transcriptional regulation of tcaps conveys a
novel inhibitory mechanotransductive response that modulates
cardiomyocyte size during both zebrafish cardiogenesis and
adult cardiac remodeling.
J. Yang: None. Y. Ding: None. X. Sun: None. X. Xu: None.
P201Not published at presenter’s request.
P202Role of Lipid Peroxidation-Derived Carbonyls in Left
Ventricular Hypertrophy
Shahid Pervez Baba, Mahavir Singh, Aruni Bhatnagar, Univ of
Louisville, Louisville, KY
Pathological left ventricular hypertrophy (LVH) is a key factor
in the development of heart failure and cardiomyopathy. It
is characterized by increased oxidative stress however the
mechanisms by which it affects pathological LVH remain
unclear. Because the effects of reactive oxygen species are
mediated by products of lipid peroxidation, we hypothesize
that myocardial injury induced by LVH is mediated by lipid
carbonyls, which form covalent adducts with proteins that
induces autophagy. To test this hypothesis, we examined
the role of aldose reductase (AKR1B3; AR) an enzyme that
catalyzes the detoxification of lipid carbonyls in modulating
the cardiac stress responses in WT and AR-null mice hearts
that were subjected to transverse aortic constriction (TAC).
Two weeks after the TAC cardiac enlargement in the AR-
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
bjective: Paracrine growth factor-mediated crosstalk
O
between cardiac myocytes and non-myocytes in the heart
is critical for programming adaptive cardiac hypertrophy in
which myocyte size, capillary density, and the extracellular
matrix function coordinately. Here we examined the role that
placental growth factor (PGF) plays in the heart as a paracrine
regulator of myocyte to non-myocyte communication and
its influence on cardiac adaptation to stress stimulation
using overexpressing and PGF knockout mice. Methods
and Results: We identified PGF as a secreted factor that
is predominantly produced in the heart during pressure
overload. We studied mice with conditional post-natal PGF
overexpression (PGF DTG). While these mice did not have
a baseline phenotype, except increased fibrosis with aging,
they responded to pressure overload stimulation induced
by transverse aortic constriction (TAC) by an increase in
hypertrophy (VW/BW (mg/g): 6.6 ± 0.2 for PGF DTG versus
5.6 ± 0.2 for controls; n ≥ 6 per group; p<0.01), capillary
density (vessels/myocyte: 1.6 ± 0.05 for PGF DTG versus
1.4 ± 0.03 for controls; n ≥ 7 per group; p<0.01) and fibrosis.
Despite a mild increase in fibrosis, cardiac function remained
intact even after 12 weeks of pressure overload. On the other
hand, PGF knockout mice (Pgf-/-) succumbed to heart failure
within a week of pressure overload (fractional shortening (%):
20.2 ± 2.3 for Pgf-/- versus 30.4 ± 1.1 for controls; n ≥ 7 per
group; p<0.01). These hearts displayed dilation and capillary
rarefaction (vessels/myocyte: 1 ± 0.05 for Pgf-/- versus 1.2
± 0.04 for controls; n ≥ 7 per group; p<0.01). Mechanistically
we show that PGF has no direct effect on the cardiomyocytes
but works through its direct actions on endothelial cells
and fibroblasts by inducing capillary growth and fibroblasts
proliferation. Conclusion: PGF is a secreted factor that
supports hypertrophy and cardiac function during pressure
overload by affecting endothelial cells and fibroblasts.
79
Poster Presentations (continued)
null TAC mice was more prominent compared with WT
TAC mice. Hemodynamic analysis of the AR-null TAC mice
demonstrated left ventricular dilatation and severe contractile
dysfunction compared with WT TAC mice. Expression levels
of molecular marker for hypertrophy (ANF) increased 2–3
fold in AR-null TAC compared with the WT-TAC. Western
blot analysis of the heart homogenates using anti-4 hydroxytrans-2-nonenal (HNE) antibody showed that the formation
of HNE-protein adduct (300KDa) increased 100-fold and the
bands corresponding to molecular weights of 75KDa, 40KDa
increased 2 to 3-fold after the TAC in WT hearts and the
intensity of these bands in AR-null TAC hearts was significantly
higher compared with WT TAC. Immuno-blot analysis of the
heart homogenates revealed an increase in the LC3-I to LC3-II
conversion in banded hearts compared with sham-operated
controls and the autophagic activity was more prominent in
the AR-null compared to the WT banded hearts. Isolated adult
cardiomyocytes from WT and AR-null hearts perfused with
HNE resulted in an increase in autophagy and the increase
in autophagic activity was robust in AR-null than in WT
myocytes. These data establish that TAC-induced hypertrophy
increases protein-HNE-adduct formation and autophagic
activity and the deletion of akr1b3 gene exacerbates these
responses to pressure overload.
S.P. Baba: None. M. Singh: None. A. Bhatnagar: None.
P203Nuclear PKA Compartmentation Manages Hypertrophic
Responses to β-Adrenergic Signaling
ABSTRACTS
Jason H Yang, Jeffrey J Saucerman, Univ of Virginia, Charlottesville, VA
Protein kinase A (PKA) directly mediates several cardiac
behaviors in response to β-adrenergic stimulation. However,
it remains unclear how PKA makes context-dependent
decisions to select between contractile and hypertrophic
β-adrenergic signaling responses. We have previously shown
PKA activation by isoproterenol is spatially heterogeneous
in neonatal rat cardiac myocytes, indicating subcellular
β-adrenergic signaling compartmentation. We hypothesize
hypertrophic β-adrenergic signaling responses are specifically
regulated by nuclear compartmentation of PKA activity. We
expressed spatially targeted FRET reporters for PKA activity
and found PKA responses to isoproterenol to be slower and
less sensitive in the nucleus than in the cytosol. To investigate
mechanisms underlying these differences, we constructed a
biochemically mechanistic computational model of cytosolic
and nuclear PKA activity in rat cardiac myocytes. Using this
model, we found that nuclear PKA dynamics are limited by
diffusional transport barriers, whereas nuclear PKA sensitivity
to isoproterenol is limited by protein kinase inhibitor (PKI)
inhibition. Moreover, our model predicts these differences in
compartmented PKA activity may help PKA select between
different substrates, sensitive to the temporal characteristics
of β-adrenergic stimulation. In the model, short, transient
isoproterenol treatment fully phosphorylates phospholamban
while CREB is only partially phosphorylated. In contrast, long,
sustained isoproterenol treatment fully phosphorylates both
phospholamban and CREB. To test the model prediction that
compartmented PKA can regulate different cardiac behaviors,
we over-expressed PKA targeted to the cytosol and nucleus.
Myocytes over-expressing PKA in the nucleus were 24.8%
larger than control (n = 908 cells, p = 0.0002), while myocytes
over-expressing PKA in the cytosol were the same size as
control cells (n = 987 cells, p = 0.9971). Together, these results
highlight an important role for nuclear PKA compartmentation
in selecting hypertrophic responses to β-adrenergic stimulation.
J.H. Yang: None. J.J. Saucerman: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
80
P204Sex Differences and the Role of Estrogen Receptor β in
Exercise-Induced Myocardial Hypertrophy
Elke Dworatzek, Shokoufeh Mahmoodzadeh, Eva Brozová, Karina
Nawrath, Carola Schubert, Inst of Gender in Med-Charité Berlin, Berlin,
Germany; Jan-Ake Gustafsson, Univ of Houston, Houston, TX; Vera
Regitz-Zagrosek, Inst of Gender in Med-Charité Berlin, Berlin, Germany
We and others found sex differences in physiological myocardial
hypertrophy (MH) in mice subjected to voluntary cage-wheel
running (VCR) and forced exercise training. Female mice
showed significantly more MH, suggesting the involvement
of estrogen (E2) and estrogen receptors (ER). We therefore
investigated the underlying mechanisms leading to sex
differences in physiological MH and the role of E2 and ER
beta (ERß). Male and female C57/Bl6 wild-type (WT) and
ERß-deficient mice (ERß-/-) at the age of 12 weeks performed
8 weeks of VCR or were kept sedentary (sed). Exercise
performance was monitored daily and left ventricular mass
(LVM) was examined by echocardiography. RNA and protein
were analyzed by Real-Time PCR and western blot. Luciferasereporter Assays were performed with PGC-1a-, MEF2A- and
MEF2C-promoter deletion constructs in a human cardiac
myocyte cell line (AC16 cells) with/without E2 treatment. Female
WT-mice run more than their male counterparts (6.7km/day
vs. 4.2km/day; p<0.001). Females showed significant greater
increase in LVM and cardiomyocyte diameter in response to
exercise compared to males. VCR led to a significant activation
of AKT and p38-MAPK signalling in female running-mice,
but not in males. Mitochondrial biogenesis associated genes
MEF2A and ATP5K mRNA expression were significantly
higher in female VCR mice. Female and male ERß-/- mice
showed similar running performance compared with WT-mice
(6.3km/day vs. 2.7km/day; respectively; p<0.001); however
they showed no changes in LVM compared to sed-controls.
In contrast to WT animals, female ERß-/- mice showed no
increase in AKT and p38-MAPK phosphorylation or upregulation of mitochondrial key enzymes. E2-treatment of AC16
cells up-regulated mitochondrial target genes (PGC-1a, MEF2A
NRF1/2 and TFAM) and led to nuclear translocation of PGC-1a.
E2 increased transcriptional activity of PGC-1a and MEF2A/C
in an ER dependent manner in cardiomyocytes. Female hearts
develop more physiological MH due to exercise, characterized
by a stronger activation of pathways and genes involved in the
regulation of mitochondrial function. ERß is necessary for the
development of physiological hypertrophy and for the activation
of p38- MAPK and AKT pathways in female mice.
E. Dworatzek: None. S. Mahmoodzadeh: None. E. Brozová: None.
K. Nawrath: None. C. Schubert: None. J. Gustafsson: None.
V. Regitz-Zagrosek: None.
P205The Role of Nuclear Nox4 in Mediating Oxidation of
HDAC4 in Response to Phenylephrine Stimulation
Shouji Matsushima, Junya Kuroda, Tetsuro Ago, Junichi Sadoshima,
Univ of Med of New Jersey, Newark, NJ
Reduction and oxidation (Redox) stress is involved in the
pathogenesis of cardiac hypertrophy and heart failure. We
have shown previously that oxidation of conserved cysteine
residues in histone deacetylase 4 (HDAC4) induces nuclearto-cytoplasmic translocation of HDAC4, thereby mediating
phenylephrine (PE)-induced cardiac hypertrophy. NAD(P)
H oxidase 4 (Nox4) is a major source of oxidative stress in
cardiomyocytes (CMs) in response to pressure overload. We
hypothesized that Nox4 plays an essential role in mediating
cardiac hypertrophy by regulating the redox state of HDAC4.
Nox4 exists on intracellular membranes, including the
mitochondria and nucleus in CMs. A subpressor dose of
phenylephrine (PE, 20 mg/kg per day) or saline alone (control)
was continuously infused into wild type (WT) and cardiac
specific Nox4 knockout (Nox4-/-) mice subcutaneously via
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Poster Presentations (continued)
an osmotic minipumps. After 14 days, mean aortic pressure
was similar between WT and Nox4-/- mice. Left ventricular
(LV) wall thickness (1.04±0.03 vs. 1.27±0.06 mm, p<0.05)
and LV weight/ tibial length (LVW/TL 5.7±0.14 vs. 6.4±0.05
mg/mm, p<0.05) were significantly lower in Nox4-/- than in
WT mice. CM cross sectional area (MCA: 223±13 vs. 258±12
μm2, p<0.05) was significantly smaller in Nox4-/- than in WT
mice. Superoxide production from the nuclear membrane, as
evaluated by lucigenin chemiluminescent assays, was also
significantly lower in Nox4-/- than in WT mice (4116±314 vs.
7057±1710 RLU, p<0.05). On the other hand, PE infusion
induced significantly greater cardiac hypertrophy in transgenic
mice with cardiac specific overexpression of Nox4 than
in control mice. In cultured neonatal rat CMs, PE (100μM)
treatment induced upregulation of Nox4 (2.8 fold vs. control,
p<0.05) within 5 min, which was accompanied by increases
in superoxide production (6 fold, p<0.01) and rapid (< 5min)
nuclear export of HDAC4. Knockdown of Nox4 with shRNANox4, but not Nox2 knockdown, attenuated superoxide
production in the nucleus and prevented PE-induced nuclear
export of HDAC4. These results suggest that Nox4 plays an
essential role in mediating PE-induced cardiac hypertrophy.
Nox4 mediates PE-induced superoxide production in the
nucleus and nuclear export of HDAC4, possibly through
cysteine oxidation.
. Matsushima: None. J. Kuroda: None. T. Ago: None.
S
J. Sadoshima: None.
P206Automated Imaging Reveals a Switch Between Reversible
and Irreversible Cardiac Myocyte Hypertrophy
Karen A Ryall, Jeffrey J Saucerman, Univ of Virginia, Charlottesville, VA
K.A. Ryall: None. J.J. Saucerman: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P207Withdrawn
Marco Hagenmueller, Hugo A Katus, Stefan E Hardt, Univ of Heidelberg,
Heidelberg, Germany
Background: Cardiac development is regulated by the
evolutionary conserved Wnt signaling pathway. While Wnt
activity is silenced in the adult heart under normal conditions
this pathway is activated during myocardial remodeling
following pathological injury such as myocardial infarction.
Among other members of this pathway Dapper-1 was
suggested to antagonize Wnt activation but its role in Wnt
signaling is poorly understood. In a murine myocardial
infarction model as well as in a cardiomyocyte hypoxia
model we found a robust over-expression of Dapper-1.
To elucidate its function in cardiomyocytes we performed
Dapper-1 specific siRNA mediated knockdown experiments.
Results: Depletion of Dapper-1 leads to reduction of
cardiomyocyte surface area (-10% ± 4%, p<0.001 vs.
control), attenuation of global protein content (-20% ± 3%,
p<0.02 vs. control) and diminishes global protein synthesis
(-27% ± 10%, p<0.02 vs. control). Augmentation of protein
synthesis by β-adrenic Isoproterenol stimulation is widely
inhibited. Wnt3a induced stimulation of protein synthesis
and enlargement of cardiomyocyte surface area is blocked
in Dapper-1 knockdown cardiomyocytes. Furthermore, we
observed reduced total- and active-β-catenin protein levels.
Moreover, Dapper-1 knockdown inhibits β-catenin from
translocation to the nucleus following Wnt3a stimulation. TCF/
LEF reporter activity was reduced in Dapper-1-diminished
-and elevated in Dapper-1-overexpressed cardiomyocytes
after Wnt3a treatment. Robust Ser9 -and weak Tyr216
phosphorylation of GSK3β suggested a GSK3β independent
inhibition of canonical Wnt signaling. In addition, activation
of non-canonical Wnt/JNK pathway by Wnt5a conditioned
medium was completely inhibited in Dapper-1 knockdown
cells. Summary: Dapper1 is an essential modulator for global
protein synthesis. Furthermore, Dapper-1 is required to
activate canonical and noncanonical Wnt signaling pathways
in cardiomyocytes.
M. Hagenmueller: None. H.A. Katus: None. S.E. Hardt: None.
P209MyD88 Mediated Inflammatory Signaling Involved in PostMI CaMKII Activation and Cardiac Hypertrophy
Madhu V Singh, Paari D Swaminathan, Beixin J He, Elizabeth E
Luczak, William Kutschke, Mark E Anderson, Univ of Iowa, Iowa City, IA
Toll-like receptors (TLRs) and calmodulin kinase II (CaMKII)
participate in pathological responses to myocardial infarction
(MI), including activation of nuclear factor kappa B (NF-κB)
transcription and complement factor B expression (Singh
JCI 2009). We tested the effect of MyD88 deficiency on the
adverse cardiac outcome after MI. MyD88 knock out (MyD88/-) hearts had significantly reduced hypertrophy, hypertrophic
gene expression, and inflammatory gene expression compared
to WT control hearts. Cultured cardiomyocytes from expressing
MyD88 and a NF-κB reporter demonstrated robust induction
of NF-κB upon TLR induction by bacterial lipopolysaccharides
(LPS). In contrast, cardiomyocytes from MyD88-/- mice failed
to induce NF-κB upon LPS treatment. An alternative MyD88independent pathway for NF-κB remained functional in WT and
MyD88-/- cells. Both LPS treatment and MI increased a Ca2+/
calmodulin independet activated form of CaMKII (CaMKIIox)
in cultured cardiomyocytes and mouse hearts, respectively.
MyD88-/- hearts, however, did not display increased CaMKIIox
upon MI. Taken together, we interpret these results to show
that: (1) MyD88 participates in MI mediated hypertrophic
and inflammatory gene expression, (2) CaMKII enhances
NF-kB activation in cardiomyocytes by a MyD88-dependent
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ABSTRACTS
Cardiac hypertrophy increases risk for heart failure, arrhythmia,
and sudden death. Little is known about the specific signaling
pathways that distinguish reversible forms of hypertrophy
from irreversible forms which lead to heart failure. This project
utilizes a novel high-throughput cell phenotype imaging and
analysis protocol to study the reversibility of cardiac myocyte
hypertrophy in a scalable cell culture model. Primary cardiac
myocytes were transfected with GFP plasmid driven under
a cardiac specific troponin T promoter. Five by five mosaic
images (~100 cells) within each well of a 96-well plate were
recorded with an automated XYZ stage and focus. Postprocessing algorithms automatically background corrected,
segmented cell edges, quantified cell phenotypes, and tracked
cells between measurements. This platform therefore has
the ability to track changes in area and shape of hundreds of
individual cells over a time period of about a week. Cell shape
changes after washout of a dose response to the hypertrophic
agonist phenylephrine (PE) showed that hypertrophy was
reversible at low but not high levels of alpha-adrenergic
signaling: a cellular “toggle switch”. Specialized alphaadrenergic receptor (αAR) antagonists were used to study a
potential mechanism for this biphasic response. Prazosin, an
αAR antagonist that could act at the sarcolemma and also be
transported inside the cell, reversed PE-induced hypertrophy.
In contrast, CGP-12177a, an αAR antagonist that acts at the
sarcolemma, did not reverse PE-induced hypertrophy. These
experimental results and a computational model support the
hypothesis that cellular uptake of PE and activity at nuclear
alpha-adrenergic receptors may explain the biphasic response
in the reversibility of PE-induced hypertrophy.
P208Depletion of Dapper-1 Acts Antihypertrophic on
Cardiomyocytes and Inhibits Wnt3a- and Wnt5a-Mediated
Activation of the Wnt Signaling Pathway
81
Poster Presentations (continued)
mechanism, and (3) CaMKIIox plays a role in MyD88
dependent signaling to induce expression of proinflammatory
genes in post-MI hearts.
and MRP4 KO with cardiac overexpression of the HCN-based
cAMP sensor were used. Echocardiography and morphology
studies showed that 3 month-old MRP4 KO mice displayed
normal cardiac function (FS: 35.7% ± 1.02 in KO vs 36% ±
1.97 in WT) and cardiac phenotype (HW/TL: 7.59 ± 0.14 in
WT mice vs 7.75 ± 0.15 in KO) in basal condition. Stimulation
of WT and MRP4 KO mice by increasing concentrations
of isoproterenol (ISO: 0.1μg/kg to 3 mg/kg) induced dosedependent positive inotropic and chronotropic effects but
there was no difference in the rate of contraction (Vmax:
11214 ± 682 in WT vs 10697 ± 691 in KO) or in the heart rate
(724 ± 21 in WT vs 711 ± 21 in KO) between both groups.
Cardiac myocytes were isolated from WT and MRP4 KO mice
with cardiac expression of the HCN-based FRET sensor and
intracellular cAMP concentrations were measured by FRET.
Application of ISO (1nM) induced a similar increase in cAMP
level in WT and MRP4 KO mice (YFP/CFP: 4.4 ± 0.15 in WT
vs 4.1 ± 0.44 in KO cells), whereas cAMP signal was greater
in MRP4 KO compared to WT mice after PDE inhibition by
IBMX (ISO 1nM + IBMX 300µM: 8.1 ± 0.6 in WT vs 11.8 ±
0.62 in KO cells; p<0.01). PDE3A and PDE4A mRNA level
was 1.6 fold higher in the myocardium of MRP4 KO compared
to WT mice. MRP4 KO mice displayed age-dependent CH
(HW/TL in 9 month-old mice: 9.8 ± 0.57 in KO vs 7.82 ± 0.13
in WT mice; p<0.01). 3 month-old MRP4 KO and WT mice
were subjected to thoracic aortic clamping (TAC) or sham
operated (Sh) and CH was assessed at 12 weeks after the
surgery by echocardiography and morphometric studies.
Heart function was also examined by hemodynamic technique
in basal conditions and upon a β-adrenergic stimulation in WT
(Sh and TAC) and MRP4 KO (Sh and TAC) mice. Alteration
of left ventricular dP/dt upon β-adrenergic stimulation after
TAC was more severe in MRP4 KO compared to WT mice.
Conclusion: Taken together, our results show that both PDEs
and MRP4 are important in cAMP homeostasis. * These
authors contributed equally to the work
M.V. Singh: None. P.D. Swaminathan: None. B.J. He: None.
E.E. Luczak: None. W. Kutschke: None. M.E. Anderson: None.
P210Human Atrial Nonmyocytes Secrete Prohypertrophic,
High-Molecular-Weight FGF-2, Which Is Upregulated by
Angiotensin II via AT-1 and AT-2 Receptors, as Well as
ERK and MMP Activation
ABSTRACTS
Jon-Jon R Santiago, Leslie J McNaughton, Barbara E Nickel, Robert
R Fandrich, Rakesh C Arora, Elissavet Kardami, Inst of Cardiovascular
Sciences, Winnipeg, MB, Canada
Background: Very little is known about the expression and
role of fibroblast growth factor-2 (FGF-2) isoforms in the
human heart. Using the rat model we have documented
that high molecular weight Hi-FGF-2 rather than the
commonly studied 18 kDa low molecular weight isoform
Lo-FGF-2 is a potent inducer of cardiac hypertrophy in vitro
and in vivo; and that Hi-FGF-2 is expressed and secreted
predominantly by cardiac non-myocytes (fibroblasts). We
have now examined (i) the expression of Hi-FGF-2 in adult
human heart (atria) and heart-derived non-myocytes (HDNM),
and; (ii) signals regulating Hi-FGF-2 expression in HDNM.
Methods and Results: Atrial tissue, obtained from patients
undergoing cardiac surgery, (blinded study), was used to
obtain extracts, and to isolate migratory cells (fibroblastic,
HDNM). All tissue extracts (n=30) contained Hi- as well as
Lo-FGF-2, assessed by Western blotting. Amounts of total
FGF-2 varied from 1.5–25.5 pg per µg of extracted protein.
Immunohistochemistry of paraffin-embedded atrial tissue
sections and immunofluorescence of HDNM illustrated that
human Hi-FGF-2 is localized mainly in the nucleus but is also
present in cytoplasm. As was the case with rat- (ventricle and/
or atria) derived fibroblasts, HDNM expressed predominantly
Hi-FGF-2 (90% of total). The expression/secretion of Hi-FGF-2
by HDNM, as well as by human embryonic heart-derived
fibroblasts, was significantly up-regulated by angiotensin
II (Ang II). Simultaneous inhibition of both AT-1 as well as
AT-2 receptors (by losartan and PD123319, respectively)
was required to fully prevent Ang II-induced Hi-FGF-2
up-regulation. In addition, both inhibition of ERK activation
(by U0126), or MMP activity (by MMP-2 Inhibitor I) fully
prevented Ang II-induced up-regulation of human Hi-FGF-2.
Conclusions: We have shown for the first time that human
heart-derived fibroblastic cells express and secrete prohypertrophic Hi-FGF-2 in culture; and thus are likely to do so
in vivo. Our data also suggest that the beneficial effects of
drugs targeting Ang II signal transduction may be due, in part
to their effects on Hi-FGF-2 accumulation.
J.R. Santiago: None. L.J. McNaughton: None. B.E. Nickel: None.
R.R. Fandrich: None. R.C. Arora: None. E. Kardami: None.
P211MRP4: A Novel Protein Involved in Camp Homeostasis in
the Heart
Aniella Abi-Gerges*, Yassine Sassi*, INSERM UMR S 956, Univ Pierre
et Marie Curie, Paris, France; Nathalie Mougenot, Adeline Jacquet, Univ
Pierre et Marie Curie-Paris 6, INSERM IFR 14 PECMV, Paris, France;
Stefan Engelhardt, Inst fur Pharmakologie und Toxikologie, Technische
Univ Munchen, Munich, Germany; Stéphane N Hatem, Jean-Sébastien
Hulot, Anne-Marie Lompré, INSERM UMR S 956, Univ Pierre et Marie
Curie, Paris, France
Rationale: Transporters of the ABCC family, called MRP4 and
MRP5, have been described to control cAMP homeostasis
by extruding it from various cell types. Objective: The aim
of our study was to determine the role of MRP4 in cAMP
homeostasis and heart function and its impact on cardiac
hypertrophy (CH). Methods and Results: MRP4 KO mice
82
. Abi-Gerges*: None. Y. Sassi*: None. N. Mougenot: None.
A
A. Jacquet: None. S. Engelhardt: None. S.N. Hatem: None.
J. Hulot: None. A. Lompré: None.
P212Haploinsufficiency of Target of Rapamycin Attenuates
Different Forms of Cardiomyopathies in Adult Zebrafish
Xiaojing Sun, Yonghe Ding, Wei Huang, Tiffany Hoage, Margaret
Redfield, Sudhir Kushwaha, Mayo Clinic, Rochester, MN; Sridhar
Sivasubbu, Inst of Genomics and Integrative Biology, Delhi, India;
Xueying Lin, Stephen Ekker, Xiaolei Xu, Mayo Clinic, Rochester, MN
In contrast to the wide application of zebrafish embryos in
developmental genetic studies, the value of an adult zebrafish
to dissect signaling pathways in human diseases such as
cardiomyopathy remains largely elusive. Previously, we
have established anemic tr265/tr265 fish as the first adult
zebrafish model of cardiomyopathy. Here, we generate the
second adult zebrafish cardiomyopathy model induced by
injection of a single bolus of doxorubicin (DOX). Despite their
different pathogenesis, cardiac enlargement induced by either
anemia or DOX can be effectively attenuated by inhibition of
target of rapamycin (TOR) signaling via rapamycin treatment.
However, along the progression of both models, we have
also detected dynamic TOR activity and distinct effects of
TOR signaling inhibition at different stages of pathogenesis.
To assess the long term effects of TOR haploinsufficiency,
we utilized a zebrafish target of rapamycin (ztor) mutant that
was identified from a mutagenesis screen. We show that
sustained TOR inhibition in ztor/+ improved cardiac function,
prevented pathological remodeling events, and ultimately
reduced mortality in both adult fish models of cardiomyopathy.
Mechanistically, these cardioprotective effects are conveyed
by the anti-hypertrophy, anti-apoptosis, and pro-autophagy
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Poster Presentations (continued)
function of TOR signaling inhibition. Together, our results
prove adult zebrafish as a novel vertebrate model for human
cardiomyopathies and provide genetic evidences for a
cardioprotective function of TOR signaling inhibition in different
forms of cardiomyopathies.
of HKII (HKII+/-) resulted in exaggerated cardiac hypertrophy
after the induction of pressure overload by trans-aortic
constriction (TAC). HKII+/- mice demonstrated increased heart
size, interstitial fibrosis, and pulmonary edema compared
to wild-type (WT) 4 weeks after TAC. This was associated
with reduced cardiac function and increased mortality in
the HKII+/- mice by the 8-week time point, indicating an
accelerated transition to heart failure. In primary cultures of
neonatal rat cardiomyocytes (NRCM), siRNA knockdown of
HKII exacerbated the hypertrophic response to Angiotensin II
(AngII) compared to control siRNA. Mechanistically, reduction
of HKII resulted in increased ROS levels after hypertrophic
stimulation relative to controls. Treatment of NRCM with
the antioxidant N-acetylcysteine (NAC) attenuated the
hypertrophic response to AngII, and abrogated the increased
hypertrophy observed with HKII knockdown. In addition to its
enzymatic activity, HKII can bind the outer membrane of the
mitochondria, and this interaction increased with hypertrophic
agonists. Dissociation of HKII from the mitochondria using
a synthetic cell permeable peptide resulted in de novo
hypertrophy compared with scrambled peptide control,
and this was also attenuated with NAC treatment. Further
investigation into the source of ROS revealed that HKII
knockdown (+AngII) or mitochondrial dissociation resulted in
increased mitochondrial permeability pore (mPTP) opening
in the absence of cell death. Conclusions: Overall, the data
suggest that HKII and its binding to the mitochondria serve
as a negative regulator of hypertrophy by decreasing ROS
production, possibly through regulation of the mPTP.
. Sun: None. Y. Ding: None. W. Huang: None. T. Hoage: None.
X
M. Redfield: None. S. Kushwaha: None. S. Sivasubbu: None. X. Lin:
None. S. Ekker: None. X. Xu: None.
P213Epidermal Growth Factor Receptor and c-Src Are Involved
in Stretch-Induced Activation of JNK1/2 and P38 in
Cardiac Myocytes and Cardiac Fibroblasts
Hao Feng, Fnu Gerilechaogetu, Honey B Golden, Ricardo Cristalis,
Kumar Dasuri, Damir Nizamutdinov, Texas A&M Health Science Ctr,
Temple, TX; Donald M Foster, Central Texas Veterans Health Care
System, Temple, TX; Carl Tong, David E Dostal, Texas A&M Health
Science Ctr, Temple, TX
H. Feng: None. F. Gerilechaogetu: None. H.B. Golden: None.
R. Cristalis: None. K. Dasuri: None. D. Nizamutdinov: None.
D.M. Foster: None. C. Tong: None. D.E. Dostal: None.
P214Reduction of Hexokinase II Exaggerated Cardiac
Hypertrophy via Increased ROS Production and
Mitochondrial Permeability Transition
Eugene J Wyatt, Rongxue Wu, Kusum Chawla, Mohsen Ghanefar,
Hossein Ardehali, Northwestern Univ, Chicago, IL
Rationale/Objective: Cardiac hypertrophy is associated with
a metabolic switch in substrate utilization from fatty acids
to glucose. Levels of Hexokinase II (HKII), a key regulator of
glucose metabolism, increase in response to hypertrophic
stimulation and we hypothesized that a reduction in HKII
could attenuate the hypertrophic response. Methods and
Results: Contrary to our hypothesis, heterozygous knockout
E.J. Wyatt: None. R. Wu: None. K. Chawla: None. M. Ghanefar:
None. H. Ardehali: None.
This research has received full or partial funding support from the
American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa,
Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota,
South Dakota & Wisconsin).
P215β3-Adrenergic Receptor Mediated NOS Signaling in
Cardiomyocytes
Vabren L Watts, Xiaolin Niu, Karen L Miller, Lili A Barouch, Johns
Hopkins Univ Sch of Med, Baltimore, MD
Beta3 -adrenergic receptors play a pivotal role in modulating
cardiac function, though their precise role in the heart remains
controversial. We have recently demonstrated alterations in
Ca2+-dependent NOS isoforms and decreased NOS activity in
left ventricular tissue of beta3-/- mice after pressure overload.
However, the exact manner in which beta3-AR signaling
regulates these isoforms to stimulate NOS activity at the
cardiomyocyte level is not well understood. In this study
we used a specific beta3-AR agonist, BRL37344 (BRL), to
assess the role of beta3-AR in eNOS and nNOS regulation in
hypertrophied isolated neonatal rat ventricular cardiomyocytes
(NRVM). To induce hypertrophy we pretreated cells with
norepinephrine for 72 hours, which resulted in a 70% increase
in cell size and a 25% increase in beta3-AR mRNA expression
as compared with non-hypertrophied cells, analyzed by
immunocytochemistry and real-time PCR. In hypertrophied
cardiomyocytes, BRL administration lead to a time-dependent
5-fold increase in NOS activity, measured by the arginineto-citrulline conversion assay. beta3-activation also caused
a 1.5-fold increase in nNOS phosphorylation at positive
regulatory site Ser1416, and dephosphorylation of negative
regulatory site Ser847 as compared with unstimulated control.
NOS activity and nNOS phosphorylation overlapped in time. In
addition BRL induced phosphorylation eNOS-Ser114, which
indicates eNOS deactivation. Pretreatment with pertussis
toxin (PTX) suppressed BRL-induced nNOS-Ser1416
phosphorylation, nNOS-Ser847 dephosphorylation, and NOS
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
Mechanical stretch is a major determinant that leads to heart
failure. Heart failure is associated with a steady increase in
myocardial angiotensinogen (Ao) expression and the biological
peptide angiotensin II (Ang II) formation. We have previously
identified key downstream effectors that couple to mechanical
stretch-induced Ao gene expression. JNK1/2 and p38α
were found to have opposing roles on stretch-induced Ao
gene expression in neonatal rat cardiac myocytes (NRVM)
and cardiac fibroblasts (NRFB). JNK negatively regulated
Ao expression in NRVM and NRFB following acute stretch,
whereas with prolonged stretch, p38α was responsible for
upregulation of Ao expression. However, the mechanisms
leading to activation of these kinases and cross-talk between
these signal molecules remain to be determined. Epidermal
growth factor receptor (EGFR) is expressed in both NRVM
and NRFB, but its precise role in mechanosensing is poorly
understood. In NRVM, stretch caused JNK1/2 activation
(phosphorylation) within 2 min, with a peak response at 15
min (3.96 ± 0.71 fold, p<0.01). Stretch induced p38 activation
within 5 min, which remained elevated (2.93 ± 0.49 fold,
p<0.05) at 60 min (final time point). Pretreatment with EGFR
blockers AG1478 (100 nM) and BIBX 1382 (200 nM) inhibited
stretch-induced activation of JNK at early time points (5 and
15 min), whereas BIBX 1382 and the Ang II receptor type
I inhibitor losartan (10 µM) inhibited p38 activation at 60
min. Stretch induced activation of c-Src (phosphorylation
of tyrosine 416) at 60 min (1.56 ± 0.21 fold, p<0.01), which
was attenuated by losartan. In NRFB, stretch-induce-JNKphosphorylation can be inhibited by both AG1478 and Src
inhibitor PP2 (100 nM) at 15 min. Stretch also increased
phosphorylation levels of c-Src at tyrosine 416 and EGFR at
tyrosine 1068 at 15 and 60 min, which were blocked in the
presence of losartan. In a summary, these studies give strong
evidence that both EGFR and c-Src are involved in stretchinduced signal cascade in NRVM and NRFB.
83
Poster Presentations (continued)
activity, suggesting Gi/o dependency. Taken together, our
data suggest that BRL regulates NOS signaling in ventricular
cardiomyocytes via phosphorylation regulation of nNOS. To
our knowledge this is first study to demonstrate a role for
nNOS phosphorylation as a key factor in beta3-AR signaling.
These results contribute significantly to our understanding
the negative inotropic properties of myocardial beta3-AR at
cellular levels during cardiac sympathetic overstimulation, and
will ultimately aid in drug discoveries that target the molecular
mechanisms associated heart failure.
V.L. Watts: None. X. Niu: None. K.L. Miller: None. L.A. Barouch: None.
P216PRAS40 Is a Critical Regulator of Cardiomyocyte Growth
Mirko Volkers, Mathias Konstandin, Natalie Gude, Shabana Din, Mark
Sussman, San Diego State Univ, San Diego, CA
ABSTRACTS
Background: Proline-rich Akt substrate of 40 kDa (PRAS40) is
a component of the mammalian target of rapamycin complex
(mTORC) 1. Whereas, PRAS40 was originally characterized as
substrate for PKB/Akt, subsequent studies identified PRAS40
both as inhibitor and substrate of mTORC1. These studies
identify PRAS40 as a negative regulator of mTORC1 activity
and cell growth. Phosphorylation of PRAS40 by AKT, which
results in dissociation of PRAS40 from mTORC1, relieves
the inhibitory constraint on mTORC1. The physiological role
and pathophysiological function of PRAS40 in the heart is
unknown. Methods and Results: PRAS40 is expressed in
cardiomyocytes and highly phosphorylated in embryonic and
neonatal hearts. PRAS40 phosphorylation and expression is
decreased in adult cardiomyocytes. Pathological challenge
in mice induced phosphorylation of PRAS40 in the border
zone after myocardial infarction and globally after transaortic
constriction. Subcellular, phosphorylated PRAS40 is localized
in the cytoplasma after pathological challenge, whereas
stimulation with Insulin or IGF1 induced nuclear localization
of PRAS40. Adenoviral mediated overexpression of PRAS40
completely blocks pathological hypertrophy after stimulation
with phenylephrine as well as physiological growth after
stimulation with IGF1 or Insulin. Molecular, mTORC1 activity
was significantly reduced after PRAS40 overexpression.
Conversely, silencing of PRAS40 resulted in significant
increased mTORC1 activity. Gain and loss of function studies
of PRAS40 in vivo will be performed to test the therapeutically
potential of PRAS40. Conclusion: PRAS40 is a novel
regulator of cardiomyocyte growth. Manipulation the activity
or expression of PRAS40 could represent a novel therapeutic
target to treat cardiac diseases.
. Volkers: None. M. Konstandin: None. N. Gude: None. S. Din:
M
None. M. Sussman: None.
P217Nuclear Protein Import as the Missing Link in
Phenylephrine-Induced Cardiomyocyte Hypertrophy
Mirna N Chahine, Maxime Mioulane, Gabor Földes, Alexander Lyon,
Sian E Harding, Imperial Coll London, London, United Kingdom
During cardiac hypertrophy, cardiomyocytes (CM) present
alterations in gene expression and increased contractile
protein content. Nuclear protein import (NPI) is critical in
regulating gene expression, transcription, and subsequently
cell hypertrophy. However, it is unknown how the nuclear
transport machinery (transport receptors and nuclear pore
complex (NPC)) functions to sustain increased demands
for nucleocytoplasmic trafficking. The aim of this study
was to determine if exposure of adult CM to phenylephrine
(PE) affects hypertrophy by altering NPI and NPC density.
Comparisons were made to adult failing rat and human CM.
Rat myocytes were enzymatically isolated from adult hearts,
and used for immunocytochemistry, qPCR and western
immunoblotting. Failing CM were obtained from explanted
84
human hearts at the time of transplant and from a rat model
of myocardial infarction-induced hypertrophy and failure. Rat
adult CM exposed for 48h to PE were injected with a protein
import substrate (Alexa488-BSA-NLS) to visually monitor
nuclear import with the confocal microscope. The effects
of P38 MAPK inhibitor, HDAC inhibitor, Exportin-1 (CRM-1)
inhibitor, and GSK-3 β inhibitor were investigated. Cell and
nuclear sizes were increased in PE treated-adult rat CM and in
the adult failing rat and human CM compared to normal CM.
In contrast, PE depressed the rate and maximal NPI (by 65 +/3.4 % (3.55 from 5.46), p<0.05) as well as nucleoporin p62
mRNA and protein expression levels in adult rat CM compared
to non-treated CM. Nucleoporin p62, cytoplasmic Ranbp1,
and nuclear translocation of importins (Imp.α and β) relative
densities were also decreased in PE treated-adult rat CM
and in adult failing rat CM and human heart tissue compared
to normal controls. On the contrary, CRM-1 nuclear export
relative density was increased during the same pathological
conditions. Thus NPI downregulation is linked to an increased
nuclear export required by CM to generate the hypertrophic
phenotype. All these effects were P38MAPK, HDAC and
CRM-1 dependent but GSK-3Beta independent in rat CM. Our
results show that alterations in NPI and NPC density occur in
failing CM as well as in CM under hypertrophic stimuli. NPI may
represent a critical therapeutic target in hypertrophic conditions.
M.N. Chahine: None. M. Mioulane: None. G. Földes: None. A. Lyon:
None. S.E. Harding: None.
P218PKCε Regulates PI3K/mTOR Complex-2-Dependent AKT
Activation Independent of c-Raf/MEK/ERK Pathway
Phillip Moschella, John McKillop, Rebecca Harston, Dhandapani
Kuppuswamy, MUSC, Charleston, SC
Mammalian target of rapamycin (mTOR) has been shown to
influence hypertrophic growth of the myocardium through
two unique complexes, mTOR complexed with Raptor
(mTORC1) and mTOR complexed to Rictor (mTORC2)
with downstream activation of S6K1 and AKT respectively.
Previously, we characterized the role of novel PKC isoforms
(epsilon and delta) on mTORC1-dependent activation of
S6K1 downstream of both PI3K-dependent and independent
pathways in adult cardiomyocytes. PKC epsilon (PKCε) has
been implicated in AKT activation during ischemia-reperfusion
injury and has been co-immunoprecipitated in transgenic
PKCε overexpressing murine myocardium. In the present
study, we explored the role of PKCε on PI3K-dependent AKT
S473 phosphorylation. As such, insulin stimulated pAKT S473
was significantly reduced by pretreatment with wortmannin, a
selective PI3K inhibitor. However, insulin stimulated activation
of AKT was not reduced by pretreatment with either Gö6976
blockade of the classical PKC isoforms (PKC alpha/beta) or
U0126 blockade of MEK. Thus, insulin stimulated activation
of AKT is mediated primarily through a PI3K-dependent
pathway independent of the classical PKC isoforms. To begin
to evaluate the role of the novel PKCs, we utilized adenoviral
expression of dominant negative PKCε (dnPKCε). After 48
hours of expression of dnPKCε, insulin stimulated pAKT S473
was significantly reduced as compared to B-gal infection
controls. Next we identified for the first time in isolated
cardiomyocytes a direct link between PKCε and AKT through
immunoprecipitation of a unique signaling complex comprised
of PKCε bound to mTORC2 and pAKT (S473) only during
insulin stimulation. These data establish for the first time in
adult isolated cardiomyocytes: i) the necessity of PKCε during
insulin stimulated PI3K-dependent AKT activation and ii) a
signaling complex containing mTOR, Rictor, PKCε, and pAKT
that forms during insulin stimulation. Given the importance of
AKT in physiologic growth and survival, these studies indicate
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Poster Presentations (continued)
that PKCε plays a pivotal role during AKT activation and for
the first time displays a direct link between PKCε, pAKT and
mTORC2 in isolated cardiomyocytes.
P. Moschella: None. J. McKillop: None. R. Harston: None.
D. Kuppuswamy: None.
P219Role of Angiotensin Type I Receptor and β1 Integrin
in Stretch-Induced Activation of Akt and JNK in
Cardiac Myocytes
FNU Gerilechaogetu, Hao Feng, Honey B Golden, Ricardo J Cristales,
Kumar Dasuri, Damir Nizamutdinov, TAMHSC-CVRI, Temple, TX;
Donald M Foster, Central Texas Veterans Health Care System, Temple,
TX; Carl Tong, TAMHSC-SBTM, Temple, TX; David E Dostal, TAMHSCCVRI, Temple, TX
. Gerilechaogetu: None. H. Feng: None. H.B. Golden: None.
F
R.J. Cristales: None. K. Dasuri: None. D. Nizamutdinov: None.
D.M. Foster: None. C. Tong: None. D.E. Dostal: None.
P220Anthrax Receptor Regulation of FAK and JNK Activation
in Cardiac Myocytes
Damir Nizamutdinov, Honey B Golden, FNU Gerilechaogetu, Hao
Feng, Donald M Foster, Carl Tong, David E Dostal, Texas A&M Univ
HSC, Temple, TX
We previously demonstrated that anthrax lethal toxin (LT,
protective antigen [PA] + lethal factor [LF]) produces acute
systolic heart failure, characterized by increased left ventricular
(LV) end-diastolic volume, reduced ejection fraction and
decreased contractility in male Sprague-Dawley (SD) rats. The
D. Nizamutdinov: None. H.B. Golden: None. F. Gerilechaogetu: None.
H. Feng: None. D.M. Foster: None. C. Tong: None. D.E. Dostal: None.
P221Retinoblastoma Protein-Associated Proteins 48 and
46 Are Novel p300/GATA4-Binding Partners Involved in
Hypertrophic Responses in Cardiomyocytes
Yoichi Sunagawa, Graduate Sch of Med, Kyoto Univ, Kyoto,
Japan; Yasufumi Katanasaka, Taishi Terada, Yuichi Watanabe, Sch
of Pharmaceutical Sciences, Univ of Shizuoka, Shizuoka, Japan;
Hiromichi Wada, Kyoto Medical Ctr, Kyoto, Japan; Akira Shimatsu,
Clinical Res Inst, Kyoto Medical Ctr, Kyoto, Japan; Masatoshi Fujita,
Graduate Sch of Med, Kyoto Univ, Kyoto, Japan; Koji Hasegawa, Kyoto
Medical Ctr, Kyoto, Japan; Tatsuya Morimoto, Sch of Pharmaceutical
Sciences, Univ of Shizuoka, Shizuoka, Japan
Background: A zinc finger protein GATA4 is one of
hypertrophy-responsive transcription factors, and increases
its DNA-binding and transcriptional activities in response to
hypertrophic stimuli in cardiomyocytes. Activation of GATA4
during this process is mediated, in part, through acetylation
by intrinsic histone acetyltransferases such as a transcriptional
coactivator p300. Here, we show that retinoblastoma protein
(Rb)-associated protein 48 and 46 (RbAp48, RbAp46),
components of NuRD (nucleosome remodeling and
deacetylase) complex that has been implicated in chromatin
remodeling and transcriptional repression associated with
histone deacetylation, are novel components of p300/GATA4
complex. However, the precise functional relationships among
p300, GATA4, RbAp48, and RbAp46 remain unknown.
Methods and Results: A series of GST pull-down assays
revealed that the C-terminal domain of RbAp48/46 bound
to the N-terminal transcriptional activation domain of GATA4
and C/H-3 domain of p300, respectively. Immunoprecipitation
followed by western blotting demonstrated that RbAp48/46
repressed p300-induced acetylation of GATA4 and histones.
While overexpressions of RbAp48/46 inhibited p300/GATA4induced atrial natriuretic factors (ANF) and endotheline-1 (ET-1)
promoter activities, knockdown of neither RbAp48 nor RbAp46
by RNAi enhanced these promoter activities in HEK293
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ABSTRACTS
Cardiac hypertrophy is a common response to mechanical
stimuli, such as increased hemodynamic load and exercise.
However, the underlying mechanotransduction processes
are poorly understood. Recent studies indicate that both β1
integrin and angiotensin type I receptor (AT1R) can serve as
mechanoreceptors in the intact myocardium and isolated
cardiac myocytes. Mechanical stress induced activation of
Akt and JNK has been associated with various aspects of
hypertrophic growth. We used primary cultures of NRVM
plated on deformable membranes to test whether static
stretch-induced activation (phosphorylation) of Akt and
JNK involves β1 integrin and/or AT1R. In these studies,
pretreatment with the AT1R antagonist Losartan (10 µM),
significantly decreased mechanical stretch induced activation
of Akt (Akt473, 47.1 ± 6.1%, p<0.01 and Akt308, 33.1 ± 3.2%,
p<0.01, n=5), but not JNK after 15 min, compared to stretch
alone. Although expression of dominant-negative β1D-integrin
(Tac-β1D) alone had no effect on stretch-induced Akt and JNK
activation, the addition of Losartan to the culture medium
decreased stretch-induced activation of both Akt (Akt473, 74.9
± 10.4%, p<0.01 and Akt308, 66.1 ± 8%, p<0.01, n=5) and
JNK (60.1 ± 2.5%, p<0.01, n=5), compared to stretch in lacZ
expressing cells. Because Rac1 is known to be activated
by mechanical stretch in NRVM, we determined whether
Rac1 is required for stretch-induced Akt and JNK activation.
Adenovirus-mediated expression of dominant-negative Rac1
decreased mechanical stretch induced activation of Akt
(Akt473, 36.3 ± 7.6%, p<0.05 and Akt308, 29.8 ± 6.2%, p<0.05,
n=5), but not JNK. Whereas, in NRVM expressing dominantnegative Rac1, Losartan pretreatment further decreased
stretch-induced activation of both Akt (Akt473, 64.9 ± 10.4%,
p<0.05 and Akt308, 50.8 ± 7.8%, p<0.01, n=5) and JNK (66.7
± 11%, p<0.05, n=5). In summary, Akt activation in acutely
stretched NRVM was mediated through the AT1R, whereas
JNK activation could be mediated through either AT1R or β1
integrin. The regulation Akt and JNK was also found to be
significantly dependent on Rac1 activation. In conclusion, this
study suggests that both AT1R and β1 integrin are important
mechanosensors that activate pathways that lead to cardiac
myocyte hypertrophy.
molecular basis for the cardiac dysfunction observed in either
an animal model or clinical setting of anthrax toxicity remains
to be examined. Previously, we tested LT dose-response
effects on isolated neonatal rat ventricular myocytes (NRVM)
at a dose of 0.05 ng/mL PA + 0.025 ng/mL LF diminished
PLB and JNK phosphorylation from 1h to 4 h. During the
LT+PA time-course, Akt phosphorylation at both Thr308 and
Ser473 were also significantly reduced at all time points from
30 min to 4 h, accompanied by elevated B56α total protein
and intracellular Ca2+, and reduced PLB phosphorylation.
This response is mediated by translocation of LF into the cell
through the anthrax receptors 1 and 2 (ATR1/2), which are
structurally similar to integrins. In this study, we tested whether
stimulation of ATR1/2 by PA alone can induce activation of
the integrin effectors, FAK and JNK. Cardiac myocytes were
treated time dependently (5 sec–30 min) by PA at 0.25 ng/
mL, and activation of FAK protein at different residues was
determined. PA induced phosphorylation of FAK-Tyr861 at
15 sec (p<0.05), whereas there was no effect on FAK-Tyr397
or FAK-Tyr925 phosphorylation for any of the time points.
Furthermore, we found that PA alone maximally increased JNK
phosphorylation at 15 min of treatment (p<0.05). This response
was not blocked with either Losartan (10 µM) or AG1479 (100
nM) indicating that neither angiotensin type I receptor (AT1)
or epidermal growth factor receptor (EGFR) transactivation
was involved. In summary, this study suggests a key role of
ATR1/2 in mediating of signal transduction across membrane
of cardiac myocytes in response to B. anthracis PA stimulation
and involvement in acute responses in regulation of FAK and
JNK activation in independent of AT1 and EGFR pathway.
85
Poster Presentations (continued)
cells. Stimulation of cardiomyocytes with phenylephrine (PE)
decreased the binding of GATA4/p300 with RbAp48/46.
RbAp48/46 repressed PE-induced hypertrophic responses
such as myofibrillar organization, increase in cell size and
promoter activation of the ANF and ET-1 in cardiomyocytes.
Conclusion: These findings demonstrate that RbAp48 and
RbAp46 form a functional protein complex with GATA4/p300
and regulated hypertrophic responses in cardiomyocytes.
Y. Sunagawa: None. Y. Katanasaka: None. T. Terada: None.
Y. Watanabe: None. H. Wada: None. A. Shimatsu: None. M. Fujita:
None. K. Hasegawa: None. T. Morimoto: None.
P222T-Type Calcium Channels Regulate NFAT Signaling in
Cardiomyocytes and Are Expressed in Chronic Hypoxic
Right Ventricle
ABSTRACTS
Leanne L Cribbs, Florentina Pluteanu, Loyola Univ Chicago,
Maywood, IL
Alterations in intracellular calcium (Ca2+) have a significant
impact on Ca2+ signaling, an integral part of the cardiac
response to pathological conditions. The calcineurin/NFAT
signaling pathway is a well established Ca2+-dependent
mechanism for transcriptional regulation of ventricular
remodeling target genes. Although low voltage-activated
T-type Ca2+ channels are not normally present in the adult
myocardium, they reappear in conditions of hypertrophy and
heart failure, where their functions remain poorly understood.
To further investigate T-type Ca2+ channels, we have employed
cultured neonatal rat ventricular myocytes (NRVM) and rat
models of pulmonary hypertension that lead to right ventricular
(RV) hypertrophy and heart failure. To assay for NFAT activation
in cardiomyocytes, NRVM were infected with NFATc1-GFP and
nuclear localization of GFP was measured. NFAT activation
was markedly stimulated by over expressing the human Cav3.2
T-type Ca2+ channel cDNA in NRVM, and this activation was
inhibited by cyclosporin A (a calcineurin inhibitor), by T-type
selective concentrations of mibefradil and NiCl2, and in the
absence of extracellular Ca2+. To investigate the up regulation
of cardiac T-type Ca2+ channels in vivo, rats were maintained
in chronic hypoxia, where they developed significantly elevated
RV systolic pressure, RV hypertrophy and reduced cardiac
output. Electrophysiological measurements revealed elevated
membrane capacitance in RV myocytes indicative of RV
hypertrophy, and T-type current density was also increased.
Myocytes isolated from the RV of pulmonary hypertensive rats
that displayed elevated T-type currents and RV dysfunction,
also showed impaired contractility with decreased sensitivity
to Ca2+. The results in NRVM suggest that T-type channels
contribute to hypertrophic signaling via their interaction with
calcineurin/NFAT signaling intermediaries. Furthermore,
T-type channels are expressed in the RV during pulmonary
hypertension where they influence RV myocyte contractility.
Thus, when expressed in pathological conditions, T-type Ca2+
channels may play a dual role as a signaling intermediary, as
well as a modifier of ventricular contractile function.
L.L. Cribbs: None. F. Pluteanu: None.
P223Not published at presenter’s request.
P224Connective Tissue Growth Factor Plays a Key Role in
Myocardial Fibrosis After Angiotensin II Exposure
Nicole L Rosin, Jean-François Légaré, Timothy D Lee, Dalhousie Univ,
Halifax, NS, Canada
Background: AngiotensinII (AngII) exposure to rodents is a
common model of fibrosis, characterized by hypertension,
hypertrophy and eventual deposition of excess extracellular
matrix (ECM) proteins resulting in organ dysfunction. We
have previously shown that cellular infiltration of bone marrow
derived progenitor cells (fibrocytes) occurs prior to ECM
deposition and is associated with production of the connective
86
tissue growth factor (CTGF) and transforming growth factor
beta (TGF-β). The objective of this project was to characterize
the role of CTGF in promoting fibrocyte recruitment and
subsequent fibrosis after AngII exposure. Methods: Mice
were treated with AngII or saline using an osmotic minipump at 2.8mg/kg/day. After 6hr to 7d hearts were excised
and embedded in paraffin or prepared for mRNA isolation.
Immunohistochemistry was used to determine extent and
localization of CTGF protein. Quantitative RT-PCR was used
to determine relative CTGF and TGF-β mRNA levels. Primary
cardiomyocyte and fibrocyte cultures were isolated from
neonatal or 3d AngII exposed animals respectively. Primary
cultures were stimulated to determine the source (qRT-PCR)
and function (proliferation, migration and differentiation) of
CTGF. Results: In animals exposed to AngII, CTGF mRNA
peaked the earliest at 6hr (21-fold; p<0.01) when compared
to TGFbeta, which peaked at 3d (5-fold; p<0.05). Concurrent
CTGF protein expression was evident by 3d of AngII exposure
and appeared localized to the cardiomyocytes. Findings were
confirmed using isolated cardiomyocytes, which significantly
increased expression of CTGF in response to AngII (2-fold;
p<0.05). While CTGF did not promote fibrocyte migration
in transwell chamber assay it promoted significant fibrocyte
proliferation in vitro (2-fold; p<0.05). Conclusion: We provide
strong evidence that AngII exposure first results in the
production of CTGF by cardiomyocytes. Furthermore, we have
shown that CTGF does not promote migration as a chemokine
but instead contributes to proliferation of fibrocytes once
recruited from the bone marrow into the myocardium.
N.L. Rosin: None. J. Légaré: None. T.D.G. Lee: None.
P225Possible Involvement of Homer-1b/c in Gq-Mediated
Hypertrophy in Cardiomyocytes
David R Grubb, Jieting Luo, Peter Iliades, Elizabeth A Woodcock,
Baker IDI Heart and Diabetes Res Inst, Melbourne, Australia
Receptor activation of Gq causes hypertrophy in
cardiomyocytes, via the activation of phospholipase Cβ 1b
(PLCβ1b). PLCβ1b, localizes to the cardiac sarcolemma
through an interaction with the multi-domain scaffolding
molecule Shank-3 (SH3 and multiple ankyrin repeat domains
protein 3; Grubb et al., 2011), which is required for PLC
activation and for hypertrophic responses. In the CNS,
Shank-3 forms higher order oligomeric complexes with three
isoforms of Homer protein homolog 1 (Homer-1), Homer1a, Homer-1b and Homer-1c. Homer-1b and Homer-1c
link G-protein coupled receptors, ionotropic receptors,
canonical transient receptor potential channel (TrpC) and
intracellular calcium store regulators into a signaling complex.
Homer-1a acts as a natural dominant negative, in dynamic
competition with Homer-1b and Homer-1c. Neonatal rat
ventricular myocytes (NRVM) infected with adenovirus
expressing either Gαq(Q209L) (constitutively active Gαq),
or its immediate down-stream effector, PLCβ1b, increased
Homer-1b/c transcription. Incubation with phenylephrine/
propranalol (α1-adrenergic agonist, PE/Pro) also increased
Homer-1b/c, but not Homer-1a, mRNA. All treatments caused
cardiomyocyte hypertrophy. There was no comparable
increase in Homer-1b/c mRNA in NRVM expressing PLCβ1a
(inactive splice variant) or incubated with fetal calf serum
to induce hypertrophy by Gq-independent mechanisms.
Homer-1b/c protein induced by PLCβ1b, Gαq or PE/
Pro was primarily localized close to the sarcolemma along
with Shank3, PLCβ1b and TrpC4. We conclude that Gαq/
PLCβ1b-mediated signaling leads to the up-regulation of
Homer-1b/c, that co-localizes with a signaling complex close
to the sacrolemma. Induction of Homer-1b/c may be critical in
facilitating localized Ca2+ signaling and thereby promoting Gq
dependent hypertrophy.
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Poster Presentations (continued)
improved cardiac function (25% increase in cardiac power).
In a separate group of animals nearly identical findings were
obtained when metformin (300 mg/kg/day) was administered
in vivo for one week. The functional improvement was also
observed when ER stress was directly relieved with sodium
4-phenylbutyrate, as well. Conclusion: Our results implicate a
critical role for glucose metabolism and AMPK in load-induced
mTOR activation. Metformin improves contractile function
in rat hearts subjected to increased workload by relieving
ER stress. The relief of ER stress by metformin suggests
previously unrecognized beneficial effects of AMPK activation
in the hemodynamically stressed heart.
D.R. Grubb: None. J. Luo: None. P. Iliades: None. E.A. Woodcock: None.
P226Small Heterodimer Partner Negatively Regulates Cardiac
Hypertrophy Through Upregulation of GATA6
Gwang Hyeon Eom, Hosouk Joung, Yoojung Kim, Hae Jin Kee, Sera
Shin, Chonnam Natl Univ Medical Sch, Gwangju, Korea, Republic of;
Don-Kyu Kim, Chonnam Natl Univ Hormone Res Ctr, Gwangju, Korea,
Republic of; Nakwon Choe, Duk-hwa Kwon, Kwang-Il Nam, Chonnam
Natl Univ Medical Sch, Gwangju, Korea, Republic of; Hueng-Sik Choi,
Chonnam Natl Univ Hormone Res Ctr, Gwangju, Korea, Republic of;.
Hyun Kook, Chonnam Natl Univ Medical Sch, Gwangju, Korea,
Republic of
Small heterodimer partner (SHP; NR0B2) is an atypical orphan
nuclear receptor that regulates a variety of cellular events such
as cell proliferation, differentiation and metabolism in liver and
bone. However, the role of SHP in heart has not yet been
elucidated. In this study, we investigated the functional roles
of SHP in cardiac hypertrophy. In rat neonatal cardiomyocytes
model, phenylephrine (PE) down-regulated expression of SHP.
Transient transfection of SHP decreased the promoter activity
of Nppa (natriuretic polypeptide precursor type A). Adenovirusmediated overexpression of SHP (Ad-SHP) blocked gene
expressions of GATA4, GATA6, and serum response factor
(SRF). The increase in [3H]-leucine incorporation induced by
PE or fetal bovine serum (FBS) was dramatically reduced by
Ad-SHP. Likewise, increases in cell size with those hypertrophic
stresses were significantly attenuated by Ad-SHP. The
expressions of atrial natriuretic factor (ANF), β-myosin heavy
chain (βMHC), and skeletal α-actin were significantly higher in
hearts of SHP null mice. SHP physically interacted with GATA6
in mammalian cells. SHP significantly decreased the activation
of -3003 Nppa promoter induced by GATA6. The action of
SHP on Nppa promoter activity was partially recovered by
GATA6. Taken together, these results suggest that SHP works
as a novel anti-hypertrophic regulator by repressing GATA6.
G. Eom: None. H. Joung: None. Y. Kim: None. H. Kee: None. S. Shin:
None. D. Kim: None. N. Choe: None. D. Kwon: None. K. Nam: None.
H. Choi: None. H. Kook: None.
P227Metformin Inhibits Glucose-Induced mTOR Activation and
ER Stress in Rodent Heart
Shiraj Sen, Patrick Guthrie, Heinrich Taegtmeyer, The Univ of Texas
Health Science Ctr at Houston, Houston, TX
P228MicroRNA Expression in Hypertension and Left Ventricular
Hypertrophy in Rat
David Kain, Neufeld Cardiac Res Inst, Tel Hashomer, Israel; Chana
Yagil, Lab for Molecular Med and Israeli Rat Genome Ctr, Ashkelon,
Israel; Nathalie Landa-Rouben, Neufeld Cardiac Res Inst, Tel
Hashomer, Israel; Jasmine Jacob-Hirsch, Chen Dor, Sheba Cancer
Res Ctr, Tel Hashomer, Israel; Yoram Yagil, Lab for Molecular Med
and Israeli Rat Genome Ctr, Ashkelon, Israel; Jonathan Leor, Neufeld
Cardiac Res Inst, Tel Hashomer, Israel
Background: Heart injury and stress can cause a hypertrophic
growth response in cardiac myocytes, which is characterized
by an increase in cell size, enhanced protein synthesis, and
activation of fetal genes, which can lead to heart failure (HF)
and death. Given the emerging roles of microRNAs (miRNAs)
in modulation of cellular phenotypes, we aimed to investigate
miRNAs that are regulated during hypertension, left ventricular
hypertrophy (LVH) and HF by using a rat model. Methods and
Results: Male salt-sensitive (SBH/y) and salt-resistant (SBN/y)
Sabra rats were obtained from the Israeli Rat Genome Center.
Animals were salt-loaded with deoxycorticosterone-acetate
(75 mg DOCA pellet s.c.) and 1% NaCl in drinking water for
16 weeks. Systolic blood pressure was determined by the
tail-cuff method. Cardiac images were obtained using cardiac
MRI using a specific small animal imaging protocol. Rats
were sacrificed at different time points. miRNA expression
was studied in the left ventricle using GeneChip miRNA
Array (affymetrix). Expression of miRNAs related to cardiac
hypertrophy or cell proliferation, including miR-132, miR-212,
miR-29, miR-31and miR-205, increased more than 1.5 fold
in salt-loaded SBH/y compared with salt-loaded SBN/y. In
contrast, expression of MiR-182, miR-17-3p and miR-130b,
known to be down-regulated in heart disease, diminished
more than -1.5 fold in salt-loaded SBH/y compared with saltloaded SBN/y. Conclusions: In animal model of salt-sensitive
hypertension and LVH, up- and down-regulation of distinct
miRNAs in the heart suggest novel therapeutic targets to
prevent and reverse LVH and HF.
D. Kain: None. C. Yagil: None. N. Landa-Rouben: None.
J. Jacob-Hirsch: None. C. Dor: None. Y. Yagil: None. J. Leor: None.
ABSTRACTS
Background: When subjected to increased workload,
the heart remodels both metabolically and structurally by
increasing glucose metabolism and activating the mTOR
signaling pathway. Specifically, glucose activates mTOR in
a TSC2-dependent manner. We now examined whether
metformin regulation of AMPK, a major upstream regulator
of TSC2, modulates mTOR activation and endoplasmic
reticulum (ER) stress when hearts are subjected to increased
workload. Methods: Working hearts from 12 week old male
Sprague Dawley rats were perfused ex vivo (5 mM glucose)
at physiologic pre- and afterload. At 30 minutes, the workload
was doubled. Hearts were freeze-clamped at 60 minutes.
Hearts received either the AMPK activator metformin (10 mM)
or the ER stress relieving agent sodium 4-phenylbutyrate
(10 mM) directly in the perfusate. Rates of glucose uptake
and oxidation were measured with radiolabeled substrate
(3H, 14C). Cardiac power, the mTOR signaling pathway, and
markers of ER stress were assessed. Results: With increased
workload rates of glucose uptake and oxidation doubled, with
rates of glucose uptake exceeding those of glucose oxidation.
Neither metformin nor sodium 4-phenylbutyrate significantly
changed those rates. In control hearts, there was a
downregulation of AMPK, an activation of mTOR, an induction
of ER stress, and a decline in contractile function. Perfusion
with media containing metformin activated AMPK, prevented
glucose-mediated mTOR activation, alleviated ER stress and
S. Sen: None. P. Guthrie: None. H. Taegtmeyer: None.
P229Nongenomic Cardioprotective Effect of
Dehydroepiandrosterone on Cardiac Hypertrophy:
Comparison with 17β-Estradiol-Induced Cardioprotection
Hideaki Tagashira, Md Shenuarin Bhuiyan, Norifumi Shioda, Kohji
Fukunaga, Graduate Sch of Pharmaceutical Sciences, Tohoku Univ,
Sendai, Japan
bjective: We recently reported a decreased σ1 receptor
O
expression in heart following abdominal aortic stenosis
in bilateral ovariectomized (OVX) rats. Here, we
demonstrated non-genemic cardioprotective effects of
dehydroepiandrosterone (DHEA) though σ1 receptor in
pressure overload (PO)-induced cardiac dysfunction.
Methods: Bilateral ovariectomy was performed in female
rats. Two weeks after the sham operation or ovariectomy,
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
87
Poster Presentations (continued)
pressure-overload was initiated by abdominal aortic
banding. 17β-estradiol (E2: 0.1 mg/kg) and DHEA (30 mg/
kg) were administered to rats subcutaneously and orally,
respectively, for 14 days starting 2 weeks after aortic banding.
Hemodynamic parameters and cardiac hypertrophy were
measured after 2 weeks of drug treatment. After dissection
out of left ventricular, gene expression and hypertrophic
signaling were analyzed by real-time RT-PCR and western
blot, respectively. Results: Both E2 and DHEA treatments
significantly inhibited pressure overload-induced increases
both in heart weight/body weight (HW/BW) ratio and
lung weight/body weight (LW/BW) ratios. Both E2 and
DHEA treatments also ameliorated hypertrophy-induced
impairment of left ventricular end diastolic pressure (LVEDP),
left ventricular developed pressure (LVDP), left ventricular
contraction and relaxation (±dp/dt) rates, heart rate (HR)
and mean arterial blood pressure (MABP). Notably, DHEA
but not E2 administration rescued PO-induced σ1 receptor
downregulation in left ventricular. Co-administration with
NE-100, a σ1 receptor selective antagonist, inhibited
DHEA-induced amelioration of heart dysfunction without
effects on E2-induced cardioprotection. Mechanistically, both
E2 and DHEA treatments significantly restored PO-induced
decreases in Akt phosphorylation and Akt-mediated eNOS
phosphorylation (Ser1179). NE-100 treatment totally abolished
DHEA-induced Akt and eNOS phosphorylation without effects
on E2-induced Akt/eNOS activation. Conclusions: Taken
together, based on these results with OVX rat heart, DHEA
but not E2 elicits cardioprotective action through σ1 receptor
activation. DHEA-induced Akt/eNOS activation through σ1
receptors likely mediates the cardioprotective activity.
H. Tagashira: None. M. Bhuiyan: None. N. Shioda: None.
K. Fukunaga: None.
P230Orchestration of Cardiac Mitochondrial Biogenesis
by Mitofusin2
ABSTRACTS
Yun Chen, Scot Matkovich, Yan Zhang, Yingqiu Liu, Gerald Dorn II,
Washington Univ in St Louis, St Louis, MO
88
Mitochondria generate energy and play central roles governing
cell signaling and programmed death. As an energy dependant
tissue, the heart is more affected by mitochondrial dysfunction
than other tissues. Mitochondrial fusion and fission are
essential for normal morphology and function. Mitofusins Mfn1
and Mfn2 are the key components to process mitochondrial
fusion. Previous data shows that Mfn1 and Mfn2 are
functional redundant in skeletal muscle and the drosophila
heart tube. But the role for the mitochondrial fusion proteins
in mammalian cardiac myocytes is unknown. The functional
role of mitofusins in the mammalian heart was investigated
by conditional gene deletion of Mfn1 and Mfn2 in mice. Mice
with myh6Cre-directed inactivation of Mfn1 exhibited normal
cardiac structure and function. In contrast, ablation of Mfn2 in
mouse heart caused progressive dilated cardiomyopathy. At
6 weeks old, the cardiac structure and function were normal,
but at 16 weeks old, Mfn2 null mice developed severe cardiac
dilation and impaired contractile performance at baseline and
in response to β-adrenergic stimulation without induction of
programmed cell death. Cardiomyocytes from young Mfn2
null mice had normal excitation-contraction coupling, whereas
older cardiomyocytes showed decreased fractional shortening
with normal calcium cycling. mRNA resequencing of Mfn2
null hearts revealed that cardiac dysfunction was associated
with induction of nuclear-encoded mitochondrial genes. Mfn2
deficient cardiac mitochondria displayed normal function
of the mitochondrial permeability transition, respiration and
expression of respiration chain complexes, but increased
mitochondrial oxidant stress. Remarkably, the development
of cardiomyopathy paralleled dramatic (60–80%) loss of
mitochondria, measured both as the ratio of mitochondrial
DNA to nuclear DNA, and as μg protein/mg cardiac protein.
Conclusion: 1. Mfn1 and Mfn2 are not redundant in the heart.
Mfn1 is dispensable for normal cardiac development and
function. 2. Mfn2 deficiency reacts in a dilated cariomyopathy
caused not by a failure of mitochondrial function, but by a
crisis of mitochondrial biogenesis.
. Chen: None. S. Matkovich: None. Y. Zhang: None. Y. Liu: None.
Y
G. Dorn: None.
P231Dispensability of Dmitofusin in Calcium Signaling in Intact
Drosophila Heart Tubes
Casey C Jowdy, Washington Univ in St Louis, St Louis, MO
Sarcoplasmic Reticulum (SR) drives cardiomyocyte excitationcontraction coupling through the release and storage of
Ca2+. Phasic fluctuations of Ca2+ require ATP. Mitochondria
occupy ~30% of the volume of cardiomyocytes and provide
cardiomyocytes with ATP. Perturbations of mitochondria
therefore have a detrimental effect on the heart. Mitochondrial
homeostasis in mammals is regulated in part through fusion
and fission by outer mitochondrial membrane proteins
mitofusin 1 and 2 (Mfn1 and Mfn2) and in Drosophila by the
homolog dMfn. Mutations in Mfn2 show impaired contractile
function in humans (hMfn), mice (mMfn) and RNAi suppression
in Drosophila (dMfn). Additionally Mfn2, independently of Mfn1,
tethers the SR to mitochondria allowing for efficient transfer
of Ca2+ between the organelles. It is currently not known
whether mitochondria-SR tethering is required for proper Ca2+
cycling in cardiomyocytes. We monitored calcium transients
in heart tubes of live flies, using the genetically encoded
calcium indicator (GECI) GCaMP3.0 to study dynamic Ca2+
oscillations. Intensity signals from GCaMP3.0 in fly heart
tubes are comparable to isolated cardiomyocytes in their
rate cycling amplitude, and their response to L-type calcium
channel antagonist, nifedipine. Additionally, non-calcium
responsive GFP does not produce a signal that mimics the
Ca2+ oscillations observed with GCaMP3.0. Comparisons
between Wt Drosophila hearts and cardiomyopathic RNAi
dMfn hearts expressing GCaMP3.0 have normal amplitude
and time to decay constant Ca2+ signals. This study shows
that suppression of dMfn with RNAi in the fly heart tube leads
to cardiomyopathy. Monitoring intercellular Ca2+ levels with
the (GECI) GCaMP3.0 demonstrates that Ca2+ signaling is not
affected when mitochondria-SR tethering is disrupted.
C.C. Jowdy: None.
P232Cardiac Excitation Contraction Coupling Stimulates
Mitochondrial Respiration and Superoxide Flash Activity
Wang Wang, Xiaoyun Liu, Guohua Gong, Univ of Washington,
Seattle, WA
Normal cardiac excitation-contraction (EC) coupling relies
on highly coordinated intracellular calcium (Ca2+) handling
and adequate mitochondrial energy metabolism. Whether
mitochondria can sense the oscillating Ca2+ signals during
EC coupling to adjust its respiration status and other functions
is largely unknown. Recently, we discovered stochastic
and transient mitochondrial superoxide production events,
named superoxide flashes, in resting cardiac myocytes.
Superoxide flashes are coupled with mitochondrial respiration
and triggered by physiological relevant transient permeability
transition pore (mPTP) openings. Using superoxide flash as
the optical determination of mitochondrial respiration status
in living myocytes, we found that EC coupling promotes
mitochondrial respiration and reactive oxygen species (ROS)
production as evidenced by the significantly increased
superoxide flash activity by electrical stimulation (pacing
frequency 0.5–2 Hz). Pre-incubation with Ru360 (50–100 uM),
a mitochondrial Ca2+ uniporter inhibitor, or cyclosporine A (1
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Poster Presentations (continued)
uM), an mPTP blocker, prevented this increase. Simultaneous
monitoring of mitochondrial Ca2+ (by rhod-2) and superoxide
(by mt-cpYFP, a mitochondrial targeted superoxide indicator)
revealed step-wise accumulation of mitochondrial Ca2+ during
cytosolic Ca2+ transients in individual mitochondria, which
is followed by superoxide flash production and matrix Ca2+
release. Importantly, mitochondrial respiration only responds to
transient Ca2+ signals (acute addition of ionophore or pacing)
but not chronic manipulations of steady state Ca2+ (depleting
or elevating resting cytosolic Ca2+). Through spatiotemporal
mapping of superoxide flash and local sarcoplasmic reticulum
(SR) Ca2+ releasing events, Ca2+ sparks, we found that
significant amount of Ca2+ sparks clustered near the
flashing mitochondria. Antioxidant pretreatment attenuated
the superoxide flash associated Ca2+ sparks. In summary,
mitochondrial Ca2+ uptake during cardiac EC coupling leads
to stochastic acceleration of mitochondrial respiration, mPTP
activity and free radical production, which in turn modulate
local Ca2+ signaling through promoting SR Ca2+ release.
W. Wang: None. X. Liu: None. G. Gong: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P233PPARα-Sirt1 Complex Mediates Metabolic Adaptation in
Response to Starvation in the Heart
Shinichi Oka, Peiyong Zhai, Takanobu Yamamoto, Dan Shao, Junichi
Sadoshima, UMDNJ, Newark, NJ
S. Oka: None. P. Zhai: None. T. Yamamoto: None. D. Shao: None.
J. Sadoshima: None.
Tiffany T Nguyen, Systems Biology Ctr, Natl Insts of Health, Bethesda,
MD; Mark V Stevens, Ctr for Molecular Med, Natl Insts of Health,
Bethesda, MD; Mark J Kohr, Charles Steenbergen, Johns Hopkins
Univ, Baltimore, MD; Michael N Sack, Ctr for Molecular Med, Natl Insts
of Health, Bethesda, MD; Elizabeth Murphy, Systems Biology Ctr, Natl
Insts of Health, Bethesda, MD
S-nitrosylation (SNO), a reversible, redox-dependent posttranslational modification, has emerged as an important
mechanism for dynamic regulation of many proteins. Our
previous studies have shown that protein S-nitrosylation (SNO)
plays a protective role in myocardial ischemia/reperfusion
(IR) injury. The primary mediator of cell death in I/R injury is
activation of the mitochondrial permeability transition pore
(mPTP). Using a proteomic approach, we have previously
found that cyclophilin D (CypD), a critical mPTP regulator,
can be SNO on cysteine 203 (C203). To investigate whether
SNO of CypD might attenuate mPTP activation, we mutated
cysteine 203 of CypD, to a serine residue (C203S) and
determined its effects on mPTP opening by assessing H2O2induced mPTP opening using the calcein AM-cobalt chloride
quenching method. Treatment of CypD-/- mouse embryonic
fibroblasts (MEFs) with H2O2 resulted loss in an ≈50 % loss
of mPTP opening as compared to WT MEFs (n=5, p<0.05),
consistent with the protective role of CypD in mPTP activation.
Addition of a nitric oxide donor, GSNO, to CypD-/- MEFs did
not further reduce mPTP opening; however, WT MEFs treated
GSNO attenuated mPTP opening by half. To elucidate the
role of SNO of C203 on CypD, we infected CypD-/- MEFs
with a C203S-CypD vector. C203S-CypD re-constituted
MEFs were also resistant to mPTP opening in the presence
or absence of GSNO. This suggests that C203 is required
for mPTP activation. To determine whether in vivo expression
of C203S-CypD would alter mPTP opening, we generated
adenovirus vectors encoding WT CypD or mutated C203SCypD and injected these viral particles into CypD-/- mice via
tail-vein. Mitochondria isolated from livers of CypD-/- mice or
mice expressing C203S-CypD were resistant to Ca2+-induced
swelling as compared to WT CypD reconstituted mice. In
summary, our results indicate that C203 of CypD is required for
mPTP opening and for the first time shows that SNO of C203
on CypD acts to attenuate mPTP activation.
T.T. Nguyen: None. M.V. Stevens: None. M.J. Kohr: None.
C. Steenbergen: None. M.N. Sack: None. E. Murphy: None.
P235Endothelium-Specific Prokineticin Receptor 1
Inactivation in Mice Leads to Endothelial Dysfunction
and Metabolic Syndrome
Canan G Nebigil, UMR 7242, CNRS, Univ of Strasbourg, ESBS,
Illkirch, France
Reciprocal relationships between endothelial dysfunction and
insulin resistance result in a vicious cycle, linking between
cardiovascular and metabolic disorders. Here we provide
the first evidence that endothelial specific loss of prokineticin
receptor-1 (PKR1) in mice leads to endothelial dysfunction and
low capillary formation, ultimately contributing cardiovascular,
kidney disorders and development of insulin resistance.
Endothelial cells derived from these mutant hearts displayed
an impaired proliferation rate and vessel-like formation. Low
capillary formation in mutant heart, kidney, pancreas and
adipose tissues was evident. The mutant aortas displayed
increased collagen deposition, fenestrated endothelium
with junctional defects, and impaired relaxation due to nitric
oxide synthesize-deficits without altering blood pressure.
Mutant hearts have thin ventricular wall due to an increase
in apoptotic cardiomyocytes. Mutant kidneys have abnormal
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ABSTRACTS
Minimizing nutritional usage is believed to be important
for prolonging survival time in organisms under starvation
conditions such as fasting and hibernation. The heart is a
major organ that constantly consumes nutrition coupling with
mitochondrial ATP production. However, it remains unknown
how mitochondrial gene expression is regulated in the heart
during starvation conditions. We have previously shown that a
PPARα-Sirt1 complex mediates downregulation of mitochondrial
gene expression in the failing heart. Since both PPARα and
Sirt1 are crucial regulators in physiological fasting responses,
we hypothesized that the same mechanism is involved in
the physiological fasting response in the heart. To investigate
this hypothesis, heterozygous knockout PPARα (PPARα+/-)
and Sirt1 (Sirt1+/-) mice were subjected to 24 hours fasting.
Several mitochondrial genes, such as Sdha and Atp5g1, were
downregulated following fasting in wild type (WT) mice, but this
downregulation was at least partly prevented in PPARα+/- and
Sirt1+/- mice, suggesting that PPARα and Sirt1 play an important
role in fasting-induced downregulation of mitochondrial gene
expression in the heart. After fasting, cardiac systolic function
was slightly reduced in WT mice, but this response was not
induced in PPARα+/- and Sirt1+/- mice (ejection fraction (%): Fed
WT 74, Fasted WT 68*, Fed PPARα+/- 76, Fasted PPARα+/75, Fed Sirt1+/- 74, Fasted Sirt1+/- 74, p<0.05 vs Fed WT).
Weight loss after 24 hours fasting increased in PPARα+/- mice,
compared to WT mice (Body weight reduction (%): WT 9,
PPARα+/- 14*, p<0.05 vs WT). Under an isolated working mouse
heart model with nutrient free perfusion buffer, spontaneous
beating terminated earlier in hearts isolated from PPARα+/- and
Sirt1+/- mice than in hearts from WT mice (Beating time (minutes):
WT 63, PPARα+/- 37*, Sirt1+/- 36*, p<0.05 vs WT). Glucose
deprivation-induced cell death was cooperatively prevented by
overexpression of PPARα and Sirt1 in primary cultured myocytes
(Cell survival rate (%): control 54, PPARα 75*, Sirt1 60, PPARα
with Sirt1 94*, PPARα with Sirt1 knockdown 52, p<0.05 vs
control). These results suggest that PPARα and Sirt1 are crucial
regulators of adaptation to starvation in the heart.
P234S-nitrosylation of Cyclophilin D Attenuates Mitochondrial
Permeability Transition Pore Opening: A Critical Role for
Cysteine 203 Residue
89
Poster Presentations (continued)
glomerulus structure with an increase in apoptotic cell
numbers and phosphate excretion. Mutant mice exhibited
low fasting blood glucose and high serum insulin levels. They
cleared the blood glucose at a considerably slower rate than
control mice accompanied with impaired insulin tolerance. The
insulin resistance induces compensatory β cell hypertrophy
but not expansion due to impaired survival signaling.
Induction of cell death and fibrosis is induced by hypoxia in
mutant adipocytes leads to increase in HIF1a and collagen
expression. Moreover, an increase in serum FFA and inhibition
of insulin-signaling pathway in adipocytes may represent
an early event in systemic insulin resistance. This mutation
provides a genetic model for metabolic syndrome and should
facilitate studies of both the pathogenesis and therapy of
cardiovascular, renal and metabolic disorders in humans.
C.G. Nebigil: None.
P236Not published at presenter’s request.
P237Exercise Training Prevents Hypercholesterolemia-Induced
Cardiac Mitochondrial Dysfunction
ABSTRACTS
Allison M McGee, Kyle S McCommis, M Harold Laughlin, Douglas K
Bowles, Christopher P Baines, Univ of Missouri, Columbia, MO
Hypercholesterolemia has been suggested to have direct
negative effects on myocardial function due to increased
reactive oxygen species (ROS) generation and increased
myocyte death. Mitochondrial permeability transition (MPT) is
a significant mediator of cell death, which is enhanced by ROS
generation and attenuated by exercise training. The purpose of
this study was to investigate the effect of hypercholesterolemia
on the MPT response of cardiac mitochondria. We
hypothesized that familial hypercholesterolemic (FH) pigs would
have an enhanced MPT response, and that exercise training
could reverse this phenotype. FH pigs were obtained from the
University of Wisconsin. Control, normolipidemic farm pigs
were maintained on standard pig chow. After 4 months on a
high-fat diet, the FH pigs were switched to the standard pig
chow, and randomized to sedentary or exercise groups. The
exercise group underwent a progressive treadmill-based training
program for 4 months. At the end of the training protocol the
animals were sacrificed and the heart removed. MPT was
assessed by mitochondrial swelling in response to Ca2+.
Protein nitrotyrosylation, GSH levels, and antioxidant enzyme
expression were also examined. FH pigs did show an increased
MPT response despite no change in the expression of putative
MPT pore components adenine nucleotide translocase (ANT),
mitochondrial phosphate carrier (PiC), and cyclophilin-D
(CypD). FH also caused increased oxidative stress, depicted
by increased protein nitrotyrosylation and decreased GSH
levels. This was associated with concomitant decreases in the
expression of mitochondrial antioxidant enzymes manganese
superoxide dismutase (MnSOD) and thioredoxin-2 (Trx2).
However, chronic exercise training was able to normalize the
MPT response in FH pigs, reduce oxidative stress, and increase
MnSOD expression. We conclude that hypercholesterolemia
causes increased oxidative stress and enhances the MPT
response in the porcine myocardium, and that exercise training
can correct for both the increased oxidative stress and MPT
alterations observed with hypercholesterolemia.
.M. McGee: None. K.S. McCommis: None. M.H. Laughlin: None.
A
D.K. Bowles: None. C.P. Baines: None.
P238Snf1-Related Kinase Is a Novel Regulator of
Cardiomyocyte Metabolism
Amy K Rines, Michael A Burke, Varun Nagpal, Hossein Ardehali,
Northwestern Univ, Chicago, IL
Background: Snf1-related Kinase (SNRK) is a serine/threonine
kinase with sequence homology to AMP-activated kinase
90
(AMPK). We have found that SNRK is upregulated in hearts
from patients with ischemic cardiomyopathy, but the function
of SNRK has not been studied in cardiomyocytes. Since
previous gene array data show that SNRK alters expression
of metabolic genes, we determined whether SNRK regulates
cardiomyocyte metabolism. Results: SNRK downregulation
reduced ATP levels by 23.34% in neonatal rat cardiomyocytes
compared to nonsilencing siRNA, and SNRK overexpression
increased ATP by 20.67% compared to GFP control. SNRK
knockdown reduced baseline oxygen consumption rate
(OCR) by 23.69%, and by 54.53% with the mitochondrial
uncoupler carbonyl cyanide 3-chlorophenylhydrazone
(CCCP). SNRK overexpression increased OCR by 9.57%
with CCCP. Mitochondrial content was unchanged as
measured by mitochondrial DNA and cardiolipin staining.
However, tetramethylrhodamine ethyl ester (TMRE) staining
was increased by SNRK, suggesting that SNRK maintains
mitochondrial membrane potential. Furthermore, the
extracellular acidification rate was reduced 19.47% with SNRK
knockdown and increased 12.57% with overexpression,
signifying enhanced glycolytic flux. SNRK also increased lactate
levels and mRNA of the glycolytic proteins hexokinase I and II.
Glucose uptake increased 29.61% with SNRK overexpression,
and expression of glucose transporters GLUT1 and GLUT4
was also increased, demonstrating an increase in cellular
glucose import. Glycogen was also decreased 40.69% with
SNRK knockdown and increased 46.03% with overexpression,
demonstrating that SNRK reduces storage of glucose into
glycogen. Additionally, OCR with palmitate over baseline was
reduced 43.56% with SNRK downregulation and increased
285.6% with overexpression, suggesting enhanced fatty acid
oxidation. Phosphorylation of acetyl-coA carboxylase was
also increased by SNRK. Fatty acid uptake, however, was
unchanged. Conclusions: Our results demonstrate that SNRK
increases ATP and oxygen consumption through augmented
mitochondrial membrane potential, glucose utilization, and
fatty acid oxidation. These findings reveal SNRK as a major
modulator of cardiomyocyte metabolism.
A.K. Rines: None. M.A. Burke: None. V. Nagpal: None. H. Ardehali: None.
This research has received full or partial funding support from the
American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa,
Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota,
South Dakota & Wisconsin).
P239Differential Proteomics of Subsarcolemmal and
Intermyofibrillar Mitochondria in Aging Rat Cardiac Muscle
Cecilia Gelfi, Univ Degli Studi di Milano/Istituto di Bioimmagine e
Fisiologia Molecolare, Consiglio Nazionale delle Ricerche, Segrate,
Italy; Roberta Leone, Daniele Capitanio, Agnese Viganò, Chiara Fania,
Univ Degli Studi di Milano, Segrate, Italy; Luigi Anastasia, Univ of Milan,
IRCCS Policlinico San Donato, San Donato Milanese, San Donato
Milanese, Italy; Annarosa Leri, Brigham and Women’s Hosp, Harvard
Medical Sch, Boston, MA
Recent studies revealed a key role of mitochondria in the
‘axis of aging’. Mitochondrial dysfunction induces ROS
production, leading to genotoxic damage, increasing erosion
of telomeres followed by the activation of p53, blocking the
mitochondrial biogenesis and generating a vicious cycle
which induces the age-related mitochondrial dysfunction.
This study, based on proteomic analysis, aims to elucidate
the role of different mitochondrial subpopulations in the aging
of heart in a rat model. Subsarcolemmal (SS) mitochondria
play a major role in the adaptation of cells to physiological
stimuli, whereas intermyofibrillar (IMF) mitochondria, are
“power plants” for cardiac muscle contraction. The two
subpopulations were isolated according to their localization in
young (6 months), old adult (22 months) and senescent (30
months) Sprague Dawley rats by differential centrifugation in
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Poster Presentations (continued)
a density gradient. The differences at proteomic level were
monitored by 2D-DIGE coupled with Maldi Tof and ESI MS/
MS. While only few significant differences were detected in
whole cardiac muscle proteome among young, adult and
old rats, we detected many differences in SS mitochondrial
proteome, particularly related to cell metabolism, cytoskeletal
organization, membrane permeability and chaperon proteins
indicating that aging may affect the SS mitochondria
to maintain heart tissue homeostasis. Proteome of IMF
mitocondria was characterized by a different pattern as
compared to SS mitochondria and the number of proteins
changed in aging were lower, even though a progression was
observed from 22 to 30 months. These preliminary results
suggest that protein dysregulation in mitochondrial proteome
could significantly contribute to age-dependent impairment of
cardiac function. However, these changes are not appreciable
when analyzing the heart muscle proteome as a whole,
highlighting the relevance of sub-cellular fractionation to
detect molecular events involved in the aging process.
C. Gelfi: None. R. Leone: None. D. Capitanio: None. A. Viganò:
None. C. Fania: None. L. Anastasia: None. A. Leri: None.
P240Mitochondrial Reactive Oxygen Species Regulate the
Cardiac Na+ Channel in Heart Failure
Man Liu, Hong Liu, Euy-Myoung Jeong, Lianzhi Gu, Samuel C Dudley,
Univ of Illinois at Chicago, Chicago, IL
M. Liu: None. H. Liu: None. E. Jeong: None. L. Gu: None.
S.C. Dudley: None.
Qing Zhao, Lawson Health Res Inst, London, ON, Canada; Futian
Tang, LAWSON HEALTH RESEARCH INST, London, ON, Canada;
Limei Shan, Inga Cepinskas, Gedas Cepinskas, Tianqing Peng,
Lawson Health Res Inst, London, ON, Canada
Objectives: Elevated levels of reactive oxygen species
(ROS) are the initial source of endothelial dysfunction in
diabetes. Calpain has been implicated in diabetic vascular
complications. The present study was to investigate the role
of calpain in mitochondrial ROS generation in endothelial
cells and vascular dysfunction in diabetic mice. Methods:
Endothelial cells cultured from human umbilical vein (HUVEC)
were stimulated with high glucose. Calpain activity and protein
were determined in mitochondria of HUVEC. Intracellular
and mitochondrial ROS generation as well as apoptosis
were measured. Type I diabetic OVE 26 mice and type II
diabetic db/db mice with calpastatin over-expression (OVE26/
CAST and db/db-CAST) were generated, respectively. Type
I diabetes was also induced in both wild-type and Tg-CAST
mice by injection of streptozocin (STZ). The endotheliumdependent relaxation of aortic ring was measured. Results:
High glucose significantly increased calpain-1 protein, calpain
activity and ROS generation in mitochondria of HUVEC.
Pharmacological inhibition of calpain or over-expression of
calpastatin abrogated high glucose-induced intracellular ROS
production, mitochondrial ROS generation and apoptosis in
HUVEC. Incubation of isolated mitochondria with calpain-1
protein significantly induced its ROS generation and the
membrane potential. In diabetic mice, calpain activity was
induced in aortic vessels, which correlated with an increase
in ROS production and protein tyrosine nitration. Overexpression of calpastatin prevented calpain activity, reduced
ROS production and inhibited protein tyrosine nitration in
diabetic mice. Aortic ring segments from diabetic mice
exhibited a significant reduction in vascular relaxation to
acetylcholine, which was reversed by over-expression of
calpastatin in Tg-CAST, OVE26/CAST and db/db-CAST mice.
Conclusions: This study has demonstrated a novel role of
calpain in mitochondrial ROS generation, which contributes
to apoptosis in endothelial cells during hyperglycemia.
Thus, over-expression of calpastatin inhibits reduces ROS
production and ameliorates endothelium-dependent vascular
dysfunction in mouse models of diabetes.
. Zhao: None. F. Tang: None. L. Shan: None. I. Cepinskas: None.
Q
G. Cepinskas: None. T. Peng: None.
P242Adenylate Kinase 2 Deficiency Disrupts Mitochondrial
Energetics, AMP Metabolic Signaling, and Metabolomic
Response to Myocardial Stress
Petras Dzeja, Song Zhang, Emirhan Nemutlu, Andre Terzic,
Mayo Clinic, Rochester, MN
ABSTRACTS
Background: Previously, we have shown that elevated
intracellular NADH causes a decrease in cardiac Na+ current
(INa) signaled by an increase in mitochondrial reactive oxygen
species (ROS). The decrease in INa can be ameliorated by
NAD+ or a mitochondrial specific anti-oxidant, mitoTEMPO. It
is known that cardiomyopathy is associated with reduced INa.
Therefore, we tested whether the NADH-mitochondrial ROS
pathway was involved in the reduction in INa in cardiomyopathic
mice. Methods: Nonischemic cardiomyopathy was induced
in C57BL/6 mice 6 weeks after unilateral nephrectomy,
deoxycorticosterone acetate (DOCA) pellet implantation, and
salt water substitution. Sham operated mice were used as
controls. Ventricular myocytes isolated from mice were utilized
for whole-cell patch clamp recording, intracellular NADH/NAD+
level measurements, and mitochondrial ROS monitoring with
confocal microscopy. Results: Compared to the sham mice,
the left ventricular volume was significantly enlarged (105.5 ±
4.4 µL vs. 91.6 ± 4.0 µL, P<0.05), and the ejection fraction was
significantly reduced (39.2 ± 1.6% vs. 48.7 ± 1.6%, P<0.05) in
DOCA mice. Intracellular NADH level was increased (2.78-fold;
P<0.01), and INa was decreased (62±8%; P<0.01) in myocytes
of DOCA mice vs. sham. NAD+ (500 μM) and mitoTEMPO (10
μM) recovered INa (90±9% and 103±9% of sham, respectively,
P>0.05). Mitochondrial ROS overproduction was observed
with DOCA mice myocytes by MitoSOX Red (~4.8-fold of
sham; P<0.01). NAD+ and mitoTEMPO decreased ROS in
DOCA mice myocytes (~20±6% of DOCA for both, P<0.01).
Conclusions: NADH and mitochondrial ROS were elevated,
and INa was decreased in nonischemic cardiomyopathy.
Maneuvers that reduced mitochondrial ROS restored INa. Since
reduced INa and the subsequent slow conduction velocity are
thought to contribute to arrhythmic risk in this condition, NAD+
and mitochondrial anti-oxidants may have anti-arrhythmic
activity in cardiomyopathy.
P241Calpain-1 Is Increased in Mitochondria and Contributes
to Mitochondrial ROS Generation in High GlucoseStimulated Endothelial Cells and Endothelial Dysfunction
in Mouse Models of Diabetes
Adenylate kinase 2 (AK2) isoform in mitochondrial
intermembrane/intracristal space provides a conduit
facilitating ATP export and delivery to cellular ATPases.
AK2+/- transgenic mice is viable while homozygous AK2-/deficiency results in midgestation embryonic lethality. AK2-/mouse embryonic fibroblasts (MEFs) have severely disrupted
mitochondrial cristae structure and display low growth and
proliferation potential. Protein knockdown using siRNA
indicates that AK2 is critical for cardiomyocyte mitochondrial
biogenesis and network formation. Transgenic AK2+/hearts display greater changes in AMP/ATP ratio and AMPK
activation during ischemia compared to wild type suggesting
deficient tuning of AMP signals. Metabolomic profile of AK2+/-
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91
Poster Presentations (continued)
hearts subjected to myocardial ischemia indicate lower ability
to maintain adenine and guanine nucleotide pools. AK2+/mice have abnormal metabolomic response to treadmill
exercise compared to wild type. Metabolomic profiling
revealed that most significant plasma metabolites critical in
the discrimination between wild type and AK2+/- phenotypes
were adenosine, tyrosine, tryptophan and hypoxanthine
indicating lower energetic potential of AK2+/- mice.
AK2+/- hearts have increased mitochondrial creatine kinase
CKmit content which could be viewed as a compensatory
mechanism. Thus, AK2 is the first phosphotransfer enzyme
which deficiency causes embryonic lethality indicating
critical significance of catalyzed nucleotide exchange and
conductive transfer in the narrow and crowded mitochondrial
intermembrane/intracristal space.
P. Dzeja: None. S. Zhang: None. E. Nemutlu: None. A. Terzic: None.
P243Simultaneous Decrease in Glucose Uptake, Increase in
Glucose Oxidation, and Improved Contractile Function by
Hearts from Sucrose-Fed Rats
ABSTRACTS
Romain Harmancey, Truong Lam, Genna Lubrano, UTHSC, Houston,
TX; Deborah Vela, Texas Heart Inst, Houston, TX; Heinrich Taegtmeyer,
UTHSC, Houston, TX
The heart responds to acute hemodynamic stress by
increasing carbohydrate oxidation. This metabolic response
is impaired in diabetes when excess fatty acid availability
inhibits glucose oxidation (Randle’s “glucose-fatty acid
cycle”). Impaired glucose uptake and oxidation are believed
to result in energy deficiency and poor contractile function.
We now propose that in the heart insulin resistance, defined
by impaired uptake of glucose in response to insulin, is an
endogenous protective mechanism which prevents excess
uptake of fuel when supply is increased. To test this we fed
Sprague-Dawley rats either a high-sucrose diet or regular
chow diet. After 5 to 8 weeks systemic insulin sensitivity
was impaired in sucrose-fed rats. Hearts were then perfused
ex vivo in the working mode to measure function and
metabolism. Rates of glucose uptake by the heart of sucrosefed animals were decreased in response to insulin. Cardiac
power remained unchanged in hearts perfused with either
normal (5mM glucose; 0.4mM oleate; 0.5ng/ml insulin) or high
(25mM glucose; 0.8mM oleate; 5ng/ml insulin) concentrations
of substrates. However, cardiac power increased for the
insulin-resistant hearts compared to controls when a
hemodynamic stress (afterload raised from 100 to 140cm
H2O; 1µM epinephrine) was superimposed on nutrient stress.
The ratio of glucose-to-oleate oxidation was also markedly
increased in these hearts, as was cardiac efficiency. Higher
glucose oxidation rates correlated with increased pyruvate
dehydrogenase (PDH) activity, and lower uncoupling protein 3
(UCP3) expression. In conclusion, insulin resistance promotes
adaptation of the stressed heart by increasing glucose
oxidation while limiting excess fuel uptake. Our findings call for a
new interpretation of the glucose-fatty acid cycle in the heart.
. Harmancey: None. T. Lam: None. G. Lubrano: None. D. Vela:
R
None. H. Taegtmeyer: None.
This research has received full or partial funding support from the
American Heart Association, South Central Affiliate (Arkansas, New
Mexico, Oklahoma & Texas).
P244Transcriptional Regulation of Cardiac Gene Expression by
Med13 Alters Global Energy Balance
Chad E Grueter, Brett A Johnson, Xiaoxia Qi, John McAnally, Rhonda
Bassel-Duby, Eric N Olson, UT Southwestern, Dallas, TX
for contractility and pump function, however little is known
about the role of the heart as a metabolic organ. Nuclear
hormone receptors, such as thyroid hormone receptor play
an important role in cardiovascular disease by significantly
altering expression of genes involved in maintaining metabolic
activity. The Mediator, a large multiprotein complex functions
as a hub to control gene expression through association
with transcriptional activators and repressors. We tested the
hypothesis that Med13, a component of the Mediator complex,
regulates cardiac function in a gain-of-function mouse model.
Trangsenic mice overexpressing Med13 in the heart are lean,
have increased energy expenditure, are resistant to high fat
diet-induced obesity and have enhanced cardiac contractility.
Microarray analysis and biochemical assays show that in
vivo and in vitro Med13 selectively inhibits nuclear hormone
receptor target genes of energy metabolism. These results
implicate the Mediator complex regulates energy balance and
cardiac contractility and suggests that the heart may function
as a key component of mammalian energy homeostasis.
C.E. Grueter: None. B.A. Johnson: None. X. Qi: None. J. McAnally:
None. R. Bassel-Duby: None. E.N. Olson: None.
P245Loss of Steroid Receptor Coactivator-2 in the Heart
Results in a Return to the Fetal Gene Program
Erin L Reineke, Brian York, Baylor Coll of Med, Houston, TX;
Christopher B Newgard, Duke Univ, Durham, NC; Heinrich Taegtmeyer,
Univ of Texas Sch of Med at Houston, Houston, TX; George E Taffet,
Mark L Entman, Bert W O’Malley, Baylor Coll of Med, Houston, TX
Steroid Receptor Coactivator-2 (SRC-2) is an integral
transcriptional mediator of metabolism throughout the body
with roles in both fatty acid and carbohydrate metabolism.
Based on the critical link between cardiac function and cardiac
metabolism, which itself has dynamic interplay between both
fatty acid and carbohydrate use, we sought to determine
if SRC-2 plays a role in controlling cardiac metabolism.
Hearts from SRC-2-/- mice were analyzed by extensive
gene expression profiling of major metabolic enzymes in
fuel usage pathways as well as metabolomic analysis of
major metabolites. Our results indicate that under normal
conditions, loss of SRC-2 compromises the ability of the
adult heart to metabolize fatty acid as its sole fuel source.
We observe a gene expression profile that mimics the fetal
heart and a metabolomic profile that suggests a reliance on
anaerobic conversion of glucose to lactate for energy. This
fetal gene switch is paralleled with changes of sarcomeric
gene expression, along with several genes altered in response
to stress. Interestingly, this long-term adaptive response is
adequate to maintain cardiac function under normal conditions.
However, SRC-2-/- mice do show impaired cardiac function
as compared to SRC-2+/+ mice under hemodynamic stress
from aortic banding. Surprisingly, this is accompanied by
the absence of hypertrophy, which may be explained by
decreased expression of serum response factor (SRF) and
several of the transcription factors with which SRF is known
to act. Taken together, loss of SRC-2 results in a cardiac
phenotype characterized by a return to the fetal gene program
that mimics the state of a stressed and/or aging heart. Further
investigation of this mouse model may play an important role in
understanding the mechanisms underlying the transcriptional
events surrounding cardiac metabolic remodeling.
E.L. Reineke: None. B. York: None. C.B. Newgard: None.
H. Taegtmeyer: None. G.E. Taffet: None. M.L. Entman: None.
B.W. O’Malley: None.
Aberrant cardiac metabolism is associated with obesity,
type 2 diabetes and heart failure. The heart requires highly
efficient metabolism to maintain the levels of ATP needed
92
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Poster Presentations (continued)
myocardial accumulation of ceramides and DAGs after HFD
compared to wt mice. APPL1 Tg mice also showed better
cardiac performance in ejection fraction and cardiac output
under these conditions. In summary, here we demonstrated
that APPL1 Tg mice are protected from HFD-induced
increases in circulating levels of FA, accumulation of lipotoxic
fatty acid derivatives and insulin resistance in myocardial tissue
and deterioration in cardiac function.
P246Inhibition of Endothelial Senescence Ameliorates Insulin
Resistance of Obese Mice
Masataka Yokoyama, Tohru Minamino, Sho Okada, Kaoru Tateno,
Junji Moriya, Ippei Shimizu, Aika Nojima, Takashi Ito, Yoko Ogasawara,
Chiba Univ Graduate Sch of Med, Chiba, Japan; Issei Komuro, Osaka
Univ Graduate Sch of Med, Osaka, Japan; Yoshio Kobayashi, Chiba
Univ Graduate Sch of Med, Chiba, Japan
Various stimuli can induce irreversible cell growth arrest,
termed cellular senescence. This response is controlled
by negative regulators of the cell cycle such as p53.
Accumulating evidence suggests a potential relationship
between cellular senescence and age-associated diseases
including type 2 diabetes. Here we show a crucial role for
endothelial p53 in the regulation of insulin resistance. We
found that treatment of endothelial cells with high glucose and
palmitate synergistically increased p53 expression. Consistent
with the in vitro results, endothelial expression of p53 was
markedly up-regulated when the mice were fed a high-calorie
diet, suggesting that excessive calorie intake promotes
endothelial senescence. To investigate the role of endothelial
p53 in type 2 diabetes, we analyzed metabolic parameters in
endothelial cell-specific p53 conditional knockout (ECp53CKO)
mice on a high-calorie diet. In spite of no difference in dietary
intake, ECp53CKO mice had a significantly smaller weight
and less fat accumulation than control mice. Moreover,
ECp53CKO mice showed better insulin sensitivity and glucose
tolerance than control littermates. ECp53CKO demonstrated
a significant increase in oxygen consumption and had a higher
core body temperature compared with control mice. Next we
considered some assumed mechanisms of relationship of
endothelial cell p53 expression and metabolic disorders. As
a result, we found that ECp53CKO mice had higher glucose
uptake in skeletal muscles than control. These results indicate
that inhibition of endothelial senescence ameliorates insulin
resistance by increasing energy consumption via glucose
uptake and suggest that endothelial p53 will be a novel
therapeutic target for type 2 diabetes.
M. Yokoyama: None. T. Minamino: None. S. Okada: None. K. Tateno:
None. J. Moriya: None. I. Shimizu: None. A. Nojima: None. T. Ito:
None. Y. Ogasawara: None. I. Komuro: None. Y. Kobayashi: None.
P247APPL1 Transgenic Mice Are Protected from High-Fat DietInduced Lipotoxic Cardiomyopathy
Increased circulating levels of free fatty acid (FA) in obesity
and diabetes lead to the accumulation of FA in non-adipose
tissue such as myocardium and this is known to be a major
contributor to cardiomyopathy. In this present study, we
investigated the role of APPL1 (Adaptor protein containing PH
domain, PTB domain and Leucine zipper motif-1), an important
regulator of insulin signaling, in a model of high fat diet (HFD)
induced cardiomyopathy. We fed APPL1 transgenic (Tg) or
wild type (wt) mice a high fat (60% kcal) or normal chow diet.
After 16 weeks, in wt mice HFD induced hyperinsulinemia and
hyperlipidemia, as well as cardiac dysfunction determined by
echocardiography system (Vevo 2100). Furthermore, HFD
induced cardiac insulin resistance and acetyl CoA carboxylase
(ACC) phosphorylation. Lipid analysis using LC/MS/MS
showed HFD significantly increased intracellular level of distinct
ceramide and diacylglycerol (DAG) species in heart tissue.
Interestingly, HFD fed APPL1 Tg mice showed a relatively
normal circulating level of FA and improved insulin sensitivity in
heart tissue, compared to the wt mice. Lipid analysis indicated
that APPL1 Tg mice had approximately 20% reduced intra-
P248The DCM-Causing D230N Tropomyosin Mutation
Produces an Age-Dependent Phenotype in Mice that Is
Influenced by Transgene Dosage
Lauren Tal, Jil C Tardiff, Albert Einstein Coll of Med, Bronx, NY
A recent study in two large multigenerational families
demonstrated a novel mutation in tropomyosin (Tm),
Asp230Asn (D230N) that caused DCM with a unique natural
history resulting in a striking bimodal disease distribution.
Infants and young children with the D230N Tm mutation
presented with a severe, often fatal DCM while adults
developed a mild to moderate clinical phenotype. Position
230 in tropomyosin is proximal to the C-terminal unwinding
of tropomyosin that is essential for the Tm-Tm overlap
and for the Tm-cardiac troponin T (cTnT) interaction. We
hypothesized that the age-dependent remodeling is a result
of temporal isoform switches in the closely linked Tmbinding partner cTnT. To directly address this hypothesis,
we used the regulated in vitro motility assay to determine
the biophysical effects of the D230N Tm mutation as well
as the effects of cTnT isoform changes on the regulatory
function of thin filaments. We have found that both wild
type and D230N Tm filaments inhibit filament sliding at
low calcium indicating that the D230N mutation does not
completely disrupt regulation. However in the presence of
high calcium the D230N Tm mutation significantly decreases
the maximal velocity of filament sliding (3.790 ± 0.1333
microns/sec, n=98) as compared to the wild type filaments
(4.900 ± 0.1044, microns/sec n=76). These findings support
an altered Tm-cTnT interaction because at maximal calcium
concentrations, the myofilament activation at the level of
cardiac troponin is not properly transmitted to tropomyosin
thus decreasing the activity of myosin-ATPase. In order
to extend our biophysical observations and to provide
translational insight, we generated a D230N Tm transgenic
mouse model. In preliminary studies we demonstrate that
at two months the D230N transgenic mice have evidence
for early dilatation. In addition, the differences in ventricular
remodeling were highly dependent on transgene dose. At six
months, we observed dilatation of the ventricle and thinning
of the walls compared to non-transgenic siblings. However,
the differences were not as marked as those at the twomonth time point. Collectively, these findings support an .
age-dependent DCM phenotype that is modulated by
transgene dosage.
ABSTRACTS
Min Park, York Univ, Toronto, ON, Canada; Tae-sik Park, Gachon
Univ of Med and Science, Incheon, Korea, Republic of; Donghai Wu,
Guangzhou Inst of Biomedicine and Health, Guangzhou, China; Aimin
Xu, The Univ of Hongkong, Pokfulam, Hong Kong; Gary Sweeney, York
Univ, Toronto, ON, Canada
M. Park: None. T. Park: None. D. Wu: None. A. Xu: None.
G. Sweeney: None.
L. Tal: None. J.C. Tardiff: None.
P249Cardiac Troponin I Pro82Ser Variant Impairs Myofilament
Cooperativity, Induces Diastolic Dysfunction, and Blunts
β-Adrenergic Response
Aisha Frazier, Genaro A Ramirez-Correa, Djahida Bedja, Xiaoxu Shen,
Wei Dong Gao, Guangshuo Zhu, Oscar H Cingolani, Eiki Takimoto,
Anne M Murphy, Johns Hopkins Univ Sch of Med, Baltimore, MD
Cardiac troponin I is a key regulatory protein for muscle
contraction and necessary for an adequate β-adrenergic
response. The Proline 82 Serine (P82S) sequence variant
of troponin I gene (TNNI3) is associated with late onset
hypertrophic cardiomyopathy (HCM). Our preliminary
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93
Poster Presentations (continued)
data has shown that cTnIP82S is heterozygous in 3% of
African Americans and is associated with increased LV
mass in hypertensive black men. We hypothesize that
cTnIP82S variant could influence the hypertrophic response
to hypertension and/or the malignancy of the phenotypic
expression when combined with known HCM disease causing
myofilament mutations. We created a transgenic cTnIP82S
mouse model. Echocardiography at baseline in older Tg and
NTg mice (64–65 wks) showed impaired ejection time and
relaxation, Tg display longer isovolumetric relaxation time
(IVRT) (Tg 29.7±0.26 vs NTg 23± 0.23 msec, n=8 vs n=7
p<0.05) and a significantly higher TEI index. In the young NTg
and Tg (17 wks) there were no echocardiographic differences.
However, preliminary data suggest that 1 wk after transverse
aortic coarctation (TAC), diastolic dysfunction was evident
in Tg, and 10 weeks after TAC LV mass was also increased
compared to NTg. Skinned fibers studies showed in the Tg
mice borderline lower maximal Force (Fmax) and myofilament
calcium sensitivity, whereas n Hill showed a marked
depression (Tg 1.72±0.17 vs NTg 3.09±0.44, n=7 vs n=5,
p<0.05), this effect was not due to the global myofilament
phosphorylation pattern. In addition, intact twitching cardiac
muscle studies in Tg mice revealed an impaired dosedependent β-adrenergic acceleration of relaxation, evidenced
by a failure to increase relaxation ([-dF/dt min]/[+dF/dt max])
and accelerate calcium transients decay after isoproterenol.
Pressure-volume (PV) loop studies confirm that Tg mice fail
to increase: Δ change in dP/dt max (NTg 4,928.3±7.7 vs Tg
843.3±10.7, n=4 vs n=4 p<0.05) in response to isoproterenol.
TnIP82S variant is near a region of TnI-TnT interaction; this
could explain its dramatic effects on myofilament cooperativity.
Overall these studies suggest that the expression of cTnIP82S
variant in the mice heart induces diastolic dysfunction, impairs
relaxation at baseline and after β-adrenergic stimulation.
A. Frazier: None. G.A. Ramirez-Correa: None. D. Bedja: None.
X. Shen: None. W. Gao: None. G. Zhu: None. O.H. Cingolani: None.
E. Takimoto: None. A.M. Murphy: None.
P250Reversal of Excessive O-GlcNAcylation Restores
Myofilament Function in Diabetic Cardiomyopathy:
Potential Role of OGT/OGA Enzymes Localization
ABSTRACTS
Genaro A Ramirez-Correa, Chad Slawson, Wengang Ding,
Xiaoxu Shen, Wei D Gao, Gerald W Hart, Anne M Murphy, JHSOM,
Baltimore, MD
94
Diabetes Mellitus impacts the heart contractile apparatus,
as well as cellular ion homeostasis, and energy production
systems. Diabetic cardiomyopathy is accompanied by
extensive changes in protein post-translational modifications
(PTMs), including phosphoylation and O-GlcNAcylation.
The cellular and molecular mechanisms of diabetic
cardiomyopathy are only partially understood. We previously
identified 32 total O-GlcNAcylation sites on MHC, Actin,
MLC 1, MLC2, and TnI from normal hearts, and showed that
exposure of skinned muscles to GlcNAc induces myofilament
Ca2+ desensitization. In addition, Actin O-GlcNAcylation
was increased when exposed to GlcNAc in vitro or to
hyperglycemia in two models of Diabetes Mellitus (DM) type
1 and type 2. Our hypothesis is that hyperglycemia directly
alters the cycling of O-GlcNAcylation on key myofilament
regulatory proteins, and that excessive O-GlcNAcylation is
responsible for defective myofilament Ca2+-activated response,
thus contributing to diabetic cardiomyocyte dysfunction.
To test this hypothesis, we have identified OGT localization
at the Z-disks where other relevant signaling molecules
reside, whereas OGA is localized along the M-band. OGT
and OGA affinity for myofilament proteins (Actin, Tm, MLC1
and MLC2) is dramatically up-regulated in the diabetic heart,
as assessed by co-immunoprecipitation and Western Blot.
We also investigated the effect of O-GlcNAc removal from
normal and diabetic skinned muscles by an engineered
hexosaminidase (CPJ). We found that 1 hour exposure to CPJ
restored myofilament Ca2+ sensitivity exclusively in diabetic
cardiac muscles (EC50 4.17±0.48 µM pre-CPJ vs 2.73±0.22
µM post-CPJ, n=5 vs n=4, p=0.029 ). These results establish
a direct link between cardiac protein hyper-O-GlcNAcylation
and diabetic altered cardiac myofilament Ca2+ sensitivity.
Furthermore, OGT and OGA are abundant, and located
at strategic signaling compartments, such as the Z-Disk.
Overall these data suggest that a specific increase of OGT/
OGA affinity towards key myofilament regulatory proteins, and
subsequent hyper-O-GlcNAcylation may lead to dysfunctional
regulation of myocardial contractility.
G.A. Ramirez-Correa: None. C. Slawson: None. W. Ding: None.
X. Shen: None. W.D. Gao: B. Research Grant; Significant; AHA0855439E. G.W. Hart: B. Research Grant; Significant; R01-DK-61671
and N01-HV-00240. H. Other; Significant; Dr. Hart receives a share of
royalty received by the university on sales of the CTD 110.6 antibody.
Terms of this arrangement are managed by JHUSOM. A.M. Murphy:
B. Research Grant; Significant; RO1-HL-63038 , R24DK084949.
P251Mechanical Stretch Induces Phosphorylation of Cardiac
Myosin Binding Protein-C
Yang Liu, Hao Feng, FNU Gerilechaogetu, Texas A&M Health Science
Ctr, Temple, TX; Sakthivel Sadayappan, Loyola Univ Chicago Stritch
Sch of Med, Maywood, IL; David E Dostal, Carl W Tong, Texas A&M
Health Science Ctr, Temple, TX
Normal hearts increase contractile force in response to
mechanical stretch caused by increasing volume. Although
this phenomenon has been extensively studied from
myofilaments’ spacing perspective, possible coupling of
mechanical-sensing signaling to modulation of myofilament
function remains unknown. Cardiac myosin binding protein-C
(MyBPC3) is a component of heart muscle thick filament.
Phosphorylation of MyBPC3 releases its inhibition on crossbridge cycling to increase cardiac contractility. Thus, we
postulate that mechanical stretch of the myocardium causes
phosphorylation of MyBPC3 to increase contractility. We
tested this hypothesis by performing static stretch of 20%
from baseline on cultured neonatal rat cardiac myocytes
(NRCM) at durations of 2, 5, 15, 30, and 60 minutes. NRCM
culture provides the advantage of cells living in an environment
free of adrenergic stimulation to avoid catecholamine
stimulation mediated phosphorylation of MyBPC3. Sitespecific phospho-serine antibodies were used to detect
phosphorylation of rat equivalent of S282 and S302 of mouse
MyBPC3. We used MyBPC3 antibody made from a different
species than site-specific phospho-serine antibodies to
account for loading. S282P transiently peaked after 5 minutes
of stretch, whereas S302P continued to increase with time
through 60 minutes (see figure). Consequently, our data show
that mechanical stretch alone can cause phosphorylation
of MyBPC3 as a mechanism that couples different signaling
pathways to myofilament function.
Y. Liu: None. H. Feng: None. F. Gerilechaogetu: None. S. Sadayappan:
None. D.E. Dostal: None. C.W. Tong: None.
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Poster Presentations (continued)
the IGFBP-1 promoter, demonstrated by ChIP assays. In
addition to insulin inhibition of IGFBP-1 levels, insulin also
dose-dependently inhibits RTEF-1 promoter activity (-2.4
± 0.3-fold) and expression. In vivo data suggest that Tie2Cre:RTEF-1 flox/flox transgenic mice, RTEF-1 EC-specific
knockout mice, show a significant decrease decrease in
IGFBP-1 mRNA levels compared with controls (0.21 ± 0.09 vs.
1.0, respectively), while VE-Cadherin/RTEF-1 overexpressing
transgenic mice have increased IGFBP-1 mRNA levels (25.4
± 0.02-fold). Increased IGFBP-1 levels relate physiologically
in our RTEF-1 EC-specific knockout by showing decreased
blood glucose tolerance as well as increased insulin resistance
as calculated by HOMA-IR compared with littermate controls,
(4.1 ± 0.5 vs. 1.1 ± 0.2, respectively). Furthermore, the RTEF1 EC-specific knockout mice showed a significant increase in
systolic blood pressure (10mmHg ± 6mmHg) compared with
littermate controls. Conclusion: These results demonstrate
that RTEF-1-stimulated IGFBP-1 expression may be central
to the mechanism by which RTEF-1 attenuates blood glucose
levels and potential cardiovascular function. These findings
provide the basis for novel insights into transcriptional regulation
of IGFBP-1 and contribute to understanding the role of vascular
endothelial cells in metabolism.
P252Loss of HMGB2 Induces Chromatin Remodeling and
Hypertrophic Growth in Cardiomyocytes
Sarah Franklin, Haodong Chen, Scherise Mitchell-Jordan, Shuxun Ren,
Peipei Ping, Yibin Wang, Thomas M Vondriska, UCLA, Los Angeles, CA
Nuclear DNA is packaged around the octameric nucleosome
core particle, constituting the basic building block of chromatin.
Non-nucleosome chromatin structural molecules have
been shown to induce higher order packaging of DNA into
structurally compact and inactive heterochromatin, or loosely
packed and active euchromatin. These chromatin remodeling
events are thought to establish a cell type specific pattern
of gene expression. During the development of cardiac
hypertrophy and failure, genes normally only expressed
during development are re-activated. While a number of
transcription factors involved in these changes in fetal gene
expression have been identified, the means for genome-wide
structural remodeling of DNA are unknown. To identify factors
controlling genomic plasticity in cardiomyocytes, we used
mass spectrometry to quantify chromatin-associated proteins
from cardiac nuclei during stages of hypertrophy and failure
in the mouse. Adult mice were subjected to cardiac pressure
overload by transverse aortic constriction. Chromatin was
fractionated from cardiac nuclei and DNA-bound proteins
were acid extracted and analyzed by mass spectrometry. We
measured chromatin occupancy patterns for >300 proteins
during distinct stages of heart failure. To explore the isoform
specific roles of individual chromatin structural proteins, we
used siRNA to knock-down expression of two high mobility
group proteins (HMGB1 and 2) exhibiting altered expression
in the hypertrophic heart. Loss of HMGB2 (but not HMGB1)
induced robust hypertrophic growth in cardiomyocytes.
qRT-PCR analyses demonstrated that HMGB2 is responsible
for some but not all changes in the fetal gene program (ANF
increased 150% and SERCA decreased 20%, whereas α- and
β-MHC were unchanged). To further explore the endogenous
regions of the genome under control of HMGB2 packing,
we performed microarrays following HMGB2 knockdown.
Hypertrophy or HMGB2 knock-down induced global chromatin
remodeling conducive to gene expression, as measured by
histone post-translational modifications and the ratio of core
to linker histones. These studies reveal a novel role of HMGB2
to inhibit hypertrophic growth and provide insights into general
principles for genome-wide chromatin remodeling.
S. Franklin: None. H. Chen: None. S. Mitchell-Jordan: None. S. Ren:
None. P. Ping: None. Y. Wang: None. T.M. Vondriska: None.
Angela F Messmer-Blust, Jiaping Wu, Yi Jin, Beth Israel Deaconess
Medical Ctr/Harvard Medical Sch, Boston, MA; Shaodong Guo, Texas
A&M Univ, Temple, TX; Jian Li, Beth Israel Deaconess Medical Ctr/
Harvard Medical Sch, Boston, MA
Background and Objective: Cardiovascular disease is the
most important complication of type 2 diabetes, however,
its underlying mechanism(s) are poorly understood. Our
lab previously reported that RTEF-1 is a transcription factor
that promotes angiogenesis via up-regulating HIF-1α and
VEGF. In addition to these studies, we recently determined
that RTEF-1 upregulates insulin-like growth factor binding
protein-1 (IGFBP-1) expression in endothelial cells in vitro and
IGFBPs are key regulators of insulin-like growth factor (IGF)
type 1 and 2 bioavailability at the cellular level. In addition,
IGFBP-1 is implicated in cardiovascular health, in terms of
blood pressure and glucose tolerance. Methods and Results:
RTEF-1 upregulates IGFBP-1 expression in endothelial cells
in vitro and in vivo through selectively binding and promoting
transcription from the insulin response element (IRE) site on
P254Protein Kinase C Binding Protein 1 Inhibits HypoxiaInducible Factor 1 Action in the Heart
Chad B Walton, David Veal, Chrisy Mafnas, Keith A MacCannell,
Cynthia D Anderson, Ralph V Shohet, Univ of Hawaii, Honolulu, HI
The response to hypoxia in tissues is regulated by the
heterodimeric transcription factor hypoxia inducible factor-1
(HIF-1). We have investigated the transcriptional effects
of hypoxia-inducible factor 1 alpha (HIF-1α) in the heart
by expressing an oxygen-stable form of HIF-1α in cardiac
myocytes of transgenic mice. The result in most cases is
regulation of an expected panoply of genes that restore
homeostasis during hypoxia, with corresponding phenotypic
changes including contractile dysfunction and increased
capillary density. In mice that do not show this phenotype,
the mRNA for protein kinase c binding protein 1 isoform 2
(PRKCBP1) was much more abundant, as was the protein,
and chromatin immunoprecipitation shows a predominant
binding of HIF to the promoter of this gene. Sequencing of
the promoter region of the PRKCBP1 gene from the two
phenotypes revealed an unexpected 480 bp insert in the HIFresistant animals containing two canonical HIF binding sites.
The protein co-immunoprecipitates with HIF and inhibits HIF
transcriptional activity in cell culture. In FVB mice, that contain
the promoter insert, PRKCBP is induced by ischemia and colocalizes with HIF in the infarct region. It may be responsible
for the greater susceptibility of this strain to heart failure after
infarction. We have confirmed with genetic, transcriptional,
biochemical, and physiological data that Prkcbp1 inhibits HIF
activity through direct interaction, in a mechanism mediated by
transcriptional control.
ABSTRACTS
P253Transcriptional Regulation of the IGFBP-1 Gene by RTEF1 in Endothelial Cells
A.F. Messmer-Blust: None. J. Wu: None. Y. Jin: None. S. Guo: None.
J. Li: None.
.B. Walton: None. D. Veal: None. C. Mafnas: None. K.A. MacCannell:
C
None. C.D. Anderson: None. R.V. Shohet: None.
P255Protein Arginine Methyltransferase 5 and Methylosome
Protein 50, Novel Components of P300/gata4
Complex, Suppress Hypertrophic Gene Transcriptions
in Cardiomyocytes
Yasufumi Katanasaka, Yukiko Nakagawa, Univ of Shizuoka, Shizuoka,
Japan; Yoichi Sunagawa, Kyoto Univ, Kyoto, Japan; Hiromichi Wada,
Akira Shimatsu, Kyoto Medical Ctr, Kyoto, Japan; Masatoshi Fujita,
Kyoto Univ, Kyoto, Japan; Koji Hasegawa, Kyoto Medical Ctr, Kyoto,
Japan; Tatsuya Morimoto, Univ of Shizuoka, Shizuoka, Japan
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
95
Poster Presentations (continued)
Introduction: One of intrinsic histone acetyltransferases (HAT),
p300, serves as a coactivator of hypertrophy-responsive
transcriptional factors such as a cardiac zinc finger protein
GATA4 and is involved in its hypertrophic stimulus-induced
acetylation and DNA binding. Disruption of this complex results
in the inhibition of hypertrophic responses in cardiomyocytes.
By tandem affinity purification and mass spectrometric
analyses, we identified protein arginine methyltransferase 5
(PRMT5) and methylosome protein 50 (MEP50), an activator
of PRMT5, as novel p300/GATA4-binding partners. However,
the precise functional relationships among p300/GATA4,
PRMT5 and MEP50 remain unknown. Methods and Results:
To confirm the binding of GATA4 with PRMT5, expression
plasmids encoding GATA4 and PRMT5 were co-transfected
into HEK293 cells. Nuclear extracts from these cells were
subjected to immunoprecipitation, followed by western blotting.
We observed that GATA4 formed a complex with p300,
PRMT5 and MEP50 in HEK293 cells. Next, to investigate
whether PRMT5 is required for p300-mediated activation of the
GATA4-dependent promoter activities, we performed reporter
assays in HEK293 cells. While overexpression of either PRMT5
or MEP50 inhibited p300/GATA4-induced atrial natriuretic
factors (ANF) and endotheline-1 (ET-1) promoter activities,
knockdown of PRMT5 by RNAi enhanced these promoter
activities in HEK293 cells. Immunoprecipitation followed
by western blotting demonstrated that PRMT5 repressed
p300-induced acetylation of GATA4 in HEK293T cells. Finally,
PRMT5 or MEP50 repressed phenylephrine-induced promoter
activation of the ANF and ET-1 in cardiomyocytes. Conclusion:
These findings demonstrate that PRMT5 and MEP50 form a
functional protein complex with GATA4/p300 and regulated
hypertrophic responses in cardiomyocytes.
Y. Katanasaka: None. Y. Nakagawa: None. Y. Sunagawa: None.
H. Wada: None. A. Shimatsu: None. M. Fujita: None. K. Hasegawa:
None. T. Morimoto: None.
P256Epigenetic Changes by Nuclear Calcium/CalmodulinDependent Protein Kinase II in the Heart
ABSTRACTS
Coralie Poizat, San Diego State Univ, San Diego, CA; Gillian Little,
Univ of Southern California, Los Angeles, CA; Mirko Volkers, Haruhiro
Toko, San Diego State Univ, San Diego, CA; Yan Bai, Univ of Southern
California, Los Angeles, CA
96
Calcium is central in the regulation of many cellular functions
and alteration of calcium signaling leads to a variety of cardiac
disorders. An important question remaining in cardiac biology
is how calcium-dependent mechanisms are regulated and
more specifically how calcium activates intracellular signaling
pathways and regulates gene activity in the cell nucleus. The
nuclear isoform of calcium/calmodulin-dependent protein
kinase II (CaMKIIσB) plays a protective role in the heart
under basal conditions, and alteration of its level or activity is
associated with pathological events such as cell death and
cardiac hypertrophy. We recently made a novel discovery
showing that CaMKIIσB couples calcium signals to the genome
by targeting specific histones in chromatin. Our preliminary
results show that CaMKIIσB selectively phosphorylates
histone H3 in vitro and in primary cardiomyocytes. Activation
of CaMK activity after α-adrenergic stimulation of primary
cardiac cells increases histone H3 phosphorylation at serine
10 (Ser-10), whereas specific elimination of the kinase has
the opposite effect. Enhanced phosphorylation of histone H3
Ser-10 is observed in primary fibroblasts and also in primary
ventricular myocytes and culminates at 24 hours of stimulation,
suggesting that signaling to histone H3 is not limited to highly
dividing cells and is not transient. Importantly, Enhanced
histone H3 Ser-10 phosphorylation can be detected in the
heart of mice subjected to myocardial infarction. These results
reveal a new role for CaMK enzymes in chromatin remodeling
the heart and that epigenetic changes may contribute to the
development cardiac diseases.
C. Poizat: None. G. Little: None. M. Volkers: None. H. Toko: None.
Y. Bai: None.
P257Isoform-Specific Effects of the Transcription Factor Sisterof-Mammalian Grainyhead on Endothelial Cells and in Vivo
Arne Mlynek, Margarete Lukosz, IUF — Leibniz Inst for Environmental
Med, Duesseldorf, Germany; Martin Graf, Christoph Winkler, Dept .
of Biological Sciences, Singapore, Singapore; Judith Haendeler,
Joachim Altschmied, IUF — Leibniz Inst for Environmental Med,
Duesseldorf, Germany
Apoptosis and reduced migratory capacity of human
endothelial cells (EC) are hallmarks for the development
of atherosclerosis. TNFalpha has been described as one
apoptotic stimulus, which is increased during cardiovascular
disease. However, recent findings support the hypothesis that
TNFalpha can induce survival genes before committing cells
to apoptosis. In a screen for anti-apoptotic genes regulated by
TNFalpha we have identified the transcription factor Sister-ofMammalian Grainyhead/Grainyhead-like 3 (SOM/GRHL3). In
humans two RNAs are transcribed from the gene, one of which
is alternatively spliced, yielding the protein isoforms SOM1
and SOM3, the latter being an N-terminally truncated version.
We have found that both isoforms are expressed in EC. Since
nothing is known about the function of these proteins in EC,
we investigated their functional properties and role in migration
and apoptosis. To analyze their transcription factor activity we
established a SOM-dependent reporter system by inserting
tandem SOM binding sites and corresponding mutants
upstream of a minimal promoter driving luciferase expression.
To assess transcriptional activation by SOM1 and SOM3 we
cotransfected these reporters with expression vectors for
both proteins. In contrast to previously published work, in
which isolated SOM domains fused to a Gal4 DNA binding
domain were used, we found that both full length proteins
are active transcription factors. We next investigated the
influence of SOM1 and SOM3 on EC functions. Surprisingly,
overexpression of isoform 1 induced migration and inhibited
apoptosis, whereas isoform 3 had opposite effects. Along the
same lines, SOM1, but not SOM3 activated endothelial nitric
oxide synthase and Akt. To investigate whether these isoforms
have different functions also in vivo, we overexpressed them in
zebrafish embryos. SOM3 but not SOM1 overexpression led
to increased lethality, a strong reduction in normal phenotype
and a 10 fold higher frequency in heavy deformations. The
effects observed on EC migration and apoptosis as well as on
zebrafish development suggest that these isoforms activate
different sets of target genes, which we are currently identifying
by microarray analysis.
A. Mlynek: None. M. Lukosz: None. M. Graf: None. C. Winkler:
None. J. Haendeler: None. J. Altschmied: None.
P258Effect of MicroRNA-223 on Hypoxic
Pulmonary Hypertension
Lei Shi, Inst for Vascular Signaling, Frankfurt am Main, Germany;
Bhola Kumar, Univ of Giessen Lung Ctr, Justus-Liebig-Univ, Giessen,
Germany; Beate Fisslthaler, Inst for Vascular Signaling, Frankfurt
am Main, Germany; Ralph T Schermuly, Univ of Giessen Lung Ctr,
Justus-Liebig-Univ, Giessen, Germany; Ingrid Fleming, Inst for Vascular
Signaling, Frankfurt am Main, Germany
Chronic hypoxia plays a key role in pulmonary hypertension
and remodeling by inducing marked changes in gene
expression. However, the regulatory effect of chronic hypoxia
on microRNA (miR) expression in the lung has not yet been
investigated. The purpose of this study was to determine
which miRs are regulated by chronic hypoxia in the mouse
lung and to determine their functional significance. We
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Poster Presentations (continued)
identified miRs altered in mouse lung after 3 weeks chronic
hypoxia (10% O2 (10 in 100) ) treatment by Microarray
screening, verified miR target genes by reporter gene assays
and Western blotting. Furthermore, we analyzed in vitro and
in vivo functional significance for mouse pulmonary vascular
artery smooth muscle cells. Among the miRs regulated, miR223 was the most significantly decreased, a phenomenon
verified by RT-qPCR. The insulin-like growth factor 1 receptor
(IGF1R) is a known target of miR-223 and the binding of
miR-223 to the 3’UTR of IGF1R was demonstrated. We found
that IGF1R protein levels (but not RNA) were increased in
lungs from hypoxic mice via a mechanism involving HIF1α/2α
and reduced C/EBPα expression and transcription activity.
Moreover, the expression of IGF1R was decreased following
introduction of pre-mir-223 into pulmonary artery smooth
muscle cells, an phenomenon coupled to attenuated IGF1induced Akt phosphorylation, cell migration and proliferation.
Decreasing endogenous miR-223 levels using a specific
antagomir (3 weeks), increased pulmonary artery pressure
and vessel muscularization. A similar phenomenon was
observed in miR-223-/y mice, which also demonstrated a
more severe response to hypoxia than wild-type mice. In
lungs from primary pulmonary hypertension (PPH) patients we
also detected a decrease in miR-223 and increase in IGF1R
expression. These data indicate that the downregulation of
miR-223 may contribute to the IGF1-induced pulmonary
smooth muscle migration by regulating the expression of its
receptor and thus contributing to pulmonary remodeling and
the development of pulmonary hypertension.
L. Shi: None. B. Kumar: None. B. Fisslthaler: None. R. Schermuly:
None. I. Fleming: None.
P259Functional Characterization of an ALOX15 Promoter
Variant Associated with Coronary Artery Calcification:
Regulation of ALOX15 by Vimentin
Susmita Samanta, Kurtis Anderson, Univ of Texas Health Science Ctr
at Houston, Houston, TX; Sean Moran, Rice Univ, Houston, TX; David
Gorenstein, Myriam Fornage, Univ of Texas Health Science Ctr at
Houston, Houston, TX
S. Samanta: None. K. Anderson: None. S. Moran: None.
D. Gorenstein: None. M. Fornage: None.
P260Not published at presenter’s request.
P261Endothelial Differentiation Gene-1 Is Involved in Related
Transcriptional Enhancer Factor-1-Induced Endothelial
Cell Aggregation
Ping He, Xiaojin An, Jiaping Wu, Angela Messmer-Blust, Melissa
Philbrick, Jian Li, Cardiovascular Inst, Beth Israel Deaconess Medical
Ctr/Harvard Medical Sch, Boston, MA
We have previously demonstrated that Related Transcriptional
Enhancer Factor-1 (RTEF-1) regulated angiogenesis through
induction of vascular endothelial growth factor (VEGF) under
hypoxic conditions. Recently, we discovered that RTEF1 accelerated endothelial cell connections and enhanced
endothelial cell aggregation. To investigate the mechanism
of RTEF-1 in regulated endothelial aggregation, a DNA array
analysis was performed in the endothelial cell (HMEC-1)
lacking RTEF-1 by its siRNA and resulted in a significant
decrease of endothelial differentiation gene -1 (Edg-1). RTEF-1
significantly increase Edg-1 promoter activity (4.10 Fold) and
the expression levels of Edg-1 gene are concordant with the
RTEF-1 gene in both mRNA and protein levels in cultured
endothelial cells as well as in endothelial specific RTEF-1
transgenic mice (VE-Cad-RTEF-1) and RTEF-1 knock out
mice (Tie2Cre:RTEF-1floxp) . In Matrigel tube formation assay,
over-expression of Edg-1 gene stimulated endothelial cell
aggregation, while siRNA of Edg-1 significantly blocked RTEF1-driven endothelial cell aggregation. Furthermore, using an
Edg-1 receptor agonist Sphingosine-1-phosphate (S1P), the
acceleration of the proliferation, migration and tube formation
were observed, whereas Edg-1 receptor inhibitor Pertussis
Toxin (PTX) could block RTEF-1-induced cell aggregation.
Our data demonstrates that Edg-1 is a potential target gene
of RTEF-1. It is involved in RTEF-1 regulated endothelial cell
angiogenesis, specifically in endothelial cell aggregation.
P. He: None. X. An: None. J. Wu: None. A. Messmer-Blust: None.
M. Philbrick: None. J. Li: None.
P262Receptor-Ligand Functional Interaction Between CD44
and Osteopontin During Calcific Aortic Stenosis: A
Molecular Mechanism for Accelerated Biomineralization
of Bicuspid Aortic Valve
ABSTRACTS
Human 12/15-lipoxygenase (ALOX15) catalyzes the oxidation
of polyunsaturated fatty acids and has been implicated in the
pathogenesis of atherosclerosis. We previously reported that
a common haplotype of the ALOX15 gene is associated with
higher prevalence of coronary artery calcification in a cohort of
middle-aged African-Americans. This haplotype was uniquely
tagged by a promoter variant (rs2255888). We carried out
an in vitro characterization of this promoter variant to further
investigate regulatory mechanisms of the ALOX15 gene. We
evaluated the activity of ALOX15 variant A-carrying and wild
type G-carrying promoter haplotypes using a luciferase assay.
We demonstrated a 65% higher activity of the A-carrying
promoter haplotype as compared to the G-carrying promoter
haplotype. Using mass-spectrometry and electrophoretic
mobility shift assay we showed that vimentin, a structural
protein, specifically binds to both A-carrying and G-carrying
promoter haplotypes in vitro. However, the A allele-carrying
promoter haplotype had an 83% increased in vitro binding
of vimentin compared to the G allele-carrying promoter
haplotype. In vivo binding of vimentin to this particular
promoter region has also been detected by chromatin
immunoprecipitation. Using non-denaturing polyacrylamide gel
electrophoresis and UV thermal melting we showed evidence
of the formation of intramolecular G-quadruplex structure in
the DNA sequence of the G-carrying promoter haplotype.
However, for the A-carrying promoter haplotype, formation
of intramolecular G-quadruplex structure was reduced
and intermolecular G-quadruplex was increased. We also
observed that vimentin increased ALOX15 gene expression.
The current studies indicate that sequence variant of the
rs2255888 polymorphism is associated with a structural
change of ALOX15 promoter region. This change may be
responsible for the increased binding of vimentin to the
promoter region. Our study is the first to show that vimentin
may act as a transcription regulator of ALOX15. It has been
reported that vimentin is secreted by activated macrophages.
Thus, the novel function identified here may be relevant to
ALOX15 pathogenesis mechanisms in atherosclerosis.
Giovanni Ferrari, Univ of Pennsylvania, Philadelphia, PA; Juan B Grau,
The Valley-Columbia Heart and Vascular Inst, Ridgwood, NJ; Rachana
Sainger, Paolo Poggio, Benjamin C Field, Emanuela Branchetti, William
F Seefried, Joseph E Bavaria, Univ of Pennsylvania, Philadelphia, PA
Introduction: Calcific aortic valve degeneration (CAVD) is the
most common indication for surgical valve replacement in
the US. The calcification starts with a Tricuspid Aortic Valve
(TAV) or with a Bicuspid Aortic Valve (BAV). Initial phases
of the disease, include thickening of the valve, a condition
known as aortic valve sclerosis (AVSc), whereas advanced
stages, aortic valve stenosis (AVS), are associated with leaflets
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97
Poster Presentations (continued)
biomineralization. The biomineralization process, occurring
in an accelerated manner in BAV, has not been described
in its molecular mechanisms. We reported that Osteopontin
(OPN) is implicated in the calcification of dystrophic and
ectopic sites. Here we investigate the interaction between
OPN, and one of its functional regulators, CD44, during
valve degeneration in TAV and BAV patients. Methods: Five
groups of 10 subjects each (TAV: control, AVSc, AVS; BAV:
control and AVS) were selected from our tissue biorepository
according to echocardiographic analysis, inclusion/exclusion
criteria, and/or intra-op observations. Histological studies,
cellular and molecular analysis, and in situ proximity-ligationassay (PLA) on surgically resected tissues were used to
investigate the functional interaction between OPN and
CD44. Results: 1) In vitro calcification assays shows that
OPN protects against calcification through CD44 interaction.
2) OPN and CD44 levels are elevated in the leafelts of BAV
patients, both stenotic patients and controls. In TAV, AVSc
and AVS patients shows elevated levels of OPN and CD44
when compared to controls. 3) In situ PLA assay displayed a
direct interaction between CD44 and OPN in BAV patients, in
both stenotic patients and controls, and in TAV AVSc and AVS
patients. Healthy, non-calcified TAV subjects, shows barely
detectible OPN-CD44 interaction. Conclusion: OPN-CD44
direct interaction correlate with the severity of biomineralization
in TAV patients. Conversely, in BAV we demonstrate a direct
interaction between OPN and CD44 in both stenotic and
non-calcified tissues. These results are aims to elucidate the
mechanisms behind the accelerated biomineralization of BAV
compared to TAV, and generate important insights into the
development of diagnostic and therapeutic approached for CAVD.
G. Ferrari: None. J.B. Grau: None. R. Sainger: None. P. Poggio:
None. B.C. Field: None. E. Branchetti: None. W.F. Seefried: None.
J.E. Bavaria: None.
P263Mechanism for Reductive Stress in Mutant Protein
Aggregation Cardiomyopathy
ABSTRACTS
Rajasekaran Namakkal Soorappan, Gayatri D Khanderao, Univ of
Utah Health Care, Salt Lake City, UT; Saradhadevi Varadharaj, DHLRI,
Ohio State Univ, Columbus, OH; Corey J Miller, Univ of Utah Health
Care, Salt Lake City, UT; Sankarnarayanan Kannan, M D Anderson
Cancer Ctr, Houston, TX; Kalavathy Ramachandran, Curtis Olson,
Christopher J Davidson, Univ of Utah Health Care, Salt Lake City,
UT; Jay L Zweier, DHLRI, Ohio State Univ, Columbus, OH; Sheldon E
Litwin, Medical Coll of Georgia, Augusta, GA; Ivor J Benjamin, Univ of
Utah Health Care, Salt Lake City, UT
98
Background: We recently demonstrated that the hR120GCryAB
associated with protein aggregation cardiomyopathy (PAC)
causes reductive stress (RS) linked to dysregulation of G6PD
in mice (Rajasekaran et.al Cell, 2007). Nrf2 (nuclear erythroid
related factor -2) is the master transcription factor that
regulates induction of genes encoding antioxidant responsive
element (ARE) dependent pathways. However, the temporal
effects of reductive stress on the molecular mechanisms of
transcriptional regulation of Nrf2 - Keap1 pathway are unknown.
Methods: Age-matched control and mutant TG mice at 3 and
6 months with RS were used to assess activation and nuclear
translocation of Nrf2 and ARE-dependent transcriptional
mechanisms. Electron paramagnetic resonance (EPR) analysis
was used to measure the ROS and RNS in heart tissues.
Results: Nuclear translocation of Nrf2 was significantly
increased in compensated hR120GCryAB transgenic mice
at 3 months compared with controls. EPR analysis revealed
significantly increased ROS (superoxide, hydrogen peroxide
and nitroxide) in 3-month old hR120GCryAB TG compared
with controls. In contrast, hR120GCryAB-TG mice at 6-months
exhibiting cardiac hypertrophy and RS (i.e., increased GSH/
GSSG ratio) had attenuated ROS/RNS levels that were
indistinguishable from age-matched controls. Conclusions:
Oxidative stress triggers the activation of redox sensitive Nrf2Keap1 pathway, which might be targeted therapeutically to
prevent pathogenic transition and reductive stress-induced
hR120GCryAB cardiomyopathy and heart failure.
R. Namakkal Soorappan: None. G. Khanderao: None. S. Varadharaj:
None. C.J. Miller: None. S. Kannan: None. K. Ramachandran: None.
C. Olson: None. C.J. Davidson: None. J.L. Zweier: None. S.E. Litwin:
None. I.J. Benjamin: None.
This research has received full or partial funding support from the
American Heart Association, Western States Affiliate (California,
Nevada & Utah).
P264Interaction of Collecting Duct-Derived Prorenin and
Soluble Prorenin Receptor Increases Intraluminal Renin
Activity and Augments Intratubular Angiotensin II
Formation in Ang II-Dependent Hypertensive Rats
Minolfa C Prieto, Renal and Hypertension Ctr and Tulane-BIRCWH
Program,Tulane Univ Sch of Med, New Orleans, LA; Liu Liu, Alexis A
Gonzalez, Dale M Seth, Tulane Univ Sch of Med, New Orleans, LA; L
Gabriel Navar, Hypertension and Renal Ctr, Tulane Univ Sch of Med,
New Orleans, LA
Upregulation of collecting duct (CD)-derived renin (CD
renin) in angiotensin II (Ang II)-dependent hypertension may
provide a pathway for intratubular Ang II formation by acting
on angiotensinogen (AGT) delivered from proximal tubule
segments. Recently, a prorenin/renin receptor (PRR) has been
cloned and shown to enhance renin and prorenin activation.
The soluble form of the PRR (sPRR) is augmented in the
renal inner medulla of chronic Ang II-infused rats. The present
study was performed to determine if renin is secreted into the
lumen by the CD cells in chronic Ang II-infused rats and to
establish the functional contribution of sPRR to the enhanced
renin activity in distal nephron segments. Accordingly, urinary
levels of renin (uRen) and Ang II (uAngII) were measured by
RIA in chronic Ang II-infused male Sprague-Dawley rats [80
ng/min, SC minipumps for 14 d, n=10] and sham-operated
rats [n=10]. Systolic blood pressure increased in the Ang II
rats by Day 5 and continued to increase throughout the study
(Day 13; Ang II: 175±10 vs. sham: 116±2 mmHg; p<0.05).
Although plasma renin activity (PRA) was suppressed in
the Ang II-infused rats, renal medullary renin content was
significantly augmented (12,605±1,343 vs. 7,956±765 ng Ang
I/h/mg; p<0.05). The excretion of uAngII was also increased
(3,813±431 vs. 2,080±361 fmol/day; p<0.05). In addition,
renin and prorenin excretion rates increased progressively and
were markedly augmented by Day 13 of Ang II infusion [renin
(8.6±1.5 vs. 2.8±0.5x10-6 Enzyme Units Excreted (EUE) /day;
prorenin: 15.8 ± 2.8 vs. 2.6 ± 0.7x10-3 EUE /day, p<0.05).
Renin and prorenin protein levels examined by Western Blot in
the urine were similarly increased. Importantly, the incubation
of urine samples of Ang II-infused rats with recombinant
human prorenin showed increased Ang I formation compared
to sham-operated rats. In conclusion, in chronic Ang II-infused
rats, the presence of sPRR in the urine reflects augmented
enzymatic activity of prorenin secreted by the principal cells of
the CD, which increase intratubular Ang II de novo formation
in the distal nephron segments thus contributing to enhanced
sodium reabsorption during Ang II-dependent hypertension.
M.C. Prieto: None. L. Liu: None. A.A. Gonzalez: None. D.M. Seth:
None. L. Navar: None.
This research has received full or partial funding support from the
American Heart Association, Greater Southeast Affiliate (Alabama,
Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).
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Poster Presentations (continued)
regulation of the TNF receptor 1 and of the MAP3K5 (aka
Apoptosis-Signaling Kinase 1, ASK1) and TNF-mediated
upregulation of the TNF receptor 2 compared to strong
responders. Promoter and mRNA stability studies revealed
that differences between strong and weak responders in
MAP3K1 and MAP3K5 regulation occurs at the transcriptional
level. Studies of promoter polymorphisms are undergoing.
We conclude that MAP3K1 and MAP3K5 are novel molecular
markers of endothelial sensitivity to TNF and future studies
on these genes could reveal novel genetic markers for
translational research.
P265Adenosine 2A Receptor Activation Regulates Expression
of Scavenger Receptors CD36 and LOX-1 in Human
Macrophages and Aortic Endothelial Cells
Iryna Voloshyna, Michael J Littlefield, Steven Carsons, Allison B Reiss,
Winthrop Univ Hosp, East Meadow, NY
Objectives of the study: A variety of scavenger receptors
(SR) have been linked to lipid accumulation in the vasculature.
Among these, CD36 and LOX-1 are proposed to play a
prominent role in atherosclerosis. Adenosine is a potent
cellular signaling molecule with anti-atherogenic properties.
Our previous work has shown that agonists specific for the
adenosine A2A receptor (A2AR) enhance cholesterol efflux,
preventing macrophage foam cell transformation. Here we
report additional atheroprotective effects of A2AR activation
through changes in SR expression. Methods: Primary
human aortic endothelial cells (HAECs) and THP-1 human
macrophages were incubated in the presence of oxLDL (20
μg/ml) and: a) growth media b) DMSO vehicle c) the A2AR
agonist CGS-21680 (1 μM) d) the A2AR antagonist ZM-241385
(10 μM, 1h) + CGS-21680 (1 μM) (n=5). SR expression was
evaluated by real-time PCR. PCR results were confirmed
by immunofluorescence analysis with CD36 and LOX-1specific primary antibodies, followed by incubation with FITC
conjugated secondary antibodies. Results: CGS-21680
downregulated CD36 and LOX-1 expression vs. untreated
HAEC (67.89±9.55% and 49.00±2.55%, respectively,
n=6, P<0.01). This effect was abrogated by ZM-241385,
resulting in upregulation of these SR to 175.9±22.67% and
172.10±39.6%, respectively, vs. control (n=6, P<0.001). The
pattern of SR expression changes in THP-1 macrophages
upon exposure to CGS-21680 and ZM-241385 was the same
as in HAEC. PCR results were confirmed by quantization of
fluorescence intensity with immunofluorescence confocal
microscopy. Summary: A2AR activation is a promising
therapeutic approach to prevent lipid overload in arterial
vasculature. Our data support an anti-atherogenic role for
the A2AR in both cholesterol influx and efflux pathways.
Several new agents that target the A2AR are in development
and they may hold promise as treatment for atherosclerotic
cardiovascular disease.
I. Voloshyna: None. M.J. Littlefield: None. S. Carsons: None.
A.B. Reiss: None.
P266Molecular Markers of Endothelial Sensitivity to TNFα
Eugenia Mata-Greenwood, Loma Linda Univ, Loma Linda, CA
E. Mata-Greenwood: None.
P267Transcriptional Profiling of Monocyte-Derived
Macrophages: Potential Mechanisms Underlying the
Anti-inflammatory and Antiatherogenic Effects of
Apolipoprotein A-I and the Mimetic Peptide 4F
Geeta Datta, David C Crossman, Manjula Chaddha, M N
Palgunachari, G M Anantharamaiah, C Roger White, Univ of Alabama
at Birmingham, Birmingham, AL
Apolipoprotein A-I (apoA-I) and the mimetic peptide, 4F,
display prominent anti-inflammatory properties, including the
ability to reduce vascular macrophage content. Macrophages
are a heterogenous group of cells, represented by two
principal phenotypes, the classically activated M1 macrophage
and an alternatively activated M2 phenotype. We recently
reported that apoA-I and 4F favor the differentiation of human
monocytes to an anti-inflammatory phenotype similar to
that displayed by M2 macrophages. In the current study,
we compared effects of apoA-I and 4F on gene expression
patterns in monocyte-derived macrophages (MDMs) by
microarray analysis. RNA isolation, labeling and hybridization
were carried out and the transcriptional profile was examined
using the Human Gene ST 1.0 Affymetrix chip. A detailed
analysis of the gene expression profiles of the MDMs was
carried out. Transcriptional profiling revealed that apoA-I and
4F modulated expression of 340 and 224 genes respectively,
out of which 135 and 113 genes regulated inflammatory
responses. Cluster heat maps revealed that apoA-I and 4F
induced similar changes in expression for 69 common genes.
Modulation of other genes, including STAT1 and PPARG, were
unique to 4F treatment. Besides modulating inflammatory
responses, both apoA-I and 4F altered gene expression in
cell-to-cell signaling, cell growth/proliferation, lipid metabolism
and cardiovascular system development. These data suggest
that the protective effects of apoA-I and 4F in a number of
disease states may be due, in part, to underlying changes in
monocyte/macrophage gene expression.
G. Datta: None. D.C. Crossman: None. M. Chaddha: None.
M.N. Palgunachari: None. G.M. Anantharamaiah: None.
C.R. White: None.
P268Nanoparticle-Mediated Delivery of Pitavastatin into
Reperfused Myocardium Reduces Myocardial Ischemia/
Reperfusion Injury
ABSTRACTS
TNF alpha mediated inflammation has been correlated with
cardiovascular diseases. Significant interindividual variability to
anti-TNF biologicals has been demonstrated in clinical trials.
The objective of this study is to unravel the molecular markers
of sensitivity to TNF which could be used as predictors of
therapeutic efficacy or disease progression. We have isolated
and tested a set of 20 HUVECs for their sensitivity to TNF
as determined by up-regulation of pro-inflammatory genes
such as the Intracellular Adhesion Molecule 1 (ICAM1) and
the NADPH oxidase subunit 4 (NOX4). We found that 75%
of HUVECs (15/20) responded strongly to TNF compared
to the remaining 25% (5/20, p<0.01). Further studies on the
TNF signaling pathway indicated that there was a positive
correlation between strong response to TNF and higher basal
mRNA and protein levels of the mitogen activated protein
3 kinase 1 (MAP3K1 or MEKK1). No further correlations
where observed with other signaling components (i.e., TNF
receptors, TRAFs, and other MAP3Ks, MAP2Ks or MAPKs).
Strong responders also showed 1) increased upregulation
of TNFR1 and TRAF5 protein and mRNA levels, 2) higher
phosphorylation of JNK and p38 MAPKs and 3) stronger
AP1-driven transcription as shown by luciferase assays.
Weak responders showed increased TNF-dependent down-
Kazuhiro Nagaoka, Tetsuya Matoba, Kaku Nakano, Kenji Sunagawa,
Kensuke Egashira, Kyushu Univ, Fukuoka, Japan
Background: In acute myocardial infarction, early myocardial
reperfusion is the most effective therapeutic strategy. The
reperfusion, however, induces ischemia-reperfusion (I/R)
injury that paradoxically cancels the beneficial effects of
reperfusion. Statins are known to activate reperfusion injury
salvage kinase (RISK) pathway, but its efficacy is not enough
to be used as cardioprotective therapy. Local delivery of
statins might optimize cardioprotective effects of statins.
Hence we tested the hypothesis that nanoparticle (NP)mediated delivery of pitavastatin into ischemic myocardium
at the time of reperfusion ameliorates myocardial I/R injury.
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99
Poster Presentations (continued)
Methods and Results: In a rat model of 30-min ischemia
followed by reperfusion, significant FITC signals were detected
in cardiomyocytes within infarct areas after intravenous (IV)
injection of FITC-NP at the time of reperfusion (Figure A). No
significant FITC signals were noted after IV injection of FITC
only. IV injection of pitavastatin-NP that contained 1.0 mg/
kg pitavastatin at reperfusion reduced infarct size (Figure B).
The therapeutic effects of pitavastatin-NP were associated
with activation of RISK pathway such as Akt signals in the
reperfused myocardium. Wortmannin blunted the therapeutic
effects of pitavastatin-NP (Figure B). On the other hand, no
therapeutic effects were noted after IV injection of pitavastatin
alone (1.0, 10 mg/kg). Conclusions: In I/R injury, NPs were
delivered selectively into reperfused myocardium possibly via
enhanced permeability and retension effects. NP-mediated
delivery of pitavastatin at the time of reperfusion reduced
myocardial infarct size by activating RISK pathway.
or IPC-IR. These proteins are likely to be reversibly oxidized,
as evidenced by the fact that they are oxidized with IPC, but
are no longer oxidized following reperfusion (IPC-IR). These
proteins may represent important targets in cardioprotective
signaling and include F-actin-capping protein β, Egl nine
homolog 1, and numerous members of the heat shock family
(HSP60, HSP70, HSP90β). Heat shock proteins are important
in the cellular stress response and have been shown to play
an important role in cardioprotection. Pathway analysis also
revealed that more than 30% (19 of 56) of the proteins uniquely
oxidized following IPC were involved with cell death pathways.
These proteins include Aminoacyl tRNA synthase complexinteracting multifunctional protein 1, Charged multivesicular
body protein 5, and Lamin-A/C. These results indicate that the
cardioprotective effects of IPC may occur, in part, through the
reversible oxidation of key protein targets.
M.J. Kohr: B. Research Grant; Significant; NRSA Fellowship.
J. Sun: None. A. Aponte: None. G. Wang: None. M. Gucek: None.
C. Steenbergen: B. Research Grant; Significant; NIH R01 Grant.
E. Murphy: None.
P271Genetically Induced Moderate Inhibition of the
Proteasome in Cardiomyocytes Exacerbates Myocardial
Ischemia-Reperfusion Injury in Mice
Zongwen Tian, Hanqiao Zheng, Xuejun (XJ) Wang, Univ of South
Dakota, Vermillion, SD
. Nagaoka: None. T. Matoba: None. K. Nakano: None.
K
K. Sunagawa: None. K. Egashira: None.
P269Not published at presenter’s request.
P270Resin-Assisted Capture Reveals Unique Targets of
Oxidation in Ischemic Preconditioning
ABSTRACTS
Mark J Kohr Jr, Johns Hopkins Medical Insts, Baltimore, MD; Junhui
Sun, Angel Aponte, Guanghui Wang, Marjan Gucek, Natl Heart,
Lung, and Blood Inst/Natl Insts of Health, Bethesda, MD; Charles
Steenbergen, Johns Hopkins Medical Insts, Baltimore, MD; Elizabeth
Murphy, Natl Heart, Lung, and Blood Inst/Natl Insts of Health,
Bethesda, MD
100
Proper maintenance of cellular thiol redox status is critical in the
regulation of many processes that dictate normal myocardial
function. Cell death signaling, in particular, is especially
susceptible to alterations in thiol redox state. Myocardial
ischemic preconditioning (IPC) is a redox sensitive process
that is thought to provide protection from ischemia-reperfusion
(IR) injury, in part, via reversible oxidative signaling. Protein
S-nitrosylation (SNO) is a reversible, thiol-based modification
that we and others have shown to provide cardioprotection
by modulating the activity of target proteins and by shielding
cysteine residues from irreversible oxidation. Additional thiolbased modifications are also thought to play an important
role in cardioprotection, and these include disulfide bridges
and glutathiolation. Therefore, it is of interest to identify these
redox sensitive targets that are unique to IPC. Langendorff
perfused mouse hearts were subjected to various perfusion
protocols (control, IPC, IR, IPC-IR) and homogenized. Specific
sites of oxidation were identified using oxidation-resin assisted
capture (Ox-RAC) with mass spectrometry. Consistent with a
role for oxidative signaling in cardioprotection, Ox-RAC analysis
identified 56 proteins that were oxidized with IPC, but not IR
Proteasome dysfunction is implicated human ischemic heart
disease and observed in experimental myocardial ischemiareperfusion (I/R) injury. Altered proteasome activities in I/R hearts
were reported but it is unclear whether proteasome function in
I/R hearts is adequate. Moreover, effects of pharmacological
inhibition of the proteasome on I/R injury remain controversial.
Hence we sought to determine the adequacy of proteasomal
function in I/R hearts and the impact of moderate cardiomyocyterestricted proteasome inhibition (CR-PSMI) on I/R injury in intact
animals. First, myocardial I/R was created by ligation (30min)
and subsequent release of the left anterior descending artery in
the transgenic (tg) mice overexpressing GFPdgn, a previously
validated surrogate proteasome substrate. Compared with the
sham controls, myocardial GFPdgn protein levels in the remote
area, the board zone, and the area at risk (AAR) of the ventricle
24hrs after reperfusion were significantly increased, indicative
of proteasome functional insufficiency (PFI) in the I/R heart. The
most important proteasome peptidase resides in β5 subunits of
the 20S proteasome. The clinically used proteasome inhibitor
bortezomib targets specially the β5 subunit. To achieve CRPSMI in intact animals, we engineered and tested a catalytically
inactive mouse β5 subunit mutant (T60A-β5) and created
multiple tg mouse lines in which T60A-β5 was overexpressed
under the control of an attenuated mouse mhc6 promoter.
Baseline analyses of the tg lines confirm the ability of T60A-β5 to
replace endogenous β5 and inhibit proteasome chymotrypsinlike activities in the heart in a dose-dependent manner. A stable
tg line with ~40% replacement was used in this study. The
same I/R procedure caused a significantly greater infarct size
in T60A-β5 tg mice (66.2% of AAR) than in the littermate Ntg
mice (53.4% of AAR). Under either the sham surgery or the I/R
condition, a decrease in phosphorylated AKT and an increase in
PKCσ proteins were evident in the tg hearts, compared with the
respective Ntg groups. These results show that I/R causes PFI in
the heart and PFI plays a pathogenic role in I/R injury. A potential
mechanism by which PFI contributes to I/R injury is to suppress
AKT activation and exacerbate PKCσ signaling.
Z. Tian: None. H. Zheng: None. X. Wang: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
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Poster Presentations (continued)
P272Physiological and Pathological Functions of Nox2 and
Nox4 in Ischemia/Reperfusion Injury
Shouji Matsushima, Junya Kuroda, Tetsuro Ago, Peiyong Zhai, Junichi
Sadoshima, Univ of Med of New Jersey, Newark, NJ
NADPH oxidases (Noxes) represent an enzyme system whose
primary function is to produce reactive oxygen species (ROS).
Both Nox2 and Nox4 play an important role in regulating
oxidative stress and growth/death of cardiomyocytes (CMs).
However, the role of Nox2 and Nox4 during ischemia/
reperfusion (I/R) is poorly understood. In order to elucidate
the function of the Nox isoforms, wild type (WT), Nox2 KO
(Nox2-/-), and cardiac specific Nox4 KO (cNox4-/-) mice
were subjected to 30 minutes ischemia followed by 24 hours
reperfusion. Myocardial infarction size/area at risk (MI/AAR) as
evaluated by TTC staining was significantly smaller in Nox2-/and cNox4 -/- than in WT mice (32 ± 3.1% and 28.0 ± 6.1%
vs. 40.3 ± 8.3%, p < 0.05). O2- production in Nox2 -/- and
cNox4 -/- hearts was significantly lower than in WT hearts,
as evaluated with the lucigenin-chemiluminescence assay
(433 ± 52, 580 ± 106 vs. 1250 ± 236 RLU, p < 0.05). The I/R
experiment was also conducted with mice in which both Nox2
and Nox4 are deleted (double KO mice). O2- production in
the double KO heart was significantly lower than in the single
KO ones (257 ± 42 RLU, p < 0.05). The MI/AAR in double KO
mice was, however, significantly greater than that in WT mice
(58.0 ± 6.3% vs. 40.3 ± 8.3%, p < 0.05). These results raised
the possibility that marked suppression of ROS by combined
downregulation of Nox2 and Nox4 exacerbates I/R injury. To
elucidate the underlying mechanism, we examined expression
of hypoxia inducible factor-1alpha (HIF-1α), using DN-Nox
transgenic (Tg) mice, which we have shown also exhibited
marked suppression of O2- and significantly greater MI/AAR
after I/R. HIF-1α is lower in DN-Nox Tg mice than in WT mice
in both the ischemic and non-ischemic areas. A genetic cross
between DN-Nox Tg and mice lacking prolyl hydroxylase 2,
an enzyme mediating hydroxylation and downregulation of
HIF-1α, partially rescued I/R injury in DN-Nox mice (35.8 ±
5.9% vs 65.2 ± 10.3%, p<0.05). Taken together, these data
show that both Nox2 and Nox4 mediate increases in oxidative
stress and myocardial injury in response to I/R. However,
combined downregulation of Nox2 and Nox4 induces marked
downregulation of ROS, which in turn exacerbates I/R injury
possibly through downregulation of HIF-1α and consequent
impairment of hypoxic adaptation.
S. Matsushima: None. J. Kuroda: None. T. Ago: None. P. Zhai: None.
J. Sadoshima: None.
Jie Li, Kathleen M Horak, Huabo Su, Univ of South Dakota,
Vermillion, SD; Atsushi Sanbe, Jeffrey Robbins, Cincinnati Children’s
Hosp Medical Ctr, Cincinnati, OH; Xuejun (XJ) Wang, Univ of South
Dakota, Vermillion, SD
The ubiquitin-proteasome system degrades most intracellular
proteins, including misfolded proteins. Proteasome
functional insufficiency (PFI) was observed in experimental
proteinopathies and implicated in many human common
diseases but its pathogenic role has not been established
because a measure to enhance proteasome function in
the cell has not been reported until very recently. We have
recently discovered that overexpression of proteasome
activator 28α (PA28α) enhances proteasome-mediated
removal of abnormal proteins in the cell and protects against
oxidative stress in cultured cardiomyocytes (FASEB J 2011;
25(3):883–93). Here we have extended the in vitro discoveries
to intact animals. First, we created inducible transgenic mice
J. Li: None. K.M. Horak: None. H. Su: None. A. Sanbe: None.
J. Robbins: None. X. Wang: None.
This research has received full or partial funding support from the
American Heart Association, National Center.
P274Cardiac-Specific Knockout of Capn4 Attenuates
Myocardial Remodeling and Improves Function After
Myocardial Infarction in Mice
Jian Ma, Lawson Health Res Inst, London, ON, Canada; Meng Wei,
Shanghai 6th People’s Hosp, Shanghai Jiaotong Univ Sch of Med,
Shanghai, China; Hao Wang, Weihua Liu, Lawson Health Res Inst,
London, ON, Canada; Wei Zhu, Shanghai 6th People’s Hosp, Shanghai
Jiaotong Univ Sch of Med, Shanghai, China; Ying Li, Lawson Health
Res Inst, London, ON, Canada; James Lacefield, Univ of Western
Ontario, London, ON, Canada; Peter Greer, Queen’s Univ, Kingston,
ON, Canada; Morris Karmazyn, Univ of Western Ontario, London, ON,
Canada; Tianqing Peng, Lawson Health Res Inst, London, ON, Canada
Background: Calpain has been implicated in myocardial injury
after myocardial infarction (MI). However, no direct evidence
is available on the role of calpain in post-MI myocardial
remodelling and dysfunction. The present study investigated
the effects of cardiomyocyte-specific deletion of Capn4,
essential for calpain-1 and calpain-2 activities on myocardial
remodelling and dysfunction following MI. Methods and
Results: A novel mouse model with cardiomyocyte-specific
deletion of Capn4 (Capn4-ko) was generated. MI was
induced by left coronary artery ligation. Deficiency of Capn4
significantly reduced the protein levels and activities of
calpain-1 and calpain-2 in the Capn4-ko heart. In vivo cardiac
function was relatively improved in Capn4-ko mice at 7 and
30 days after MI compared with their wild-type littermates.
Deletion of Capn4 reduced cardiac apoptosis, limited infarct
expansion and infarct zone thinning, and prevented left
ventricle dilation in Capn4-ko mice. Furthermore, myocardial
collagen deposition and cardiomyocyte cross-sectional
areas were significantly attenuated in Capn4-ko mice, which
were accompanied by down-regulation of pro-fibrotic genes
and hypertrophic genes. These effects of Capn4 knockout
correlated with down-regulation of inflammatory mediators and
normalization of matrix metalloproteinase (MMP)-9 activity in
the non-infarct area of Capn4-ko mice after MI. In vivo mouse
model of endotoxemia confirmed that calpain activation
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ABSTRACTS
P273Enhancement of Proteasomal Function Protects Against
Proteinopathy and Myocardial Ischemia-Reperfusion
Injury in Mice
with cardiomyocyte-restricted PA28α overexpression (CRPA28αOE). CR-PA28αOE does not alter the homeostasis
of normal proteins and cardiac function but increases the
degradation of a surrogate misfolded protein in the heart.
This marks the establishment of the first animal model of
benign enhancement of proteasomal function. Second, by
breeding CR-PA28αOE mice with a well-established mouse
model of mutant αB-crystallin (CryABR120G) based desminrelated cardiomyopathy, a bona fide cardiac proteinopathy, we
demonstrate that CR-PA28αOE markedly reduced aberrant
protein aggregation and cardiac hypertrophy and the lifespan
of the animals significantly increased. Furthermore, PA28α
knockdown promoted, while PA28αOE attenuated, CryABR120G
protein accumulation in cultured cardiomyocytes. Finally,
we tested the effect of proteasome functional enhancement
on myocardial ischemia/reperfusion (I/R) injury induced by
ligation and subsequent release of the left anterior descending
artery. We found that the infarct size was significantly reduced
and cardiac function recovery during reperfusion was
markedly facilitated in the CR-PA28αOE mice, compared
with their littermate control mice. We conclude that (1)
benign enhancement of cardiac proteasome function can be
achieved by CR-PA28αOE; (2) PFI plays a major pathogenic
role in proteinopathy and myocardial I/R injury; and (3)
upregulating PA28α is potentially a novel therapeutic strategy
for proteinopathies and disease alike.
101
Poster Presentations (continued)
resulted in inflammatory gene expression and MMP-9 activity
in the heart. Conclusions: Cardiomyocyte-specific knockout
of calpain attenuates myocardial adverse remodelling and
improves myocardial function after MI. These beneficial effects
of calpain disruption may result from inhibition of cardiac
apoptosis, inflammation and MMP-9 activity.
J. Ma: None. M. Wei: None. H. Wang: None. W. Liu: None. W. Zhu:
None. Y. Li: None. J. Lacefield: None. P. Greer: None. M. Karmazyn:
None. T. Peng: None.
P275Time-of-Day-Dependent Variation in Myocardial Ischemia/
Reperfusion Injury Is Lost in Mice Lacking the Regulator
of Calcineurin 1 Gene
Poster
Program
ABSTRACTS
D Bennett Grinsfelder, David Rotter, Nita Sachan, Beverly A
Rothermel, Univ of Texas Southwestern Medical Ctr, Dallas, TX
Many important components of the cardiovascular system
display circadian rhythmicity. In humans, the incidence of
myocardial infarction peaks in the morning. Furthermore,
studies in mouse models have demonstrated that damage
from ischemia/reperfusion (I/R) occurring during the transition
to waking is greater than at the end of the active period. In
the hearts of wild type mice we have observed large circadian
oscillations in the transcript and protein levels of the exon
4 isoform of the calcineurin regulatory protein (Rcan1.4).
This 15 to 20-fold oscillation peaks around circadian time
0 (CT:0) when mice enter a period of rest. Using an in vivo
murine model of myocardial I/R injury we subjected wild type
and Rcan1 KO mice to 45 minutes of myocardial ischemia
followed by reperfusion for 24 hrs. Mice were housed in
hermetic light boxes with opposing light cycles such that the
surgeon was blinded as to the light entrainment of individual
mice. I/R was performed at the beginning (CT:0 to CT:2) and
the end (CT:10 to CT:12) of the mouse’s light entrainment
cycle. Twenty-four hours after reperfusion Evan’s Blue and
2,3,5-Triphenyltetrazolium chloride staining were used to
quantify the area-at-risk per left ventricle (AAR/LV) and the
extent of myocardial infarction per area-at-risk (INF/AAR).
Serum troponin-I levels were quantified as an indication
of muscle damage. AAR/LV was the same in all animals,
however, I/R injury in wild-type mice at CT:12 was 30% higher
than in mice subjected to I/R at CT:0. Plasma troponin-I
levels were also higher. Remarkably, there was no circadian
difference in the extent of I/R injury or troponin-I levels in the
Rcan1 KO mice. Damage in the Rcan1 KO mice at both time
points was comparable to the level of damage observed in
wild type mice at CT:12, when Rcan1.4 expression is low.
Importantly, expression of all central clock genes remained
circadian in the hearts of Rcan1 KO mice suggesting that
circadian changes in the vulnerability of the myocardium to I/R
damage is directly dependent upon Rcan1 function.
D.B. Grinsfelder: None. D. Rotter: None. N. Sachan: None.
B.A. Rothermel: None.
This research has received full or partial funding support from the
American Heart Association, South Central Affiliate (Arkansas,
New Mexico, Oklahoma & Texas).
P276Cardiac-Specific Knockout NF-kB Mice Resist
Dysfunction Induced by Global Ischemia-Reperfusion
Xiu Q Zhang, Ling Li, Yubin Deng, Lensey Scott, Craig Selzman, Univ
of Utah, Salt Lake City, UT
102
Subjective: Nuclear factor Kappa B (NF-kB) is a pluripotent
transcriptional factor that has been implicated as mediator
of cardiac injury after ischemia-reperfusion (IR). Multiple
approaches have been used to target inhibition of NF-kB,
yet these strategies are often non-specific. P65-NF-kB exists
as a heterodimer with its p50 subunit and is the dominant
transcriptional subunit of NF-kB. In order to better understand
its role in the heart, we have recently generated mice that
have cardiomyocyte (CMC) specific deletion of p65 NF-kB
and hypothesized that these animals would be protected from
ischemic stress. Methods: Transgenic mice were generated
with (WT) or without CMC p65 (KO). NF-kB p65 expression
was assessed with Western Blots. We subjected the mice
to global IR using Langendorf system with baseline 20 min,
ischemia 45 min and reperfusion 60 min. Cardiac contractile
functional recovery was analyzed using rate pressure product
(RPP), contractility (+dp/dt), and relaxation (-dp/dt). Infarct
size was assessed with TTC staining. LDH and CK were
measured in reperfused solutions. Results: 1. No differences
were found between groups of WT (n=7) and KO (n=8) in
body weight and heart weight. 2. KO mice express significant
less cardiac-specific NF-kB p65. 3. During reperfusion,
RPP, +dp/dt, -dp/dt recovery to base line were all better in
p65 KO mice (RPP: 0.841±0.074 in KO vs 0.635±0.080
in WT; +dp/dt: 0.997±0.096 in KO vs 0.731±0.108 in WT;
-dp/dt: 0.869±0.096 in KO vs 0.630±0.090 in WT; p<0.05
respectively). 4. After ischemia, both LDH and CK release
were markedly less in solutions collected from p65 KO heart
(LDH: 45.2±28.0 u/L; CK: 28.1±23.6 u/L) than those in p65
WT heart (LDH: 143.2±25.9 u/L; CK: 107.2±26.2 u/L; p,0.01
respectively). 5. TTC staining shown significant less necrotic
tissue in KO heart vs WT heart (0.314±0.052 vs 0.653±0.061,
p<0.05). Conclusion: CMC-specific knockout NF-kB p65 mice
have improved cardiac recovery function, less heart necrosis,
and decreased CMC injury compared to WT after global heart
ischemia. These studies confirm many of the previous studies
suggesting a protective role for anti-NF-kB strategies for
ischemia. It furthermore provides a template to further define
the mechanisms of p65-NF-kB afforded cardioprotection.
X.Q. Zhang: None. L. Li: None. Y. Deng: None. L. Scott: None.
C. Selzman: None.
P277Cardioprotection Enabled by Proteomic Remodeling of
the Mitochondrial Respiratory Chain
Christopher Lotz, Ning Deng, Jun Zhang, Yueju Wang, Chenggong
Zong, David Liem, Ji-Youn Youn, Hua Cai, Univ of California at Los
Angeles, Los Angeles, CA; Christine Wu, Univ of Colorado, Aurora, CO;
John Yates III, The Scripps Res Inst, La Jolla, CA; James Weiss, Peipei
Ping, Univ of California at Los Angeles, Los Angeles, CA
The mitochondrial respiratory chain is a collection of five
multi-protein complexes, whose unobstructed functionality
represents a pivotal element at the crossroads of cell
death or survival. However, its molecular composition and
stochiometric information remains elusive and the adaptive
abilities of the chain remain largely unknown. We employed a
quantitative proteomic approach to investigate the hypothesis
that cardioprotection against ischemic injury is afforded by a
salutary proteomic remodeling of the mitochondrial respiratory
chain. The respiratory chain of cardiac mitochondria
isolated from wild type (WT) mice and from mice expressing
a constitutively active protein kinase Cε (AE-PKCε) were
characterized using 15N-stable isotope labeled murine models
(SILAM), as well as a label-free method in conjunction with
high resolution LC-MS/MS, respectively. Enzymatic function of
electron transport chain and the ATP synthase were evaluated
(n=7/group); and mitochondrial superoxide production was
examined by ESR-spectroscopy (n=3/group). Three novel and
important observations are made: (i) five individual respiratory
complexes exhibited a molar ratio of 1:1:1.4:1.2:4.5 in the
WT-heart; (ii) subunits within the five complexes encoded by
the mitochondrial genome were expressed at much lower
abundance (p<0.05) than those encoded by the nuclear
genome; and (iii) Genetic cardioprotection by AE-PKCε elicited
a proteomic remodeling of complex I and III, mitochondria
from AE-PKCε exhibited an increased expression of multiple
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Poster Presentations (continued)
cardiomyocytes (CMs), thereby mediating reperfusion injury
and heart failure. YAP, a transcription factor co-factor, is
negatively regulated by the Hippo pathway, and controls
cell survival, proliferation and tissue regeneration. The role
of YAP in regulating growth and death of CMs is poorly
understood. YAP overexpression in CMs induced cardiac
hypertrophy, as indicated by increases in cell size (+1.2
fold, p;0.01), protein content (+1.1 fold, p;0.01) and ANF
(luciferase reporter activity +1.7 fold, mRNA +2.2 fold, and
staining +2.7 fold, p;0.01). Lats2 phosphorylates YAP at
Serine 127, which induces cytoplasmic translocation of YAP,
whereas YAP(S127A) is localized constitutively in the nucleus.
Expression of YAP(S127A) enhanced hypertrophy in cultured
CMs compared to that of wild type YAP (+1.87 fold ANF
staining, p;0.05), suggesting that the Mst1/Hippo pathway
negatively regulates cardiac hypertrophy through YAP. YAP
inhibited cell death induced by H2O2 treatment, as evaluated
with TUNEL staining (-65%, p;0.05) and CellTiter Blue assays
(+34.9%, p;0.01), indicating that YAP plays an essential role
in mediating CM survival. Interestingly, YAP also significantly
increased Ki67 positive cells in cultured CMs compared to
LacZ (+2.65 fold, p;0.05). We used a mouse model of chronic
myocardial infarction (MI) to evaluate the function of YAP in
the heart in vivo. Although YAP is diffusely localized both in
the nucleus and cytosol in CMs in control hearts, CMs in
the border zone of MI exhibited nuclear localization of YAP
whereas YAP was excluded from the nucleus in CMs in the
remodeling area four days after MI (+6.52 fold and +1.28 fold).
Some of the YAP positive CMs in the border zone exhibited
positive co-staining with Ki67, suggesting that YAP potentially
induces CM proliferation. A significant increase in nuclear YAP
and Ki67 positive CMs (+2.95 fold, p;0.01 and +2.18 fold,
p;0.05) was also observed in neonatal rat hearts whose apex
was surgically resected three days before euthanasia. These
results suggest that YAP plays an important role in mediating
not only hypertrophy and survival, but also proliferation of CMs
in response to myocardial injury.
subunits, including the catalytic complex III subunit Cyc1.
This finding was accompanied by a preserved complex III
activity (p<0.05), as well as tempered superoxide production
(p<0.01) subsequent to Ca2+-induced damage. This is the
first study documenting a salutary proteomic remodeling of
the mitochondrial respiratory machinery in cardioprotection.
Quantitative proteomics technology enabled novel information
and new insights into mitochondrial biology.
C. Lotz: None. N. Deng: None. J. Zhang: None. Y. Wang: None.
C. Zong: None. D. Liem: None. J. Youn: None. H. Cai: None. C. Wu:
None. J. Yates: None. J. Weiss: None. P. Ping: None.
This research has received full or partial funding support from the
American Heart Association, Western States Affiliate (California,
Nevada & Utah).
P278Fibroblast Growth Factor 21 Mediated Protection of
Ischemic Myocardium
Shu Q Liu, Brandon Tefft, Northwestern Univ, Evanston, IL; Alexei
Kharitonenkov, Lilly Res Labs, Indianapolis, IN; Yupeng Ren, Li-Qun
Zhang, Yu H Wu, Northwestern Univ, Evanston, IL
S.Q. Liu: None. B. Tefft: None. A. Kharitonenkov: None. Y. Ren:
None. L. Zhang: None. Y.H. Wu: None.
P279YAP, a Transcriptional Cofactor of the Hippo Pathway,
Regulates Cardiomyocyte Growth, Survival, and Proliferation
Yanfei Yang, Noritsugu Nakano, Junichi Sadoshima, UMDNJ-New
Jersey Medical Sch, Newark, NJ
Mst1 and Lats2, components of the mammalian Hippo
pathway, stimulate apoptosis and inhibit hypertrophy of
Y. Yang: None. N. Nakano: None. J. Sadoshima: None.
P280Deletion of Cardiac Ankyrin Repeat Kinase Reduces
Ischemia/Reperfusion Injury in the Heart in Vivo
Ronald J Vagnozzi, Thomas Jefferson Univ, Philadelphia, PA;
Gregory Gatto, Lara Kallander, Victoria Ballard, Brian Lawhorn, .
Patrick Stoy, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA;
Erhe Gao, Thomas Force, Thomas Jefferson Univ, Philadelphia, PA
Ischemic heart disease impacts millions of Americans and can
progress to heart failure. Current therapies do not address
this progression and new therapeutic targets are needed.
One novel potential target is cardiac ankyrin repeat kinase
(CARK, also troponin I interacting kinase; TNNI3K). CARK is
expressed only in the heart and is significantly up-regulated
in failing human hearts. Beyond this, little is known about
CARK’s biological roles. To determine CARK’s function in the
injured heart we subjected transgenic (Tg) mice expressing
wild-type or kinase-inactive (KI) CARK to 30 minutes of LV
ischemia followed by 24 hours of reperfusion (I/R). CARK-Tg
mice had significantly larger infarcts (32.2% AAR vs 16.1%
in WT littermates, p<0.05) following I/R. Cardiac troponin I
(cTnI) serum levels were also significantly elevated in CARK-Tg
mice after 24h, consistent with increased injury. Conversely,
infarct size was decreased in mice expressing KI CARK and
levels of cTnI were reduced, suggesting that blocking CARK
activity may protect against acute injury. To test this, we
employed an inducible, cardiac-specific knockout mouse
(CARK-KO). CARK-KO mice showed a significant reduction
in infarct size (20.52% vs 32.9%, p=0.01) as well as cTnI
levels post-I/R. To confirm these findings, wild-type mice
were treated with a small molecule CARK inhibitor and then
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
Poster
Program
ABSTRACTS
Myocardial ischemia is a prevalent disorder causing heart
failure. As cardiomyocyte death is the principal cause of
cardiac functional deficits, a recognized treatment is to
minimize cardiomyocyte death post myocardial ischemia.
While extensive investigations have been conducted, few
effective cardioprotective agents have been developed for
clinical applications. The goal of this investigation is to test
and establish a cardioprotective agent based on fibroblast
growth factor 21 (FGF21), a secretory protein participating
in innate cardioprotective responses. In myocardial ischemia
induced by LAD coronary artery ligation in the mouse,
hepatocytes upregulated FGF21 mRNA by 11 folds at 12
hrs, followed by an increase in the FGF21 protein level in
hepatocytes and the serum. Administration of recombinant
FGF21 to mice immediately post MI (50 ng/gm, IV, twice/day
for 3 days) resulted in a significant reduction in the fraction
of myocardial infarction (MI) with reference to the LV wall
volume below the LAD ligation (38+/-5 and 21+/-4%, n=7,
vs. albumin administration 51+/-8 and 31+/-5%, n=6, at day
1 and 10, respectively, p;0.05 for both times) in association
with improved LV dp/dt. In FGF21 overexpressing mice, the
volume fraction of MI (36+/-6%, n=6) was significantly lower
than that in wildtype mice (48+/-6%, n=5, p;0.05) at day 1
post MI. Furthermore, FGF21 administration to normal mice
induced phosphorylation of FGFR1, PI3K, Akt, and BAD in
cardiomyocytes within 10–30 min. These molecules were
also phosphorylated in cardiomyocytes within 1 day post MI.
Injection of siRNA specific to FGFR1, PI3K, or Akt to the LV
anterior wall at 6 locations about 2 mm apart (diffusion range
of FITC-siRNA tested by fluorescence microscopy) 3 days
prior to MI resulted in a reduction in the protein level of FGFR1,
PI3K, or Akt by 65+/-9, 71+/-11, or 68+/-12%, respectively
(n=3), within the region of siRNA injection at 1 day post MI in
association with a reduction in BAD phosphorylation and an
increase in the fraction of MI by 9+/-3, 8+/-2, or 11+/-3%,
respectively (n=3). These observations suggested that FGF21
contributed to myocardial protection possibly via the FGFR1PI3K-Akt-BAD signaling mechanisms and recombinant FGF21
may be potentially used as a cardioprotective agent.
103
Poster Presentations (continued)
were subjected to I/R. CARK inhibition significantly reduced
infarct size (10.92% vs 21.74% p<0.01) as well as serum
levels of cTnI. These data indicate that loss of CARK reduces
myocyte injury and death after I/R. To examine the mechanism
of this effect, primary NRVM were either transduced with a
CARK adenovirus or treated with one of two selective CARK
inhibitors, and then subjected to oxidative stress using H2O2.
CARK over-expression worsened, while CARK inhibition
significantly blunted H2O2-induced apoptosis. Taken together,
these data suggest that CARK plays an adverse role in the
heart’s response to ischemia, in part by increasing apoptosis.
Furthermore, inhibition of CARK may protect the ischemic
heart by limiting initial cell loss and thus reducing infarct size.
These findings enhance understanding of CARK’s role in the
heart and provide evidence for CARK as a novel therapeutic
target for ischemic injury.
.J. Vagnozzi: None. G. Gatto: None. L. Kallander: None. V. Ballard:
R
None. B. Lawhorn: None. P. Stoy: None. E. Gao: None. T. Force: None.
This research has received full or partial funding support from the
American Heart Association, Great Rivers Affiliate (Delaware, Kentucky,
Ohio, Pennsylvania & West Virginia).
P281Bcl-2 Limits Ischemia-Reperfusion Injury of the Naive
Myocardium by Preserving Mitochondrial Integrity and
Respiratory Complex Functionality and Is Modulated
by PKCε
ABSTRACTS
David A Liem, Jun Zhang, Christopher Lotz, Ding Wang, Peipei Ping,
UCLA, Los Angeles, CA
Over-expression of Bcl-2 protects against myocardial
ischemia/reperfusion (I/R) injury. Nevertheless, the participation
of Bcl-2 in basal myocardium, and its subcellular targets
under such conditions, remains elusive. Using a mouse
line with an ablation of the Bcl-2 gene, we found that
myocardial infarct size (IS) was exacerbated vs. wild type
(WT) mice, demonstrating that Bcl-2 limit IS in basal I/R
injury. The exacerbated IS in Bcl-2 KO was abolished by in
vivo treatment with the selective Mitochondrial Permeability
Transition (MPT) inhibitor cyclosporine A (10 mg/kg, iv) , while
isolated cardiac mitochondria from Bcl-2 null mice exhibited
increased matrix swelling in response to CaCl2, showing
an increased susceptibility to MPT. However, recombinant
Bcl-2 or PKCε were both sufficient to attenuate the increased
susceptibility to MPT. Interestingly, spectrophotometric
analysis of baseline activities of Mitochondrial Electron
Transport Chain Complexes (ETC) I and V (but not of ETC II,
III and IV), were increased in cardiac mitochondria from Bcl-2
null mice as compared to mitochondria from normal WT mice,
demonstrating an altered mitochondrial respiratory complex
functionality. In addition, immunoprecipitation with PKCε in
AE-PKCε mouse hearts (i.e. mice with an increased activity
of PKCε) followed by immunoblotting for Bcl-2, showed
an 2-fold increased interaction between PKCε and Bcl-2.
Similarly, immunoprecipitation with Phospho-Serine followed
by immunoblotting for Bcl-2 indicated a 2-fold increased
Serine residue phosphorylation of Bcl-2. Mass spectrometry
analysis further showed that PKCε can phosporylate Bcl-2
at its Serine24 residue site in vitro, indicating that PKCε
can directly interact and phosphorylate Bcl-2. These data
suggest that Bcl-2 is pivotal in limiting IS in basal I/R injury by
counteracting MPT and preserving mitochondrial respiratory
complex functionality, and implicate a direct interaction and
phosphorylation of Bcl-2 by PKCε in this process.
D.A. Liem: None. J. Zhang: None. C. Lotz: None. D. Wang: None.
P. Ping: None.
104
P282Increased Nuclear Localization of Fyn May Limit
the Cardioprotective Effects of Hydrogen Sulfide by
Interfering with Nrf2 Signaling
Bridgette F Moody, Chad K Nicholson, Rebecca L Hood, John W
Calvert, Emory Univ Sch of Med, Atlanta, GA
Background: Hydrogen sulfide (H2S) therapy reduces
myocardial infarct size by up to 56% in non-diabetic mice. Here,
we sought to examine if H2S could provide cardioprotection
in the setting of diabetes. Methods and Results: Diabetic
mice (db/db) were subjected to 30 min of left coronary artery
occlusion (LCA) followed by reperfusion for 24 hours at
which time the extent of myocardial infarction was evaluated.
Sodium sulfide (Na2S, 100 μg/kg) administered at the time
of reperfusion decreased infarct size relative the area-at-risk
by 18% compared to vehicle treated animals (p<0.001).
Previously, Na2S has been shown to increase the nuclear
localization of Nrf-2 and upregulates its downstream target,
heme oxygenase (HO)-1, in non-diabetic mice. To investigate
if this cascade contributed to the observed cardioprotection,
Na2S was administered (tail vein) to non-diabetic and diabetic
mice and heart tissue was excised 1 hr and 24 hrs later. One
hr following the administration of Na2S, the nuclear expression
of Nrf2 was increased in both groups. In contrast, Na2S only
increased the expression of HO-1 (24 hrs) in the non-diabetic
heart. Further studies investigating Fyn, a tyrosine kinase known
to inhibit Nrf2 signaling, revealed an upregulation of nuclear Fyn
expression in the diabetic heart compared to the non-diabetic
heart. Conclusion: This data demonstrates the complexity
of therapeutic intervention for diabetics following myocardial
ischemia, as the robust cardioprotective effects of H2S in the
non-diabetic state were found to be diminished in the diabetic
state. This data also suggests that interference of Nrf2 signaling
by Fyn may be responsible for the loss of protection.
B.F. Moody: None. C.K. Nicholson: None. R.L. Hood: None.
J.W. Calvert: B. Research Grant; Significant; RO1 from NIH.
P283Parkin Mediates Mitophagy in Cardioprotection
Allen M Andres, Chengqun Huang, Eric P Ratliff, Genaro Hernandez,
Pamela Lee, Roberta A Gottlieb, San Diego State Univ, San Diego, CA
Autophagy-dependent mitochondrial turnover in response
to cellular stress is necessary for maintaining cellular
homeostasis. However, the mechanisms that govern the
selective targeting of damaged mitochondria are poorly
understood. Parkin, an E3 ubiquitin ligase, has been shown
to be essential for the selective clearance of damaged
mitochondria. Parkin is expressed in the heart, yet its function
has not been investigated in the context of cardioprotection.
We previously reported that autophagy is required for
cardioprotection by ischemic preconditioning (IPC). In the
present study, we used simulated ischemia in vitro and
IPC in hearts (in vivo and ex vivo) to investigate the role of
Parkin in mediating cardioprotection. In HL-1 cells, simulated
ischemia induced Parkin translocation to mitochondria and
mitochondrial elimination. Mitochondrial loss was blunted in
Atg5-deficient cells, revealing the requirement for autophagy
in mitochondrial elimination. Consistent with previous reports
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Poster Presentations (continued)
implicating p62/SQSTM1 in mitophagy, we found that
downregulation of p62 attenuated mitophagy and exacerbated
cell death in HL-1 cardiomyocytes subjected to simulated
ischemia. While wild type mice showed p62 translocation
to mitochondria after IPC, Parkin knockout mice exhibited
attenuated translocation of p62 to mitochondria. Importantly,
ablation of Parkin in mice abolished the cardioprotective
effects of IPC. These results reveal for the first time the crucial
role of Parkin and mitophagy in cardioprotection.
A.M. Andres: None. C. Huang: None. E.P. Ratliff: None.
G. Hernandez: None. P. Lee: None. R.A. Gottlieb: None.
P284Inhibition of the Rheb/mTORC1 Pathway During
Prolonged Ischemia Is Protective Through Autophagy
Activation in Cardiomyocytes
Sebastiano Sciarretta, Peiyong Zhai, Junichi Sadoshima, UMDNJ,
New Jersey Medical Sch, Newark, NJ
S. Sciarretta: None. P. Zhai: None. J. Sadoshima: None.
P285Impaired Recovery of Left Ventricular Function After Acute
Myocardial Reperfusion Injury Is Rescued by the Antiinflammatory Peptide Annexin-A1 ex vivo
Rebecca H Ritchie, Keith Buxton, Baker IDI Heart & Diabetes Inst,
Melbourne, Australia; Salvatore Pepe, The Univ of Melbourne and
Murdoch Childrens Res Inst, Melbourne, Australia; Anh H Cao, Kylie
Venardos, Jane Love, Baker IDI Heart & Diabetes Inst, Melbourne,
Australia; Yuan Yang, Eric Morand, Monash Univ, Melbourne, Australia;
David Kaye, Baker IDI Heart & Diabetes Inst, Melbourne, Australia
.H. Ritchie: None. K. Buxton: None. S. Pepe: None. A.H. Cao:
R
None. K. Venardos: None. J. Love: None. Y. Yang: None. E. Morand:
None. D. Kaye: None.
P286Acetylation Affords Enhanced Proteolytic Function in
Normal and Diseased Myocardium
Nobel Zong, Caiyun Fang, Ding Wang, Hongxiu Yu, Jun Zhang,
Pengyuan Yang, Peipei Ping, Univ of California, Los Angeles, Los
Angeles, CA
Proteasome complexes have essential implications on protein
homeostasis and myocardial viability under stress. In the heart,
the dynamics of proteasome complexes is regulated by posttranslational modifications. In particular, acetylation contributes
to the adjustment of cardiac functional proteome. However,
the regulation of proteasome complexes by acetylation
remains largely unknown. We characterized the acetylome
(N-terminal and lysine acetylation) of cardiac 20S proteasomes
using proteomic technology. With targeted enrichment,
eight sites of lysine acetylation and nine sites of N-terminal
acetylation were captured, which constituted the first
comprehensive acetylome map of cardiac 20S proteasomes.
Among them, six sites of lysine acetylation were inducible
with pharmacological inhibitors of histone de-acetylases
(HDACs). Both in vitro and in vivo assays demonstrated
that HDACs inhibition led to elevated proteasome function.
With purified 20S proteasomes, this impact directly related
to acetylation modifications. Significantly, this endogenous
regulatory mechanism manifests in both clinical and murine
model of diseased myocardia. Proteasome biology is
modulated by acetylation modifications in the heart. The
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
The mTOR complex 1 (mTORC1) mediates the cellular
response to energy stress. However, the role of mTORC1
signaling in mediating cardiomyocyte (CM) tolerance of
nutrient starvation and ischemia remains unclear. mTORC1
activity, as evaluated by the phosphorylation status of
p70S6K and 4E-BP1, was decreased in CMs during glucose
deprivation (GD) in vitro, and was accompanied by decreases
in the activity of Rheb, a positive regulator of mTORC1
(31.7% reduction in Rheb-bound GTP, p<0.05). Exogenous
Rheb was sufficient to restore mTORC1 activity, suggesting
that Rheb regulates the activity of mTOR during GD. Rheb
overexpression reduced CM survival during GD (42.6%
reduction at 18 hours, p<0.01), whereas knock-down of Rheb
increased CM survival (158.6% increase at 18 hours, p<0.05).
Rheb overexpression significantly inhibited autophagy,
reduced expression of autophagic genes, including Atg7,
reduced the ATP content, and increased ER stress in CMs
during GD. Under these conditions, restoration of autophagy
through Atg7 overexpression or trehalose inhibited cell death,
increased ATP content, and reduced ER stress. The activity of
Rheb/mTORC1 is also inhibited during myocardial ischemia
in vivo. After 3 hours of ischemia, transgenic mice with
cardiac-specific and inducible Rheb overexpression (Tg-Rheb)
exhibited a larger myocardial infarction (MI) (59.7% vs. 37.8%,
p<0.05) and a significant reduction in autophagy compared
to control mice, both of which were reversed by rapamycin, a
selective mTORC1 inhibitor. Interestingly, both Rheb (+135% in
Rheb-bound GTP, p<0.05) and mTORC1 (+158% in phosphop70S6K, p<0.05) were activated in the hearts of mice with
high fat diet-induced obesity. Prolonged ischemia induced less
autophagy and a larger MI (60.6% vs. 37.2%, p<0.05) in the
obese mice, effects which were reversed in the presence of
rapamycin. However, rapamycin failed to protect the obese
mice in the presence of genetic downregulation of Beclin 1
(beclin1+/- mice fed with high fat diet). In summary, inhibition
of the Rheb/mTORC1 pathway during energy deprivation
is protective through activation of autophagy. Rheb and
mTORC1 represent potential therapeutic targets to reduce
myocardial damage during prolonged myocardial ischemia in
obese patients. .
The glucocorticoid-regulated protein annexin-A1 (ANX-A1) is
a key effector molecule of anti-inflammatory glucocorticoid
actions. We have shown that the N-terminal derived peptide
ANX-A1(2–26) preserves cardiomyocyte viability after
metabolic inhibition in vitro. Our hypothesis was that ANX-A1
preserves the myocardium post-ischemic injury. ANX-A1(2–26)
(0.3μM) prevented adult rat cardiomyocyte injury, whether
present for hypoxia-reoxygenation (LDH release reduced
from 4.1±0.7- to 1.1±0.2-fold n=11 P<0.001) or only on
reoxygenation (LDH release reduced to 1.4±0.4-fold n=11,
P<0.001). Similar protection at both time points was also
evident on cardiomyocyte viability (trypan blue exclusion).
The recovery of post-ischemic left ventricular (LV) function
in intact rodent hearts was also protected. After 20mins
reperfusion, the recovery of LV developed pressure (LVDP)
remained significantly impaired in untreated rat hearts (47±5%
baseline, n=11) whereas addition of ANX-A1(2–26)(0.3μM)
on reperfusion improved LVDP recovery (to 66±7% baseline,
n=13, p<0.05). Phosphorylation of both Akt (1.9±0.2-fold,
P<0.05) and phospholamban (3.4±0.4-fold, P<0.05) was also
enhanced by ANX-A1(2–26). Similar ANX-A1(2–26)-induced
rescue of LV function was observed in mouse hearts after
acute myocardial reperfusion injury (57±7% of baseline, n=11
untreated hearts vs 78±8% baseline, n=6 ANX-A1(2–26)treated hearts, p<0.05). ANX-A1(2–26) cardioprotection
was associated with attenuated cardiac enzyme release
(LDH, CK) and Akt phosphorylation (3.1±0.8-fold, P<0.005).
In contrast, deficiency of endogenous ANX-A1 further
exacerbated recovery of post-ischemic LV function, across
LVDP (to 29±4% baseline, P<0.001), LV end-diastolic pressure
(to 5.5±1.0-fold baseline, P<0.001), LV+dP/dt (to 33±4%
baseline, P<0.001), LV-dP/dt (to 36±4% baseline, P<0.001)
and rate-pressure product (to 24±4% baseline, P<0.001). This
is the first evidence that ANX-A1 or its mimetic ANX-A1(2–26)
preserve post-ischemic recovery of LV function. ANX-A1 is
thus an endogenous regulator of LV function. Furthermore,
ANX-A1-based therapies may thus represent a novel clinical
approach for the prevention and treatment of reperfusion injury.
105
Poster Presentations (continued)
potency of this regulatory mechanism was demonstrated
in both mouse and human species. Moreover, this inherent
rheostat effects in both acutely pathological murine model
of ischemia-reperfusion and chronically pathological model
of end-stage heart failure. With the novel observation made
in this investigation, restoration of proteasomal function via
pharmacological application after stress turns feasible.
and upregulated on myocyte sarcolemmal membranes during
acute ischemia. We hypothesize that the channel is part of a
protein complex that plays a role in downstream cell signaling
post myocardial infarction (MI). Methods: Using a canine
model of MI, where the left anterior descending coronary
artery is occluded (CO) we examined epicardial tissue from
hearts subjected to 0–3 hrs of CO. Immunoprecipitation (IP)
with subsequent silver stain studies revealed novel binding
partners for Panx1. Co-immunoprecipitation studies (Co-IP)
were performed to confirm Panx1 binding partners. In vitro dye
uptake studies and ATP assays were used to determine the
function of Panx1 channels under hypoxic conditions. Results:
IP followed by silver staining revealed multiple potential
binding partners for Panx1 in the heart. Following 3 hrs CO
some interactions increased while others decreased. Coimmunoprecipitation identified one of these partners for Panx1
as the MAGUK scaffolding protein SAP97, an interaction that
is significantly increased following 3 hr CO (n=3, p<0.05).
Immunostaining showed that CO increased trafficking and
stabilization of Panx1 channels at the cell membranes. In
vitro dye uptake studies show that cells increase dye uptake
following hypoxia, an event that is blocked by the Panx1
channel blocker, Probenecid. Hypoxia also causes increased
ATP release likely due to opening of Panx1 channels. ATP
release activated fibroblasts and stimulated early fibrosis
signaling. Conclusions: Our data show the presence of a
Panx1 protein complex in cardiac myocytes that is scaffolded
at the cell membranes by SAP97. Under cellular stress Panx1
channels release ATP to induce early fibrotic events. We We
conclude that Panx1 plays a key functional role in ATP signaling
and formation of fibrosis in the post-MI heart.
N. Zong: None. C. Fang: None. D. Wang: None. H. Yu: None.
J. Zhang: None. P. Yang: None. P. Ping: None.
P287Therapeutic Hypothermia Cardioprotection During
Cardiac Arrest Alters Reperfusion-Associated mTORC1
and AMPK Signaling
ABSTRACTS
Willard W Sharp, David Beiser, Zuo-Hui Shao, Mei Han, Chang-Qing
Li, Jing Li, Huashan Wang, Kimm Hamann, Univ of Chicago, Chicago,
IL; Terry Vanden Hoek, Univ of Illinois at Chicago, Chicago, IL
Introduction: Therapeutic hypothermia (TH) is highly
cardioprotective in cellular and animal models of ischemia/
reperfusion (I/R) and cardiac arrest and is dependent
upon Akt. The mammalian target of rapamycin complex 1
(mTORC1) is a downstream target of Akt and an important
regulator of cellular energy utilization and survival but its
relationship to TH is not clear. We hypothesized that TH
attenuates mTORC1 activation while simultaneously activating
the cell survival kinases AMP activated protein kinase
(AMPK) and Stat3. We further hypothesized that inhibitors
of mTORC1 or activators of AMPK would replicate and/or
enhance TH cardioprotection. Methods: Cardiomyocytes
isolated from 1–2-day old C57BL6/J mice, were exposed to
simulated I (90 min)/R (3 h). For TH, cells were cooled from
37C to 32C during ischemia and the first hour of reperfusion.
Heart samples were also obtained from C57BL/6 mice that
underwent an 8-min cardiac arrest. Following 6 min, the mice
were randomized to normothermia (NT, 37C) or TH (30C)
extended during CPR and for 1 h after resuscitation. Protein
lysates were collected at serial time points for Western blot
analyses. Results: Cardiomyocyte death during ischemia
was minimal, but accelerated during reperfusion (4% vs.
46%,p<.05 n=5). TH attenuated this cell death (24% p<.05
n=5) and attenuated reperfusion-induced mTOR activation
seen within 30 min as measured by differential phosphorylation
of the mTOR targets 70S6K and eEF2. Compared to NT, THtreated cardiomyocytes and hearts demonstrated increased
p-AMPK after resuscitation, consistent with mTOR inhibition.
Stat3 also showed enhanced phosphorylation on tyrosine
705. The mTOR inhibitor rapamycin (100nM) given during
I/R similar to TH affected 70S6K and eEF2 and significantly
reduced cell death (28% vs. 42%, p<.05 n=5). The AMPK
activator metformin (100uM) also significantly reduced cell
death (22%, p<.05 n=4). Conclusion: TH cardioprotection is
associated with altered signaling events suggesting mTORC1
inhibition and activation of the cell survival kinases AMPK and
Stat3. Pharmacologic strategies that mimic these TH effects
are available and could be useful as TH adjuncts or mimetics
that improve survival after cardiac arrest.
W.W. Sharp: None. D. Beiser: None. Z. Shao: None. M. Han: None.
C. Li: None. J. Li: None. H. Wang: None. K. Hamann: None.
T. Vanden Hoek: None.
P288ATP Signaling Stimulates Fibroblast Activation: The Role
of Pannexin1 Channels in Fibrosis
Heather S Duffy, Elena Dolmatova, Jennifer Baum, Kimberly Keith,
Beth Israel Deaconess Medical Ctr, Harvard Medical Sch, Boston, MA;
Daniella Boassa, Gina Sosinsky, Univ of California, San Diego, CA;
Maria I Kontaridis, Beth Israel Deaconess Medical Ctr, Harvard Medical
Sch, Boston, MA
106
Background: We have shown that Pannexin1 (Panx1), a
novel ATP channel, is found in ventricular cardiac myocytes
.S. Duffy: B. Research Grant; Significant; NIH RO1083205.
H
E. Dolmatova: None. J. Baum: None. K. Keith: None. D. Boassa:
None. G. Sosinsky: None. M.I. Kontaridis: None.
P289Cardiomyocyte-Specific, but Not Systemic, Rassf1A
Deletion Is Protective Against Ischemia-Reperfusion Injury
in the Heart
Dominic Del Re, Peiyong Zhai, Junichi Sadoshima, Univ of Med and
Dentistry, Newark, NJ
Our previous work demonstrated Rassf1A to be a critical
mediator of Mst1 activation, the chief component of the
mammalian Hippo pathway, in heart failure. In the setting of
ischemia/reperfusion (I/R) injury, Mst1 is robustly activated in
the heart; however, its regulation remains unclear. Further, the
role of Rassf1A in I/R injury has not been investigated. Using
genetically modified mice in which expression of Rassf1A is
altered in a cell type-specific manner, we demonstrate that
systemic deletion of rassf1a (KO) does not alter infarct size
(36±4% vs 33±3%) or cardiac myocyte apoptosis (1.7±0.2%
vs 2.1±0.4%) following I/R versus WT. Conversely, mice
harboring deletion of rassf1a in cardiac myocytes (CKO)
have smaller infarcts (38±3% vs 23±2%, p<0.05) and less
apoptosis (2.0±0.2% vs 0.9±0.2%) after I/R. Importantly,
attenuation of Mst1 activation in ventricular homogenates
was observed in both deletion models, implicating Rassf1A
as a positive regulator of Mst1 during I/R. Langendorff global
I/R injury yielded similar results — no protection in KO, yet
significant protection in CKO hearts versus control mice,
suggesting that native cardiac cells are sufficient to mediate
this response. A candidate molecule screen found exaggerated
TNF-α expression in KO hearts compared to WT (4.8±0.7%
vs 1.0±0.3%), whereas TNF-α expression was attenuated
in CKO hearts (0.9±0.2% vs 0.3±0.1%). Finally, WT and KO
mice were administered TNF-α neutralizing or control IgG
and subjected to I/R. KO hearts treated with TNF-α Ab, but
not IgG, had reduced infarcts (19±4% vs 34±5%), but no
significant reduction in infarct size was observed in WT mice
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Poster Presentations (continued)
given TNF-α Ab. Taken together these data suggest that TNF-α
blockade prevents the deleterious consequences of Rassf1A
deletion in non-myocytes while unmasking the protective effect
of Rassf1A deletion in cardiac myocytes following I/R. Further,
these results demonstrate the importance of non-myocytes in
modulating cardiac myocyte survival and I/R injury.
D. Del Re: None. P. Zhai: None. J. Sadoshima: None.
P290RhoA Protects Against Myocardial Ischemia/
Reperfusion Injury
Sunny Y Xiang, Shigeki Miyamoto, UCSD, La Jolla, CA; Davy
Vanhoutte, Jeffery D Molkentin, Cincinnati Children’s Hosp Medical
Ctr, Cincinnati, OH; Gerald W Dorn II, Washington Univ in St Louis, St
Louis, MO; Joan Heller Brown, UCSD, La Jolla, CA
The small GTPase RhoA has established effects on
cytoskeletal dynamics and gene expression but its role
in regulating cardiac physiology and disease remains
elusive. To characterize the in vivo role of RhoA signaling in
cardiomyocytes, we generated conditional cardiac-specific
RhoA transgenic mice (CA-RhoA) with 2–5 fold increases in
RhoA activation in the adult heart. CA-RhoA mice show no
overt cardiomyopathy but when challenged by in vivo or ex
vivo I/R, these mice exhibit strikingly increased tolerance to
injury. Compared to control mice, myocardial infarct size in
CA-RhoA mice is reduced by 60–70% (20% vs. 50%, ex vivo;
10% vs. 37%, in vivo) and recovery of contractile function
is significantly improved. Protein kinase D (PKD) is robustly
activated in CA-RhoA hearts and inhibiting PKD reverses the
cardioprotection afforded by RhoA. Both RhoA and PKD are
also activated during I/R and blocking PKD augments I/R
injury in WT mouse hearts. To further confirm that RhoA and
PKD play a protective role during I/R, cardiac-specific RhoA
knockout mice generated in the Molkentin laboratory were
tested and demonstrated to show decreased tolerance to I/R
injury, manifests as increased infarct size (42% vs. 23%) and
lactate dehydrogenase release relative to control mice. This
was accompanied by attenuated PKD activation during I/R.
Taken together, our data indicates that RhoA signaling in adult
cardiomyocytes promotes survival and reveals an unexpected
role of PKD as a downstream mediator of RhoA and on
cardioprotection against I/R.
.Y. Xiang: None. S. Miyamoto: None. D. Vanhoutte: None.
S
J.D. Molkentin: None. G.W. Dorn: None. J. Heller Brown: None.
P291The Ubiquitin Ligase Siah2 in Cardiac Response
to Ischemia
Background: The E3 ubiquitin ligases SIah1 and 2 mediate
a cellular response to hypoxia through its control of prolylhydroxylases and consequently HIF1α stability. Given the
impact of hypoxia on heart function under stress, we have
evaluated the effect of genetic deletion of Siah1 and 2 on
cardiac function in response to myocardial infarction. Objective:
To determine the physiological significance of the ubiquitin
ligase Siah genes under in vivo ischemia condition. Materials
and Methods: To substantiate the role of Siah2 in ischemiainduced cell death, we tested difference in the response of
myocardial tissue to ischemia using Siah1a+/-/Siah2-/- mutant
mice and their wildtype littermates. Analysis of heart tissue from
Siah1a+/-/Siah2-/- mice subjected to a model of 24 hours anterior
descending coronary artery occlusion revealed their resistance
to ischemic injury when compared with their littermates. Cardiac
function, measured as EF %, was significantly less impaired in
the mutant mice. Heart tissue obtained from Siah1a+/-/Siah2-/mice subjected to myocardial ischemia, exhibited a significantly
.C. Scimia: None. H. Kim: None. D. Bowtell: None. M. Mercola:
M
None. Z.A. Ronai: None.
P292Possible Mechanism and Potential Markers for OzoneInduced Cardiac Toxicity
Rajat Sethi, Texas A&M Univ Health Science Ctr, Kingsville, TX; Rama
Surya Perepu, Magdalena Ramirez, Ashoka Bandala, Texas A&M Univ
HSC, Kingsville, TX; Shubham Manchanda, Univ of Dallas, Dallas, TX;
Carlos Garcia, Texas A&M Univ, Kingsville, Kingsville, TX; David Dostal,
Texas A&M Univ HSC, Temple, TX
A significant number of deaths each year in the US have
been linked to environmental pollutants such as ozone (O3).
Earlier studies from our laboratory have shown that myocardial
dysfunction, subsequent to chronic O3 exposure, in normal
adult rats may be associated with a decrease in antioxidant
reserve and with an increased activity of inflammatory mediators.
The present study tested the hypothesis that O3 induced
cardiac dysfunction in healthy normal adult rats may be due to
changes in caveolin-1 and caveolin-3 levels. Sprague Dawley
rats were exposed 8 hr/day for 28 and 56 days to filtered
air or 0.8 ppm O3. In order to assess the chronic effects to
O3, in-vivo cardiac function was assessed by measuring left
ventricular developed pressure (LVDP), 24 hr after termination
of O3 exposure. Compared to rats exposed to filtered air, LVDP
values significantly decreased in all O3 exposed animals. This
attenuation of cardiac function was associated with increased
myocardial TNF-alpha (TNF-α) levels and decreased myocardial
activities of superoxidase dismutase (SOD). Progressive
increases in the expression of myocardial TNF-α in 4 and 8
week O3 exposed animals were followed by decreases in
cardiac caveolin-1 levels. However, differential changes in
the expression of caveolin-3 in hearts from 4 and 8 week O3
exposed animals were independent of intra-cardiac TNF-α
levels. These novel findings suggest the interesting possibility
that a balance between caveolin-1 and caveolin-3 may be
involved in O3- mediated cardiac toxicity. Furthermore, differential
changes in caveolin-3 content may serve as a marker that
predicts moderate and chronic stages of cardiac injury specific
to exposure to O3 in human populations residing in urban areas
with unhealthy levels of O3. The study is timely and has clinical
significance related to environmental causes of cardiovascular
disease. This novel study will form a basis for future studies
to understand and define the various components of the
mechanistic cascade responsible for generation of cell death
signaling subsequent to O3 exposure. The long-term goal of this
study is in guiding regulatory policies to the USEPA regarding air
quality standards pertaining to O3 levels.
ABSTRACTS
Maria Cecilia Scimia, Sanford Burnham Inst/UCSD, San Diego, CA;
Hyungsoo Kim, Sanford Burnham Inst, San Diego, CA; David Bowtell,
Peter McCallum Cancer Ctr, Melbourne, Australia; Mark Mercola, Ze’ev
A Ronai, Sanford Burnham Inst, San Diego, CA
lower degree of apoptosis (Tunel Staining), and myocardial
infarct size (Evans blue, TTC). Conclusions: Collectively, these
data establish that reduced expression of Siah1/2 exerts a
cardioprotective function under ischemic conditions. The
underlying molecular mechanisms linking cardioprotection to
ischemic challenge will be discussed.
. Sethi: None. R.S.P. Perepu: None. M. Ramirez: None. A. Bandala:
R
None. S. Manchanda: None. C. Garcia: None. D. Dostal: None.
P293Endogenous Lats2 Plays an Important Role in Mediating
Myocardial Injury Caused by Ischemia/Reperfusion
Through Inhibition of FoxO3
Dan Shao, Peiyong Zhai, Junichi Sadoshima, UMDNJ, Newark, NJ
Lats2 is a tumor suppressor and a serine/threonine kinase,
acting downstream of mammalian sterile 20 like kinase1
(Mst1), which stimulates apoptosis and inhibits hypertrophy
in cardiomyocytes (CM). We investigated the role of Lats2 in
mediating myocardial injury after ischemia/reperfusion (IR).
Phosphorylation of YAP, an in vivo substrate of Lats2, was
increased after 45 minutes ischemia followed by 24 hours
reperfusion in control mouse hearts compared with sham,
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
107
Poster Presentations (continued)
but not in dominant negative (DN) Lats2 transgenic mouse
(Tg) hearts, suggesting that Lats2 is activated by IR. The
size of myocardial infarction (MI)/area at risk was significantly
smaller in Tg mice than in NTg mice (19% and 49%, p<0.01).
And there were fewer TUNEL positive cells in Tg than in NTg
mice (0.04% and 0.11%, p<0.05). Following 30 min of global
ischemia and 60 min of reperfusion in Langendorff perfused
heart preparations, left ventricular (LV) systolic pressure (100
vs 71mmHg, p<0.05) and LV developed pressure (79 vs 47
mmHg, p<0.05) were significantly greater in Tg than in NTg
mice, indicating that suppression of Lats2 induces better
functional recovery after IR. Oxidative stress, as evaluated by
8-OHdG staining, was attenuated in Tg mice. In cultured CMs,
DN-Lats2 significantly decreased H2O2-induced cell death.
Overexpression of Lats2 significantly downregulated (51% and
75%, p<0.05), whereas that of DN-Last2 upregulated (100
and 70%, p<0.05), MnSOD and catalase, suggesting that
Lats2 negatively regulates expression of antioxidants. Reporter
gene assays showed that overexpression of Lats2 significantly
inhibits (-70%), whereas knocking down Lats2 by sh-Lats2
increases (+60%), FoxO3-mediated transcriptional activity.
Overexpression of Lats2 in CMs inhibited FoxO3 expression,
whereas that of DN-Lats2 significantly inhibited FoxO3
downregulation after IR in vivo, suggesting that Lats2 negatively
regulates FoxO3 protein expression, which may lead to the
downregulation of MnSOD and catalase. Taken together, these
results suggest that endogenous Lats2 plays an important role
in mediating myocardial injury in response to IR, In part through
downregulation of FoxO3 and consequent downregulation of
antioxidants and increased oxidative stress in the heart.
D. Shao: None. P. Zhai: None. J. Sadoshima: None.
This research has received full or partial funding support from the
American Heart Association, Founders Affiliate (Connecticut,
Maine, Massachusetts, New Hampshire, New Jersey, New York,
Rhode Island, Vermont).
P294Cardiomyocyte-Specific RBPJ Regulates Angiogenesis
and Stress Response in the Adult Heart
ABSTRACTS
Ramon Diaz Trelles, Maria Cecilia Scimia, Burnham Inst for Medical
Res, La Jolla, CA; Pilar Ruiz Lozano, Stanford Univ, Palo Alto, CA; Mark
Mercola, Burnham Inst for Medical Res, La Jolla, CA
Regulation of adult myocardial angiogenesis is critical for an
appropriate cardiac function. We found that lack of RBPJ on
isolated mouse adult cardiomyocytes increases angiogenic
factors gene expresión and promoter hyperacetylation and
hypermethylation of Notch target gene promoters (HEY2) and
angiogenic factors. Accordingly, cardiomyocyte specific RBPJ
KO adult mice show a denser microvasculature. RBPJ KO
mice suggest an angiogenesis repressive role of RBPJ during
homeostasis. Stress induced by myocardial infarction (MI) or
cardiac overload (TAC) activate an angiogenic response to
compensate the increased oxygen demand. We have found
that Notch pathway is activated and RBPJ accumulated
in the nucleus after MI and TAC. On isolated mouse adult
cardiomyocytes, RBPJ is also able to control angiogenesis by
mediating a hypoxia-induced response independent of Notch
receptor activation. Mice lacking RBPJ in cardiomyocytes
present a blunted angiogenic response. Suprisingly RBPJ KO
mice had a better cardiac performance alter MI. We propose
that RBPJ is a critical effector of the hypoxic response in the
heart and the mechanisms by which RBPJ KO could protect
from injury will be discussed on the presentation.
R. Diaz Trelles: None. M. Scimia: None. P. Ruiz Lozano: None.
M. Mercola: None.
This research has received full or partial funding support from the
American Heart Association, Western States Affiliate (California,
Nevada & Utah).
108
P295The Inflammasome Is Involved in Myocardial IschemiaReperfusion Injury
Masafumi Takahashi, Jichi Medical Univ, Tochigi, Japan; Masanori
Kawaguchi, Shinshu Univ, Matsumoto, Japan; Fumitake Usui, Hiroaki
Kimura, Jichi Medical Univ, Tochigi, Japan; Shun’ichiro Taniguchi, Uichi
Ikeda, Shinshu Univ, Matsumoto, Japan
Background: Accumulating evidence indicates that inflammation
is involved in the pathophysiology of myocardial ischemiareperfusion (I/R) injury. However, the mechanism of I/R-initiated
inflammation remains to be determined. The inflammasome is
a multiprotein complex consisting of nod-like receptor (NLR),
apoptosis-associated speck-like adaptor protein (ASC), and
caspase-1, and regulates caspase-1-dependent maturation of
IL-1beta and IL-18. In the present study, we investigated the
role of inflammasome in myocardial I/R injury. Methods and
Results: Wild-type (WT), ASC-/-, and caspase-1-/- mice were
subjected to 30 min LAD ligation, followed by reperfusion. ASC
and caspase-1 were expressed at the site of myocardial I/R
injury. Deficiency of ASC and caspase-1 reduced inflammatory
responses, such as inflammatory cell infiltration and cytokine
expression, and subsequent injuries such as infarct development,
myocardial fibrosis, and dysfunction in myocardial I/R injury. To
determine the contribution of inflammasome in bone marrow
cells, we produced bone marrow transplant mice and found that
inflammasome activation was critical not only in bone marrow
cells but also in myocardial resident cells. Since myocardial
damage was observed before the inflammatory cell infiltration
after I/R, we hypothesized that myocardial resident cells are
responsible for an initial activation of inflammasome. To test
this hypothesis, we examined whether hypoxia/reoxygenation
(H/R) stimuli could induce inflammasome activation in
cardiac fibroblasts and cardiomyocytes in vitro. Interestingly,
inflammasome activation was detected only in cardiac fibroblasts,
but not in cardiomyocytes, and mediated through reactive
oxygen species (ROS) and potassium efflux. Conclusion:
These findings indicate that inflammasome activation in cardiac
fibroblasts is essential for inflammation and injury after myocardial
I/R, and suggest that the inflammasome is a potential novel
therapeutic target for myocardial I/R injury.
. Takahashi: None. M. Kawaguchi: None. F. Usui: None. H. Kimura:
M
None. S. Taniguchi: None. U. Ikeda: None.
P296Intralipid Protects the Heart Against Ischemia/Reperfusion
Injury More Efficiently than Cyclosporine-A
Jingyuan Li, Ji-Youn Youn, Hua Cai, Mansoureh Eghbali, UCLA, Los
Angeles, CA
It has been shown that intralipid protects the heart against
ischemia reperfusion (I/R) injury. Here we compared the
cardioprotective effect of Intralipid (ILP) with cyclosporine-A
(CsA), a potent inhibitor of the mitochondria permeability
transition pore (mPTP) opening. Isolated hearts (Langendorff)
from male mice were subjected to 20 minutes of global
ischemia followed by reperfusion with Krebs Henseleit buffer
(CTRL), additional 1% ILP (ILP group) or 1.5µM CsA (CsA
group). The reperfusion duration was 40 min for measurement
of cardiac function and infarct size, 10 min for measurement
of mitochondrial calcium retention capacity and 5 min for
superoxide production measurements using electron spin
resonance. Post-ischemic administration of ILP improved the
heart functional recovery even better than CsA as Rate pressure
product (RPP) at the end of 40 min was significantly higher in
ILP group compared to CsA group (13676±611 mmHg*beats/
min (n=6) in ILP group vs. 7426±1158 mmHg*beats/min (n=3)
in CsA, P<0.01). Consistent with the heart function, the infarct
size was also significantly smaller in ILP compared to CsA group
(18.3±2.4% (n=9) in ILP vs. 29.9±2.0% (n=3) in CsA, P<0.01).
The heart functional recoveries were significantly higher and the
infarct sizes were significantly smaller in both groups compared
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Poster Presentations (continued)
to CTRL (RPP=2999±863 mmHg*beats/min (n=6), the infarct
size=54.8±2.9% (n=10)). ILP was as efficient as CSA in inhibiting
mPTP opening as calcium retention capacity (CRC) was not
significantly different between the mitochondria isolated from
the heart which were reperfused with ILP or CSA (274.3±8.4
nM/mg-mitochondria protein (n=6) in ILP vs. 260.3±2.9 nM/
mg-mitochondria protein (n=6) in CsA). The CRC in both groups
were higher than CTRL (168.6±9.6 nM/mg mitochondria protein
(n=7)). ILP prevented superoxide production in mitochondria
similarly as CSA (0.344±0.1104 (n=4) in ILP vs. 0.5744±0.1445
(n=4) in CsA) which were significantly lower than CTRL
(1±0.1197 (n=4), normalized to CTRL). In conclusion, although
ILP inhibits the opening of the mPTP as efficiently as CsA,
ILP is more effective than CsA in reducing the infarct size and
improving the heart functional recovery.
J. Li: None. J. Youn: None. H. Cai: None. M. Eghbali: None.
P297Not published at presenter’s request.
P298Probucol Inhibits Oxidized Low-Density LipoproteinInduced Immune Maturation of Dendritic Cells via
Heme Oxygenase 1
Aijun Sun, Xueting Jin, Keqiang Wang, Yunzeng Zou, Junbo Ge,
Shanghai Inst of Cardiovascular Diseases, Shanghai, China
A. Sun: None. X. Jin: None. K. Wang: None. Y. Zou: None. J. Ge: None.
P299Gender-Based Differences in Ozone-Induced
Cardiac Dysfunction
Rajat Sethi, Rama Perepu, Texas A&M Health Science Ctr, Kingsville,
TX; Carlos Garcia, Texas A&M Univ Kingsville, Kingsville, TX; David
Dostal, Texas A&M Health Science Ctr, Temple, TX
R. Sethi: None. R. Perepu: None. C. Garcia: None.
: None.
P300A Novel Highly Selective Adenosine A1 Receptor Agonist
(VCP28) Reduces Ischemia/Reperfusion Injury
at Concentrations that Do Not Affect Heart Rate
Paul J White, Vijay Urmaliya, Colin W Pouton, Shane Devine, Peter
Scammells, Monash Univ, Parkville, Australia
Whilst adenosine A1 receptor agonists have repeatedly been
shown to protect the ischaemic myocardium, the clinical
use of these agents is limited by strong cardiodepressant
effects. The cardioprotective effects of a novel adenosine A1
receptor agonist N6-(2,2,5,5-tetramethylpyrrolidin-1-yloxyl-3ylmethyl) adenosine (VCP28) were compared with the selective
adenosine A1receptor agonist N6-cyclopentyladenosine
(CPA) in a H9c2(2-1) cardiac cell line-simulated ischemia (SI)
model (12 hours) and a global ischemia (30 minutes) and
reperfusion (60 minutes) model in isolated rat heart model.
H9c2(2-1) cells were treated with CPA and VCP28 at the start
of ischemia for entire ischemic duration, whereas isolated
rat hearts were treated at the onset of reperfusion for 15
minutes. In a H9c2(2-1) cell SI model, CPA and VCP28 (100
nM) significantly (P , 0.05, n = 5–6) reduced the proportion
of nonviable cells (30.88% 6 2.49% and 16.17% 6 3.77% of
SI group, respectively) and lactate dehydrogenase efflux. In
isolated rat hearts, CPA and VCP28 significantly (n = 6–8, P ,
0.05) improved postischemic contractility (dP/dtmax, 81.69%
6 10.96%, 91.07% 6 19.87% of baseline, respectively), left
ventricular developed pressure, and end diastolic pressure and
reduced infarct size. The adenosine A1 receptor antagonist
DPCPX abolished the cardioprotective effects of CPA and
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
The anti-atherosclerotic mechanism of Probucol remains to be
elucidated. Studies confirmed the key role of Dendritic Cells
(DCs) and cytoprotective role of heme oxygenase 1(HO1) in the
development of atherosclerosis. Here, we tested the hypothesis
that probucol exerts is anti-atherosclerotic effect by inhibiting
maturation of DCs via HO1. [Method]Immature DCs were derived
from purified human monocytes; HO1 siRNA, probucol, ox-LDL
or LDL was added to different groups of medium. The maturation
markers of DCs were analyzed by Flow cytometric analysis,
including CD1a, CD40, CD86, HLA-DR, endocytosis function
and the cytokines secretions of culture supernatants (IL-4,TNF-α)
by ELISA. The expression of HO1, STAT1 (signal transducers and
activators of transcription 1) and STAT1(Tyr 701) phosphorylation
were analyzed by Westernblot and the expression of CIITA(MHC
II transactivator) by Real Time PCR.[Results] Ox-LDL promoted
immune maturation of DCs : MIF of CD86,CD 40, CD1a and
HLA-DR was significantly increased after ox-LDL stimulation
(7697±672 vs 2120±662, 7844±754 vs 843±109 ,323±31
vs 272±14,2636±173 vs 1443±45,respectively); decreased
endocytosis function (26256±354 vs 34511±3078); increased
TNF-α(43±5 vs 30±0.3 pg/ml), suppressed IL-4 (852±9 vs
793±11 pg/ml)2.Probucol inhibited immune maturation of DCs
,which was most obviously in ox-LDL treated group : MIF of
CD 86 CD 40, CD1a and HLA-DR was significantly decreased
after probucol treatment (7697±672 vs 3202±660,7844±754 vs
843±109,323±31 vs 56±31, 2636±173 vs 304±60), endocytosis
(26256±1689 vs 109568±4195), TNF-α(43±5 vs 14±0.6 pg/ml),
IL-4(793±11 vs 1037±17 pg/ml) (P<0.05). 3.After silencing of the
expression of HO-1 by siRNA, immune maturation markers of
DCs were increased than respective non-silencing group ,such
as CD86, CD 40, HLA-DR (549±19 vs 353±23, 2645±307vs
524±119,1179±66 vs 766±23,respectivly), endocytosis function
were suppressed (7408±229 vs 10860±706), TNF-α was
increased (22±0.2 vs 14±0.6 pg/ml), IL-4 was decreased (926±2
vs 1037±17 pg/ml) (P<0.05), STAT1(701) phosphorylation and
CIITA expression level were increased.[Conclusion]Probucol
significantly inhibited immune maturation of DCs and is
associated with HO1, STAT1/CIITA signaling pathway.
Sex related differences have been noted in cardiovascular
disease where females have a lower incidence of heart failure,
and a higher rate of heart failure survival. On-the-other-hand,
some studies have reported increased mortality in women
compared with men. Recent data also suggest that women
have an increased risk of death due to O3 air pollution, which
is in contrast to reports that short-term variations in gaseous
pollutants are associated with an increase in hospitalization
for cardiac disease that is not modified by gender. It has been
speculated that because of the nature of the photochemical
equilibrium of O3 in the ambient environment and due to
other confounding factors epidemiological investigations
of the health effects of O3 may be using O3 as an exposure
surrogate for other oxidants or co-pollutants, some of which
may be interfering with response to O3. The present study
in a controlled exposure protocol tested the hypothesis that
female rats compared to male have decreased sensitivity
to cardiac injury subsequent to O3 exposure. Age matched
male/female rats were exposed 8 hrs/day for 28 and 56 days
to filtered air or 0.8 ppm O3. In order to assess the chronic
effects to O3, in-vivo cardiac function was assessed, 24 hrs
after termination of the O3 exposure. Compared to female
rats, LVDP values significantly decreased in O3 exposed male
rats. This enhanced attenuation of cardiac function in male
animals was associated with increased myocardial TNFalpha (TNF-α) levels and decreased myocardial activities of
superoxidase dismutase (SOD). These novel findings suggest
that decreased attenuation of cardiac function in O3 exposed
females compared to males exposed to similar conditions was
associated with decreased inflammatory mediator production
and decreased oxidative stress. Identifying the underlying
factors for gender based variations in ozone response is very
important to recognize at-risk groups who would benefit from
preventive strategies. In addition identification of those at
risk, their degree of sensitivity will assist with the cost-benefit
analysis of “safe” exposure levels in the public health setting.
The long-term goal of this study is in guiding regulatory
policies for reduced environmental related health costs.
109
Poster Presentations (continued)
VCP28 in both models. At the concentrations used in the
ischaemia models, VCP28 had no effect on heart rate, unlike
CPA. In conclusion, the adenosine A1 receptor agonist VCP28
has cardioprotective equal effects to the prototype A1 agonist
CPA at concentrations that have no effect on heart rate.
two major isoforms expressed in the heart, CaMKIIδ and
CaMKIIγ, respectively. Both single and double KO models
were examined. In addition, we developed a reliable approach
to express CaMKII δB and δC splice variants in CaMKIIδnull mice using adeno-associated-virus serotype 9 (AAV9)
to investigate their specific roles in I/R. Methods and
Results: A surgical in vivo I/R model was used following two
protocols: 30 minutes ischemia with 48 hours of subsequent
reperfusion (30/48) and 60 minutes ischemia followed by a 24
hour reperfusion period (60/24). Planimetric measurements
of infarct size after staining with Evans Blue and Triphenyl
tetrazolium chloride showed no significant difference between
CaMKII KO and control groups (infarct-zone/area-at-risk ratio
for 30/48-protocol: CaMKIIγ-/- vs. C57/Bl6: 0.49 ± 0.04 vs.
0.43 ± 0.05, p=0.43, n≥6; CaMKIIδ-/- vs. C57/Bl6: 0.42 ± 0.04
vs. 0.44 ± 0.04, p=0.69, n≥19; infarct-zone/area-atrisk ratio
for 60/24-protocol: CaMKIIδ-/- vs. C57/Bl6: 0.52 ± 0.02 vs.
0.56 ± 0.03, p=0.33, n≥8; CaMKIIγloxP/loxPδloxP/loxP; alphaMHCCre vs. CaMKIIγloxP/loxPδloxP/loxP: 0.52 ± 0.03 vs. 0.52 ± 0.04,
p=0.86, n≥9). Consistently, no differences in high-sensitive
serum troponin T levels 24 hours after onset of ischemia
could be detected. Neither histologic TUNEL-staining nor
Caspase3/7-acivity assays revealed any significant differences
in apoptotic cell death processes. Importantly, specific
expression of CaMKII δB or δC splice variants via AAV9 gene
transfer — at approximate endogenous protein levels — in
CaMKIIδ-/- mice had no effect on infarct size and necrotic or
apoptotic markers. Conclusion: Our findings indicate that
CaMKII is not critical for myocardial infarct size and apoptosis
in response to I/R.
P.J. White: None. V. Urmaliya: None. C.W. Pouton: None. S. Devine:
None. P. Scammells: None.
P301Investigation of the Mechanism of the Cardioprotective
Effects of Flavonols in Langendorff Perfused Rat Hearts
ABSTRACTS
Owen L Woodman, RMIT Univ, Bundoora, Australia; Cheng X Qin,
Spencer J Williams, Univ of Melbourne, Parkville, Australia
We investigated the mechanism(s) of flavonol-induced
cardioprotection using a flavonol which inhibits calcium
utilization and has antioxidant activity 3’,4’-dihydroxylflavonol,
a flavonol which only affects calcium activity 4’-OH-3’-OCH3
flavonol and a water-soluble flavonol with selective antioxidant
activity DiOHF-6-succinamic acid in rat isolated hearts. Hearts
were perfused with physiological solution using a Langendorff
apparatus and subjected to global, no-flow 20-min ischaemia
followed by 30-min reperfusion. Hearts (n = 6–8 per group)
were randomly treated with vehicle (0.05% DMSO), DiOHF,
4’-OH–3’-OCH3 flavonol or DiOHF-6-succinamic acid (all
10 µM). Flavonols were included in the perfusate for 10 min
before ischaemia and during the entire duration of reperfusion.
In vehicle treated hearts, LV+dP/dt was significantly reduced
at the end of reperfusion (60±8% decrease in LV+dP/dt)
compared to the pre-ischaemic level. Furthermore, LDH release
(895±121 U/L) was significantly elevated during reperfusion
compared to shams (70±10 U/L) and total eNOS expression
was significantly lower in vehicle-treated hearts. In comparison,
DiOHF treatment significantly improved LV function upon
reperfusion (35±6% reduction in LV+dP/dt), significantly
decreased the level of LDH (265±66 U/L) and preserved total
eNOS expression. Hearts treated with the antioxidant DiOHF6-succinamic acid had a similar preservation of contractility,
resulting in a 36±5% reduction in LV+dP/dt, and a significantly
lower release of LDH (388±57 U/L) and preserved total eNOS
expression. In contrast, hearts treated with 4’-OH–3’-OCH3
showed similar impairment of contractility to the vehicle group.
DiOHF-6-succinamic acid also exerted cardioprotection when
given only during reperfusion but not when administered only
prior to ischaemia. This study demonstrated that flavonolinduced cardioprotection relies on the antioxidant activity and is
mainly exerted during the reperfusion phase. The water-soluble
DiOHF-6-succinamic acid with selective antioxidant activity
may lead to the development of a new cardioprotective agent
effective when applied at reperfusion and therefore appropriate
for clinical use.
O.L. Woodman: G. Consultant/Advisory Board; Modest; Neuprotect
Pty Ltd. C.X. Qin: None. S.J. Williams: None.
P302CaMKII Is Not Critical for Ischemia/Reperfusion Injury
Martin Weinreuter, Lorenz H Lehmann, Michael Kreußer, Univ of
Heidelberg, Heidelberg, Germany; Hermann-Josef Gröne, German
Cancer Res Ctr, Heidelberg, Germany; Oliver J Müller, Univ of
Heidelberg, Heidelberg, Germany; Eric N Olson, UT Southwestern
Medical Ctr, Dallas, TX; Hugo A Katus, Johannes Backs, Univ of
Heidelberg, Heidelberg, Germany
110
bjectives: The role of the Calcium/calmodulin-dependent
O
protein kinase II (CaMKII) in Ischemia/Reperfusion of the
heart (I/R) has not completely been clarified yet — partly due
to lacking evidence from animal studies with specific gene
knockout (KO) strategies. Moreover, the contribution of the
nuclear and cytosolic CaMKIIδ splice variants δB and δC to
I/R-induced myocardial damage have been controversially
discussed. To investigate the role of CaMKII in myocardial
I/R we used several KO mice with genetic deletion of the
. Weinreuter: None. L.H. Lehmann: None. M. Kreußer: None.
M
H. Gröne: None. O.J. Müller: None. E.N. Olson: None. H.A. Katus:
None. J. Backs: None.
P303Panaxnotogensengsaponins Promotes Angiogenesis and
Enhances the Buildup of Effective Coronary Collateral
Circulation in Ischemic Myocardium
Teng Zhang, Wen-jian Wang, Clinical Inst of Integrated Western and
Chinese Med, Yueyang Hosp, Shanghai Univ of Traditional Chinese
Med, Shanghai, China
Background: Ischemic heart disease (IHD) is a condition
that the blood vessels are narrowed or blocked due to the
deposition of cholesterol plaques on the blood vessel walls.
This affects the supply of blood to the heart and reduces
the supply of oxygen and nutrients to the heart musculature,
which has been regarded as the largest threat to the public
health worldwide. PNS is a purified natural product extract
that has been proved effective and widely used clinically in
preventing and treating IHD in China. However, the detailed
mechanisms of action of PNS remain to be addressed. The
present study aims to explore the effect of PNS on modulating
angiogenesis in vitro and in vivo to better understand its
mechanism of action in treating IHD. Methods and Results:
In this study, myocardial ischemia rat model and endothelial
cell derived from human capillary blood vessel (ECV304)
were utilized. Our data revealed that PNS significantly
increased capillary blood vessel density in isoproterenolinduced rat ischemic myocardium model. It also decreased
the pathological damage and ameliorated the ultrastructure
of myocardial cells in rat ischemic myocardium as assessed
by light microscopy, transmission electron microscopy and
immunohistochemistry. At cellular level, PNS significantly
decreased the number of apoptotic cells in rat ischemic
myocardium model. Mechanistic study utilizing microarray
and real-time PCR were further performed to examine
the gene expression profile. The data revealed that PNS
significantly upregulated the mRNA expression of a panel of
angiogenic growth factors and their receptors such as VEGF,
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Poster Presentations (continued)
VEGFR2 and FGFR1. In vitro study revealed that PNS did
not exhibit significant effect on the proliferation of ECV304
cells under normoxia culture condition; however, it facilitated
the proliferation of ECV304 cells under hypoxia condition,
suggesting the selective effect of PNS on angiogenesis
under ischemic/hypoxic conditions. Conclusions: Our study
demonstrated for the first time that PNS exerts its therapeutic
effect on IHD through the mechanisms that in part involve
regulating the expression of important angiogenic growth
factor, which coordinately enhance the build-up of effective
coronary collateral circulation in ischemic myocardium.
TNF-α in 4 and 8 week O3 exposed animals were followed by
decreases in cardiac caveolin-1 levels. Acute activation of the
serine-threonine kinase Akt is cardioprotective and reduces
both infarction and dysfunction after ischemia/reperfusion
injury (IRI). Akt is activated in samples from patients with
chronic heart failure, however, less is known about the chronic
effects of Akt activation in the hearts from ozone exposed
patients. Although caveolin-1 has been shown to negatively
regulate AKT expression; a cell survival pathway, regulatory
role of AKT in hearts from ozone exposed subjects has never
been studied. Sprague Dawley rats were exposed 8 hr/day for
28 and 56 days to filtered air or 0.8 ppm O3. In order to assess
the chronic effects to O3, expressions of AKT and p-AKT were
determined by wetern blotting in hearts extracted from rats,
24 hr after termination of air or O3 exposure. Compared to
rats exposed to filtered air, total AKT levels were significantly
increased in all O3 exposed animals. Similar results were
seen with pAKT levels. Since earlier studies have determined
detrimental effects of chronic activation of AKT in ischemia
reperfusion injury, our novel findings suggest the interesting
possibility of a mechanism by which chronic Akt activation
can become maladaptive after exposure to chronic levels of
ozone. It is possible that PI3K-dependent but Akt-independent
effectors are required for full cardioprotection. Studies are
currently in progress to prove this hypothesis and complete
study will be presented at the meeting in New Orleans in 2011.
T. Zhang: None. W. Wang: None.
P304Salvianolic Acid B Suppresses Human Monocyte-Derived
Dendritic Cell Maturation via PPARγ Activation
Aijun Sun, Hongying Liu, Shijun Wang, Dazhuo Shi, Lei Xu, Yong
Cheng, Keqiang Wang, Keji Chen, Yunzeng Zou, Gunbo Ge, Shanghai
Inst of Cardiovascular Diseases, Zhongshan Hosp, Fudan Univ,
Shanghai, China
Background and purpose: Salvianolic acid B (Sal B), a watersoluble antioxidant derived from a medicinal herb, is known
to be effective in the prevention of atherosclerosis. Here, we
tested the hypothesis that the anti-atherosclerotic effect of Sal
B might be mediated by suppressing human monocyte-derived
dendritic cells (DCs) maturation. Experimental approach: DCs
was derived by incubating purified human monocytes with
GM-CSF and IL-4. DCs was pre-incubated with or without Sal
B and stimulated by oxidized low density lipoprotein (ox-LDL) in
the presence or absence of PPAR-γ siRNA. Expression of DCs
membrane molecules were analyzed by FACS, cytokines were
measured by ELISA, and TLR4 associated signaling pathway
was determined by Western blotting. Key results: Ox-LDL
promoted DCs maturation, stimulated CD40, CD86, CD1a,
HLA-DR expression and IL-12, IL-10, TNF-α production and
upregulated TLR4 signaling. These effects could be significantly
inhibited by Sal B. Sal B also triggered PPAR-γ activation and
promoted PPAR-γ nuclear translocation, attenuated ox-LDLinduced upregulation of TLR4 and myeloid differentiation
primary-response protein 88 (MyD88), and inhibited
downstream p38-MAPK signaling cascade. Knocking down
PPAR-γ through siRNA transfection significantly blocked above
mentioned effects of Sal B. Conclusions and implications:
Our data suggested that Sal B effectively suppressed ox-LDLinduced DCs maturation through PPAR-γ activation.
P305Detrimental Effects of Chronic Activation of AKT in Hearts
from Ozone-Exposed Healthy Adult Rats
Rajat Sethi, Ashoka Bandla, Magdalena Ramirez, Shubham
Manchanda, Texas A&M Health Science Ctr, Kingsville, TX; Carlos
Garcia, Texas A&M Univ Kingsville, Kingsville, TX; David Dostal, Texas
A&M Health Science Ctr, Temple, TX
Death of cardiomyocytes results in decreased cardiac
function, which correlates with overall morbidity and mortality
in many different clinical settings. For these reasons,
understanding the intracellular signaling pathways that control
cardiomyocyte survival has significant clinical implications.
Each year in the US 60,000 cardiovascular deaths have
been linked to environmental pollutants such as ozone
(O3). Earlier studies from our laboratory have shown that
myocardial dysfunction, subsequent to chronic O3 exposure,
in normal adult rats. This attenuation of cardiac function was
associated with increased myocardial TNF-alpha (TNF-α)
levels. Progressive increases in the expression of myocardial
. Sethi: None. A. Bandla: None. M. Ramirez: None. S. Manchanda:
R
None. C. Garcia: None. D. Dostal: None.
P306Maintenance of Intracellular Zinc Homeostasis
Contributes to the Mechanism by Which Postconditioning
Protects the Heart from Ischemia/Reperfusion Injury
Zhelong Xu, Seok Jai Kim, Juan Zhou, Jin Huh, Rachel McIntosh,
David Zvara, UNC Chapel Hill, Chapel Hill, NC
Myocardial ischemia/reperfusion causes zinc depletion and
supplementation of zinc can attenuate ischemia/reperfusion
injury. The purpose of this study was to determine the role
of the intracellular homeostasis in the cardioprotective
mechanism of ischemic postconditioning. Isolated rat hearts
were subjected to 30 min regional ischemia followed by 2
h of reperfusion. Postconditioning was elicited by six cycles
of 10 second reperfusion and 10 seconds ischemia. Zinc
concentration was measured with inductively coupled plasma
optical spectroscopy (ICPOES). Upon reperfusion cytosolic
zinc levels were dramatically decreased but this was prevented
by postconditioning, indicating that postconditioning
can prevent reperfusion-induced zinc loss. Moreover,
postconditioning could also attenuate mitochondrial zinc loss
upon reperfusion. In agreement with these observations, the
anti-infarct effect of postconditioning was reversed by the zinc
chelator N,N,N’,N’-tetrakis-(2-pyridylmethyl) ethylenediamine
(TPEN), and exogenous zinc given at reperfusion mimicked the
effect of postconditioning by reducing infarct size, suggesting
that intracellular zinc account for the cardioprotective effect
of postconditioning. Finally, postconditioning enhanced
phosphorylation of Akt, ERK, and GSK-3β upon reperfusion,
and these effects of postconditioning were inhibited by
TPEN, indicating that zinc may mediate the cardioprotective
effect of postconditioning by activating the RISK pathway. In
conclusion, these data suggest that postconditioning may
protect the heart by maintaining intracellular zinc homeostasis
and activation of the RISK pathway may account for zincmediated cardioprotection of postconditioning.
ABSTRACTS
. Sun: None. H. Liu: None. S. Wang: None. D. Shi: None. L. Xu:
A
None. Y. Cheng: None. K. Wang: None. K. Chen: None. Y. Zou:
None. G. Ge: None.
Z. Xu: None. S. Kim: None. J. Zhou: None. J. Huh: None.
R. McIntosh: None. D. Zvara: None.
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
111
Poster Presentations (continued)
were female, 70% (28/40) hyperlipidaemic, 64% (25/40) had
history of hypertension and 60% (24/40) were smoker, 27%
(11/40) patients had previous PCI. Using MPI as the reference,
PRIME Delta Map was more sensitive for the detection of
TRMI than the 12-Lead ECG 77% vs. 15%, p 0.0001 and was
also more specific for TRMI 96% vs. 84%, p 0.0001. The PPV
& NPV for Delta Map was 91% & 89% respectively, where as
33.3% & 66.7% for 12-Lead ECG. Conclusion: The PRIME
ECG Delta map is a near patient, rapid, accurate, objective
and intuitive method for detecting TRMI and is superior to the
12-Lead ECG. The Delta Map could potentially facilitate more
rapid identification and appropriate treatment in patients with
coronary artery disease. A larger clinical study is now required.
P307A Novel Protective Effect of the Antidiabetic Drug
Voglibose via GLP-1 Receptor in the Infarcted Heart
Masamitsu Iwasa, Yoshihisa Yamada, Shinji Yasuda, Hiroyuki
Kobayashi, Hiroaki Ushikoshi, Takuma Aoyama, Kazuhiko Nishigaki,
Genzou Takemura, Takako Fujiwara, Hisayoshi Fujiwara, Shinya
Minatoguchi, Gifu Univ, Gifu, Japan
Glucagon-like peptide 1 (GLP-1) reportedly exerts a protective
effect against cardiac ischemia. We hypothesized that
the α-glucosidase inhibitor voglibose, an unabsorbable
antidiabetic drug with cardioprotective effects, may act via
stimulation of GLP-1 receptors. The results of the present
study suggest oral administration of voglibose reduces
myocardial infarct size and mitigates cardiac dysfunction
in rabbits following 30 min of coronary occlusion and 48 h
of reperfusion. Voglibose increased basal and postprandial
plasma GLP-1 levels and reduced postprandial plasma
glucose levels. The infarct size-reducing effect of voglibose
was abolished by treatment with exendin(9–39), wortmannin,
L-NAME or 5-HD, which inhibit GLP-1 receptors, PI3K,
NOS and KATP channels, respectively. Western blot analysis
showed that treatment with voglibose upregulated myocardial
expression of phospho-Akt, phospho-eNOS, phospho-ERK
and HSP27 following myocardial infarction. The upregulation
of phospho-Akt was inhibited by exendin(9–39) and
wortmannin, while the upregulation of phospho-ERK and
HSP27 was inhibited by wortmannin but not by exendin(9–39).
These findings suggest that voglibose reduces myocardial
infarct size through stimulation of GLP-1 receptors, activation
of PI3K-Akt-eNOS and ERK pathways, and the opening of
mitochondrial KATP channels. These findings may provide
new insight into therapeutic strategies for the treatment of
diabetic patients with coronary artery disease.
M. Zeb: B. Research Grant; Modest; Unrestricted research grant from
Medtonic UK and Verathon UK Limited. F. Garty: None. W. Banister:
None. N. Nagaraj: None. N. Curzen: B. Research Grant; Modest;
Unrestricted research grant from Medtronic UK and Verathon UK Limited.
P309ZnT-1 Protects HL-1 Cells from Simulated IschemiaReperfusion Through Activation of Ras-ERK Signaling
Shani Dror, Ofer Beharier, Shiry Levy, Merav Mor, Joy Kahn, BenGurion Univ, Beer-sheva, Israel; Amos Katz, Barzilai Medical Ctr,
Ashkelon, Israel; Arie Moran, Ben-Gurion Univ, Beer-sheva, Israel;
Yoram Etzion, Soroka Univ Medical Ctr, Beer-sheva, Israel
Background: ZnT-1 is a protein that confers cellular resistance
from heavy metal toxicity, but its function in the myocardium
is not clear. Our group recently demonstrated that ZnT-1
regulates calcium influx into cardiomyocytes by interacting
with the regulatory β-subunit of the L-type calcium channel.
In addition, in non-cardiac cells ZnT-1 was found to interact
with Raf-1 kinase leading to downstream activation of MEK/
MAPK signaling. In the present study we investigated the
role of ZnT-1 in myocardial ischemia\reperfusion injury (I\R).
Methods and Results: Cultured cells of cardiomyocyte origin
(HL-1 cells) were exposed to ischemic conditions for 60 min
using sodium cyanide and 2-Deoxi Glucose, followed by 60
min of washout mimicking reperfusion. I\R injury was detected
by measuring LDH release and staining for pro-apoptotic
proteins activation (caspase 3 and 7). Overexpression of ZnT-1
reduced the LDH release following I\R injury to 50.1±2.5 %
of control (n=6, p<0.01) and markedly reduced caspases
staining. Consistently, ERK phosphorylation was increased in
the ZnT-1 transfected cells to 266±27.8 % of control (p<0.01).
Knockdown of endogenous ZnT-1 by shRNA blocked the
phosphorylation of ERK and markedly augmented LDH
release following I/R injury to 287.4±36.9 % of control (n=5,
p<0.01). The protective effect of ZnT-1 following I\R injury
was apparent following pretreatment of HL-1 cells with the
L-type calcium channel blocker nifedipine (1 μM). In contrast,
pretreatment with the MEK inhibitor PD98059 (10 μM)
completely abolished the protective effect of ZnT-1 following
I\R injury (n=3). Moreover, a mutated form of ZnT-1 lacking the
ability to bind Raf-1, failed to protect HL-1 cells from I\R injury
(n=3). Conclusions: ZnT-1 confers cellular resistance from I\R
injury trough its ability to interact with Raf-1 and its ability to
stimulate the Raf/MEK/MAPK signaling pathway. Our findings
suggest an important new role for ZnT-1 in the myocardium.
. Dror: None. O. Beharier: None. S. Levy: None. M. Mor: None.
S
J. Kahn: None. A. Katz: None. A. Moran: None. Y. Etzion: None.
M. Iwasa: None. Y. Yamada: None. S. Yasuda: None. H. Kobayashi:
None. H. Ushikoshi: None. T. Aoyama: None. K. Nishigaki: None.
G. Takemura: None. T. Fujiwara: None. H. Fujiwara: None.
S. Minatoguchi: None.
P308Detection of Transient Regional Myocardial Ischemia with
80-Electrode Body Surface Delta Map and Its Comparison
with Myocardial Perfusion Imaging and 12-Lead ECG
ABSTRACTS
Mehmood Zeb, Wessex Cardiothoracic Unit, Univ of Southampton,
Southampton, United Kingdom; Florence Garty, Univ of Southampton,
Southampton, United Kingdom; Wendy Banister, Wessex
Cardiothoracic Unit, Univ of Southampton, Southampton, United
Kingdom; Nirmala Nagaraj, Southampton Univ Hosp, Southampton,
United Kingdom; Nick Curzen, Wessex Cardiothoracic Unit, Univ of
Southampton, Southampton, United Kingdom
112
ackground: The diagnosis of transient regional myocardial
B
ischaemia (TRMI) in patients presenting to the Chest Pain
Clinics is a challenge. Traditionally EET has been most
commonly employed test for diagnosing TRMI, however
due to the limitations of 12-Lead ECG this is no longer
recommended. Thus the clinicians have to rely on more
expensive and time consuming test including Myocardial
Perfusion Imaging (MPI) and Cardiac MRI. Delta Map was
described by this group and is an intuitive colour display
of digitally subtracted ST segment shift derived from two
80-electrode BSM recordings, one at baseline and one at
peak stress. Objectives The purpose of this pilot study was
to asses the diagnostic ability of Delta-Map & 12-Lead ECG
in detection of TRMI, and to compare the results with MPI.
Method: Forty consecutive patients who were referred by two
consultant cardiologist for MPI with a history of angina-like
symptoms were consented and recruited in to this study. The
Delta Map was derived from two 80-electrode PRIME BSM
carried out simultaneously with MPI at rest and peak stress,
using dobutamine.12-Lead ECGs were also recorded at the
same time. Results: The mean age was 68+7.1, 50% (20/20)
P310Connective Tissue Growth Factor Antibody Protects
from Adverse Left Ventricular Remodeling Following
Myocardial Infarction
Laura Vainio, Zoltan Szabo, Raisa Serpi, Heikki Ruskoaho, Risto
Kerkelä, Univ of Oulu, Oulu, Finland
Ischemic heart disease (IHD) is the most common cause
of death in most Western countries, but the therapies for
IHD are lacking. We aimed to determine the potential of
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Poster Presentations (continued)
connective tissue growth factor (CTGF) antibody in protecting
myocardium from long-term deleterious effects of myocardial
infarction. Healthy 8-week old mice were subjected to
myocardial infarction (MI) and at 1 week post-MI the animals
were randomized for treatment with vehicle, metoprolol or
CTGF monoclonal antibody. After six weeks of treatment,
CTGF antibody treated animals showed significantly better left
ventricular (LV) systolic function than the vehicle-treated animals.
LV diastolic function or LV dimensions were not affected by
treatments. Analysis of cardiac sections revealed no difference
in infarct scar size between the experimental groups. However,
RT-PCR analysis revealed a decrease in the expression levels
of fibrosis-related genes in non-ischemic area in the hearts of
CTGF antibody treated animals. In addition, cardiomyocyte
size was significantly reduced in LV sections of CTGF antibody
treated animals compared to vehicle-treated animals. This was
accompanied with a significant decrease in B-type natriuretic
peptide mRNA levels in the LV, whereas expression levels
of atrial natriuretic peptide or beta-myosin heavy chain were
similar in MI groups. There was also a slight decrease in mRNA
levels of interleukin-6 and CTGF in the hearts of CTGF antibody
treated animals. Treatments had no effect on mRNA levels of
transforming growth factor-β1, tumor necrosis factor-α and
vascular endothelial growth factor A following MI. In conclusion,
CTGF antibody treatment attenuates LV hypertrophy and
improves LV systolic function following MI. Blocking CTGF
function by a specific antibody may be useful for prevention of
adverse myocardial remodeling and heart failure.
. Vainio: None. Z. Szabo: None. R. Serpi: None. H. Ruskoaho:
L
None. R. Kerkelä: None.
P311Differential Changes in Caveolin Levels in Lung and Heart
from Ozone-Exposed Adult Healthy Rats
Rajat Sethi, Magdalena Ramirez, Ashoka Bandla, Shubham Manchanda,
Texas A&M Health Science Ctr, Kingsville, TX; Carlos Garcia, Texas A&M.
Univ Kingsville, Kingsville, TX; David Dostal, Texas A&M Health Science
Ctr, Temple, TX
R. Sethi: None. M. Ramirez: None. A. Bandla: None. S. Manchanda:
None. C. Garcia: None. D. Dostal: None.
P312Tracheal Instillation of Diesel Exhaust Particles Exacerbates
Myocardial Ischaemia and Reperfusion Injury in Rats
Sarah Robertson, Ashleigh L Thomson, Catherine A Shaw, Mark R
Miller, David E Newby, Patrick W Hadoke, Gillian A Gray, Univ of
Edinburgh, Edinburgh, United Kingdom
Episodes of increased air pollution are associated with
higher cardiovascular mortality. The adverse effects of air
pollution have been attributed to particulate matter, especially
ultrafine particles. This study addresses the hypothesis that
ultrafine diesel exhaust particles (DEP) exacerbate myocardial
ischaemia reperfusion (I/R) injury secondary to induction of a
systemic inflammatory response. Wistar rats (n=5-6/group)
received DEP (0.5 mg) or saline vehicle by intratracheal
instillation. 6h later I/R was induced either in vivo or ex
vivo in isolated buffer perfused hearts. Lung inflammation
was confirmed 6h after DEP instillation by increased levels
of neutrophils, total protein and IL-6 in bronchoalveolar
lavage fluid. However, there was no evidence for systemic
inflammation as assessed by plasma cytokine levels (IL-6,
TNF-α, and CRP) or by neutrophil priming (CD11b expression)
or activation (CD62L expression). In vivo, systolic blood
pressure was significantly higher in DEP-instilled rats (129
± 7 mmHg) than in saline controls (92 ± 3 mmHg, P<0.01),
consistent with increased autonomic activation. Arrhythmias
occurred intermittently after induction of ischaemia (in total
8.2±1.2 s in the saline group) and were more prevalent in
DEP-instilled rats (32.9±5.0s, P<0.001). Fatal arrhythmias
occurred in 60% of rats receiving DEP but not at all in
saline controls. Following reperfusion, infarct size (extent
of triphenyltetrazolium chloride staining) was significantly
increased after DEP (34.7 ± 1.2 % left ventricle) vs salineinstilled (10.3 ± 1.2 %, P<0.001). Infarct size was similarly
potentiated in hearts isolated from DEP instilled rats and
perfused ex-vivo. Histological examination confirmed the
absence of inflammatory infiltrate in hearts prior to I/R. Prior
exposure to pollution in vivo thus renders the heart more
vulnerable to I/R injury, either in situ in the body or ex vivo
when the heart is isolated from systemic mediators and cells.
Systemic inflammation does not appear to be necessary for
this ‘priming’ effect of DEP. The role of autonomic activation in
promoting cardiac arrythmia in vivo after DEP instillation and
in determining the ability of the heart to withstand subsequent
I/R injury merits further investigation.
S. Robertson: None. A.L. Thomson: None. C.A. Shaw: None. M.R. Miller:
None. D.E. Newby: None. P.W.F. Hadoke: None. G.A. Gray: None.
P313Genetic Deletion of Pim Causes Premature
Cardiac Senescence
ABSTRACTS
Acute exposure to ozone (O3) has been reported to possess
beneficial effects in the injured myocardium. We have
demonstrated that chronic O3 exposure elicits cardiac
dysfunction in rats. These results suggest that acute and
chronic exposures to O3 have different effects on myocardial
function. On the contrary exposure to acute ozone levels
has shown to cause lung injury. Although effects of chronic
exposure to ozone on the lung has not been determined, it
is reasonable to suggest that ozone mediated toxicity may
be tissue specific. It appears that in chronic cardiac injury
subsequent to an ischemic insult, lipid raft proteins such as
caveolin-1 and caveolin-3 play vital roles in the generation
of death and survival signaling in the heart. In lung injury
subsequent to acute O3 exposure, dramatic reductions in
caveolin-1 levels were associated with increases in lung
TNF-α. Our previous studies showed increased expressions
of myocardial TNF-α after chronic O3 exposure. In the present
study we wanted to determine the regulatory role of caveolin-1
and caveolin-3 in hearts and lungs from rats exposed to
chronic O3 levels. Sprague Dawley rats were exposed 8 hrs/
day for 28 and 56 days to filtered air or 0.8 ppm O3. In order
to assess the chronic effects to O3, levels of caveolin-1 and
caveolin-3 in the lung and heart tissue were measured 24
hrs after termination of the O3 exposure. Caveolin-1 levels
in hearts from the ozone exposed groups were significantly
decreased compared to air exposed rats; while caveolin-1
levels in lungs from the same animals were not different from
the air exposed group. On the other hand caveolin-3 levels
in hearts from the ozone exposed groups increased and
decreased after 4 and 8 week of ozone exposure compared
to air exposed rats; while caveolin-3 levels in lungs from the
same animals were not different from the air exposed group.
These novel findings suggest the interesting possibility that
caveolin expressions subsequent to chronic ozone exposure
may be tissue specific.
Shabana Din, Michael McGregor, Brandi Bailey, Sadia Mohsin, Daniele
Avitabile, Natalie Gude, Mark A Sussman, SDSU, San Diego, CA
Rationale: Cardiac aging is defined as increased
cellular senescence and loss of survival signaling within
cardiomyocytes and the cardiac stem cell population. To
prevent myocardial, aging it is important to reverse the
characteristic aging phenotype. Pim-1 is a cardioprotective
kinase downstream of AKT, and has been shown to be proproliferative and anti-apoptotic. Pim-1 mediated antagonism of
myocardial aging will lead to a decrease in cardiac senescence,
maintain contractile performance, and prolong the lifespan
of an organism. Objective: Demonstrate that myocardial
senescence is antagonized by Pim-1 mediated signaling
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113
Poster Presentations (continued)
and loss of Pim-1 activity leads to premature aging of the
myocardium and associated decline in cardiac performance.
Methods and Results: Pim Triple KnockOut (PTKO) mice, in
which all three isoforms of Pim are genetically deleted, develop
premature phenotypic hallmarks of myocardial senescence.
Elevated levels of the tumor suppressor p53 and senescence
marker p16 are present in hearts of PTKO mice at two months
of age. Increased p16 expression is consistent with observed
induction of Ets-1, a transcription factor that activates p16
transcription in PTKO mice. Levels of Id1 and Id2, positive
transcriptional regulators of the cell cycle, are decreased
in hearts of PTKO mice. Hemodynamic analyses reveal
impaired cardiac function in PTKO mice at two months of age.
Conversely, cardiac specific overexpression of Pim1-kinase
attenuates the senescent phenotype. Hypoxia treatment of
neonatal rat cardiomyocytes infected with Pim-1 encoding
adenovirus results in reduction of p16 accumulation as seen
by qRT-PCR. Pim-1 overexpression increases expression of
c-myc, thereby upregulating Id2 and inhibits p16 induced
senescence. Cardiac Pim-1 transgenic mice show a delay in
the onset of senescence as evidenced by decreased frequency
of p16 positive cells in the myocardium. Conclusion: Pim-1
inhibits cardiac senescence and is important for maintaining
cardiac function. Therefore, selective manipulation of Pim-1
should be pursued as a therapeutic target to promote and
extend myocardial cellular lifespan and function.
. Din: None. M. McGregor: None. B. Bailey: None. S. Mohsin:
S
None. D. Avitabile: None. N. Gude: None. M.A. Sussman: None.
This research has received full or partial funding support from the
American Heart Association, Western States Affiliate (California,
Nevada & Utah).
P314Grainyhead-like 3 Plays an Essential Role in Endothelial
Cell Function in a NO-Dependent Manner
ABSTRACTS
Margarete Lukosz, Arne Mlynek, Joachim Altschmied, Judith
Haendeler, IUF, Duesseldorf, Germany
114
Aging of human endothelial cells (EC) is associated with
reduced nitric oxide (NO) bioavailability, decreased migratory
capacity and an increase in Src kinase activation and
apoptosis sensitivity. We recently identified the transcription
factor grainyhead-like 3 (GRHL3) as a pro-migratory
transcription factor in EC. A role for GRHL3 in aging processes
was suggested by reduced expression in brains from old
mice. Therefore, we wanted to investigate the regulation
of GRHL3 by NO and Src kinase and GRHL3 effects on
NO-bioavailabilty, apoptosis and migration. We treated EC
either with physiological concentrations of NO or the Src
kinase inhibitor PP2. In both cases GRHL3 expression was
increased (3.75 fold and 4.50 fold, respectively). In addition,
both treatments induced migration and inhibited apoptosis.
Interestingly, overexpression of GRHL3 activated endothelial
nitric oxide synthase (eNOS), its upstream regulator Akt
and subsequently increased the S-NO content of EC. This
demonstrates that GRHL3 enhances NO-bioavailability in
EC, which is inseparably tied to apoptosis protection and
migration. Along this line, GRHL3 overexpression reduced
apoptosis of EC (1.89 fold reduction of basal apoptosis
vs. empty vector transfected cells). Interestingly, this antiapoptotic effect was dependent on NO synthesis by eNOS,
since the eNOS inhibitor L-NMMA completely abrogated the
protective effect of GRHL3. Having demonstrated a promigratory effect of GRHL3, we wanted to know whether this
effect is mediated by induction of vascular endothelial growth
factor (VEGF) expression. Surprisingly, GRHL3 overexpression
did not change VEGF protein levels. To exclude a bystander
effect of GRHL3 in EC migration, we knocked down
expression with shRNA. Reduction of GRHL3 mRNA levels
decreased basal and NO-induced EC migration (scr: 73 +/- 16
migrated cells; shGRHL3: 26 +/- 12 migrated cells; scr+NO:
149 +/- 19 migrated cells; shGRHL3+NO: 36 +/- 10 migrated
cells) demonstrating an essential role in this process. Taken
together, these data suggest that GRHL3 is essential for EC
functions compromised during aging.
M. Lukosz: None. A. Mlynek: None. J. Altschmied: None.
J. Haendeler: None.
P315S-Glutathiolation of Low-Molecular-Weight PTP Regulates
VEGF-Angiogenic Signal
Mohammed A Abdelsaid, Azza El-Remessy, Univ of Georgia,
Augusta, GA
While reparative angiogenesis improves outcome of stroke and
myocardial infarction; pathological angiogenesis aggravates
tumors, psoriasis and diabetic retinopathy. We have previously
shown that low levels of peroxynitrite (PN, 1µM) are required to
mediate VEGF’s angiogenic signal. Prior studies showed that
low molecular weight protein tyrosine phosphatase (LMWPTP) can regulate activation of VEGFR2 and focal adhesion
kinase (FAK), key proteins involved in VEGF angiogenic signal.
Here, we test the hypothesis that VEGF-induced peroxynitrite
regulates LMW-PTP activity via reversible S-glutathiolation.
Our results using human microvascular endothelial (HME)
cells showed that VEGF (20 ng/ml) caused immediate (1,5
min) and transient negative shift in redox-state assessed
by reduced-glutathione (GSH) level that was restored back
to baseline by 15–30 minutes. In parallel, total free thiols of
LMW-PTP; assessed by 5-iodoacetamide; were immediately
oxidized (1, 5 min) and recovered after 15–30 minutes.
PN (1μM) mimicked VEGF’ actions by inducing oxidative
inhibition of LMW-PTP and FAK phosphorylation that peaked
at 15 min. Studies using anti-GSH antibody demonstrated
strong s-glutathiolation of LMW-PTP thiols that peaked at
(5–10min) under non-reducing conditions and were completely
abrogated in DTT-treated samples. These effects were
associated with impaired tyrosine phosphorylation of LMWPTP. PN caused concentration-dependent significant inhibition
of LMW-PTP phosphatase activity reaching maximum
at 0.5mM. Under pathological conditions recapitulated
by combining with peroxynitrite (0.5mM), VEGF caused
immediate yet permanent negative shift in cellular redox-state,
LMW-PTP inactivation and sustained FAK activation in HME
cells. Modulating cellular redox-state to reductive stress using
N-acetyl cysteine (NAC, 1mM) or FeTPPS (2.5μM) prevented
VEGF-induced angiogenesis in vitro and in vivo using hypoxiainduced neovascularization mouse model. Taken together,
shifting redox-state to oxidative stress or reductive stress can
impair VEGF’s signal and achieving a balanced redox-state is
critical to facilitate VEGF’ angiogenic signal.
M.A. Abdelsaid: None. A. El-Remessy: None.
This research has received full or partial funding support from the
American Heart Association, Greater Southeast Affiliate (Alabama,
Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).
P316Cysteine-Independent Interactions of Thioredoxin-1
and Apex1: Implications for Endothelial Cell Apoptosis
and Aging?
Tim-Christian Zschauer, Lisa Beissel, Joachim Altschmied, Judith
Haendeler, IUF, Duesseldorf, Germany
Aging processes are associated with increased intracellular
formation of reactive oxygen species. One redox regulator
is the ubiquitously expressed oxidoreductase Thioredoxin-1
(Trx). It contains two active site cysteines and has antioxidative and anti-apoptotic properties. Trx levels are
reduced in senescent endothelial cells (EC), leading to
increased apoptosis sensitivity. Several functions of Trx
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Poster Presentations (continued)
are enhanced by its interaction with AP-endonuclease 1
(APEX1). APEX1 is a multifunctional protein and in concert
with Trx-1 a reductive activator of e.g. the transcription
factor AP-1, suggesting at least an interaction of Trx and
APEX1 in vivo. Recent evidence suggests that also APEX1
levels are decreased in senescent cells. Therefore, the aim
of this study is to understand the function of APEX1 in
concert with Trx in EC. We first investigated whether APEX1,
similar to Trx has anti-apoptotic properties in EC. Indeed,
overexpression of APEX1 inhibited apoptosis. To analyze
where the Trx/APEX1 interaction occurs, we performed
fluorescence microscopy demonstrating a colocalization
throughout the whole cell. To study the interaction in more
detail, we carried out coimmunoprecipitations from nuclear
and cytosolic extracts and demonstrated their association
in both fractions. To determine whether disulfide bridges are
involved in the Trx/APEX1 association, we mutated APEX1
cysteines 65, 93 and 310 previously shown to interact
with cysteines in other proteins, to serines. This mutant
bound Trx in the nucleus and cytosol of EC with the same
efficiency as APEX1 wildtype. Moreover, a Trx mutant with
serine mutations of active site cysteines (Trx(C32S/C35S))
was still able to bind to endogenous APEX1. These data
demonstrate that the Trx/APEX-1 interaction is independent
of disulfide bridge formation. We are currently analyzing
a set of APEX1 deletion mutants for their interaction with
Trx-1 and a potential loss of anti-apoptotic properties Exact
delineation of the Trx/APEX1 interaction surface will in the
future allow the development of small peptides containing
the interaction domains of Trx or APEX1 respectively, in
order to enhance transcription factor activity and bypass the
loss of antioxidative capacity during aging processes.
T. Zschauer: None. L. Beissel: None. J. Altschmied: None.
J. Haendeler: None.
P317Acute Exercise Stress and ROS Signaling: Activation of
Nrf2/ARE-Dependent Antioxidant Defense Mechanisms in
the Mouse Myocardium
Corey J Miller, Kalavathy Ramachandran, Gayatri D Khanderao, Univ
of Utah Health Care, Salt Lake City, UT; Sankaranarayanan Kannan,
M D Anderson Cancer Ctr, Houston, TX; Vasanthi Rajasekaran, Univ
of Utah Health Care, Salt Lake City, UT; Christoph Boheme, Univ of
Utah, Salt Lake City, UT; J David Symons, Coll of Exercise Physiology,
Univ of Utah Health Care, Salt Lake City, UT; Christopher J Davidson,
Univ of Utah Health Care, Salt Lake City, UT; Rajasekaran Namakkal
Soorappan, Univ of Utah Sch of Med, Salt Lake City, UT
C.J. Miller: None. K. Ramachandran: None. G. Khanderao: None.
S. Kannan: None. V. Rajasekaran: None. C. Boheme: None. J. Symons:
None. C.J. Davidson: None. R. Namakkal Soorappan: None.
This research has received full or partial funding support from the
American Heart Association, Western States Affiliate (California,
Nevada & Utah).
P318Vasoprotective Effects of an Apo A-I Mimetic Peptide
C White, Geeta Datta, Dale Parks, G Anantharamaiah, Univ of Alabama
at Birmingham, Birmingham, AL
Hepatic ischemia/reperfusion (I/R) injury is associated with
several pathologic states and contributes to the development
of systemic vascular complications. High density lipoprotein
(HDL) and apolipoprotein (apo) A-1 possess antioxidant and
anti-inflammatory properties and reduce tissue injury in several
models of I/R. In the current study, we tested the hypothesis
that the synthetic apoA-I mimetic peptide 4F, similar to HDL
and apoA-I, exerts cytoprotective effects in a murine model of
hepatic I/R injury. C57BL/6 mice were randomized to receive
4F (5 mg/kg, IP) or an equivalent volume of saline vehicle.
Mice were then subjected to 45 min of segmental hepatic
ischemia, followed by 24 hr reperfusion. Sham-operated mice
served as a control. Hepatic I/R injury was associated with
an increase in alanine transaminase (ALT: 980±95U/mL) and
xanthine oxidase (XO: 700±100U/mL) release compared to
sham-operated controls (ALT=27±8U/mL; XO=100±10U/mL).
Endothelium-dependent relaxation was impaired in aortae of
I/R mice (Rmax=20%) compared to controls (Rmax=72%). This
response was associated with a decrease in eNOS protein
and an increase in iNOS and plasma levels of nitric oxide
metabolites (NOx). Further, immunohistochemical studies
revealed an increase in the nitration (3-NT) and chlorination
(3-CT) of protein tyrosine residues in aortic sections from
I/R mice that was associated with an increase in tissue
myeloperoxidase (MPO) content. 4F treatment significantly
improved the relaxation response in the aorta of I/R mice
(Rmax=60%), prevented the upregulation of iNOS and NOx and
reduced tissue protein nitration/chlorination. 4F treatment was
further associated with a reduction in hepatocellular injury in
I/R mice, as revealed by a decrease in ALT (110±15U/mL) and
XO (270±50U/mL) release. Collectively, these data suggest
that 4F reduces systemic vascular injury via cytoprotective
effects at the level of the liver. ApoA-I mimetic peptide-based
therapies may be effective in reducing vascular complications
associated with clinical procedures such as transplantation.
ABSTRACTS
Background: Cellular defense mechanisms are crucial for
maintaining intracellular redox state and mitigating free radical
accumulation with aging. Nuclear Erythroid 2 p45 related
factor-2 (Nrf2) regulates basal and inducible expression of
numerous cytoprotective/antioxidant genes. We hypothesize
that acute exercise will induce ROS, which triggers Nrf2/ARE
signaling and promotes myocardial defense mechanisms.
Methods: Age-matched wild-type (WT) and Nrf2-/- (KO)
mice at 2 and >20 months were subjected to acute exercise
stress (AES) and then we assessed the activation of Nrf2/
ARE-dependent transcriptional mechanisms in the heart.
Myocardial ROS was measured by electron paramagnetic
resonance (EPR) analysis. Results: Under basal conditions,
total ROS and GSH levels were identical at 2 months,
whereas they were significantly impaired in Nrf2-KO when
compared to Wt myocardium at ~20 months indicating that
Nrf2-deficiency is coupled with blemished redox potential.
Upon AES, the young WT and KO mice exhibited oxidative
stress (OS), but the WT were able compensate for the stress
by increasing Nrf2 nuclear translocation and subsequent
upregulation of cytoprotective genes. However, the aged (WT
& KO) mice developed OS in response to AES. The degree of
OS was several fold higher in the aged Nrf2-KO mice when
compared with WT, suggesting an important age dependent
function for Nrf2 in the myocardium. Western blot analysis
revealed significant down regulation of major antioxidants
(GCS, Nqo1, Ho1, catalase, G6pd and Gsr) in KO mice, while
WT mice exhibited compensatory antioxidant response to
the AES. Gene expression (qPCR) analysis revealed profound
upregulation of major antioxidants in WT, but there was no
such response occurred in KO mice after AES, suggesting
Nrf2 independent mechanisms are inadequate to protect the
myocardium. Conclusions: Acute exercise induces ROS
and thereby activates Nrf2 in the myocardium. However,
disruption of Nrf2 increases susceptibility of the myocardium
to OS induced damage. Thus Nrf2 signaling might be a
potential therapeutic target to protect the heart from ROS
and/or age dependent ischemia/reperfusion (I/R) injury and
myocardial infarction (MI).
. White: None. G. Datta: None. D. Parks: None.
C
G. Anantharamaiah: None.
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
115
Poster Presentations (continued)
with Ang-II) by echocardiography to elucidate the degree of
pathologic hypertrophy caused by Nrf2 deficiency and Ang-II
infusion. We then analyzed the redox state, ROS levels (by
EPR), protein/mRNA expression for hypertrophic markers, and
Nrf2-dependent antioxidants including enzymes involved in
glutathione metabolism in the heart. Results: Disruption of Nrf2
expression augmented ROS generation and hastened the Ang-II
induced cardiac hypertrophy. Echo analysis of Ang-II infused
mice (at day 15) revealed significantly decreased FS (22%) in
Nrf2-KOs. Though the myocardial glutathione (GSH) content
was cognate between the Wt and Nrf2-KO mice under basal
conditions, it was significantly depleted in the Ang-II treated Nrf2KOs when compared to treated Wts. Protein and mRNA levels
for major antioxidants (NQO1, catalase, G6PD, GCL, etc.) were
significantly diminished in Nrf2-KOs while Wt mice demonstrated
signs of antioxidant compensation — suggesting that Nrf2independent pathways are unable to maintain an adequate
antioxidant capacity under stressful conditions. Conclusions:
Our results demonstrate a critical role for Nrf2 underlying
antioxidant potential in the murine myocardium and loss of Nrf2
may be detrimental to heart. Instigating the Nrf2 antioxidant
pathway would be a potential target for therapeutic intervention.
P319Protein Modulation in Mouse Heart Under Acute and
Chronic Hypoxia
Cecilia Gelfi, Univ degli Studi di Milano/Istituto di Bioimmagini e
Fisiologia Molecolare, Consiglio Nazionale delle Ricerche, Segrate,
Italy; Agnese Viganò, Univ degli Studi di Milano, Segrate, Italy; Michele
Vasso, Istituto di Bioimmagini e Fisiologia Molecolare, Consiglio
Nazionale delle Ricerche, Segrate, Italy; Daniele Capitanio, Roberta
Leone, Univ degli Studi di Milano, Segrate, Italy; Anna Caretti, Michele
Samaja, Ospedale San Paolo, Univ degli Studi di Milano, Milano, Italy
Exploring cellular mechanisms underlying beneficial adaptive
processes and detrimental responses to hypoxia represents
the object of the present study. Signaling molecules controlling
adaptation to hypoxia (HIF-1α), energy balance (AMPK),
mitochondrial biogenesis, (PGC-1α) and the regulation of
autophagic/apoptotic processes, combined with proteomic
dysregulation were assessed. Responses to normobaric
acute (48 hrs FiO2 = 0.08) and chronic (10 days FiO2 = 0.08)
hypoxia in mouse heart proteome were detected applying 2-D
DIGE, mass spectrometry and antigen antibody reactions.
Both in acute (AH) and chronic (CH) hypoxia, results indicated
a deregulation of proteins related to sarcomere stabilization
and muscle contraction. Neither in AH nor in CH HIF-1α
stabilization was detected. In AH, the metabolic adaptation to
oxygen lack is controlled by AMPK activation and sustained by
the massive up-regulation of adenosylhomocysteinase and of
acetyl coenzyme A synthetase. AH is also characterized by an
up-regulation of Bnip 3 which is known to induce mitophagy
and decreasing oxidative damage. PGC-1α, a master
regulator of mitochondrial biogenesis, was down- regulated.
CH was characterized by up-regulation of enzymes involved
in antioxidant defense, in aldehyde bio-product detoxification
and in misfolded protein degradation. In addition, a massive
down-regulation of enzymes controlling anaerobic metabolism
was observed. After 10 days of hypoxia, cardioprotective
molecules were substantially decreased whereas proapoptotic signals were correlated with the increment of
apoptotic program and sustained by the down-regulation
of specific target proteins. In conclusion, differential analysis
provided new insight into target molecules sensing lack of
oxygen in vivo and could open new possibilities for protection
from damages induced by long term hypoxia exposure.
C. Gelfi: None. A. Viganò: None. M. Vasso: None. D. Capitanio:
None. R. Leone: None. A. Caretti: None. M. Samaja: None.
ABSTRACTS
P320Deficiency of Nrf2 Promotes Angiotensin II-Induced
Cardiac Hypertrophy in Mice
116
Jeremy R Chidester, Univ of Utah Health Care, Salt Lake City, UT;
Anuradha Guggilam, Ohio State Univ, Columbus, OH; Curtis Olson,
Gayatri D Khanderao, Kalavathy Ramachandran, Corey J Miller,
Vasanthi Rajasekaran, Univ of Utah Health Care, Salt Lake City, UT;
Sankarnarayanan Kannan, M D Anderson Cancer Ctr, Houston, TX;
Sheldon E Litwin, Medical Coll of Georgia, Augusta, GA; Rajasekaran
Namakkal Soorappan, Univ of Utah Sch of Med, Salt Lake City, UT
Background: Nuclear erythroid 2-related factor 2 (Nrf2), a
basic leucine zipper protein, is a redox-sensitive transcription
factor that regulates a majority of antioxidant and cytoprotective
genes in the heart. Augmentation of reactive oxygen species
(ROS) is implicated in the development of cardiac hypertrophy.
We have identified sustained activation of Nrf2/ARE signaling
in protein aggregation cardiomyopathy and hypothesize that
genetic abscission of Nrf2 will promote oxidative stress and
hasten angiotensin-II (Ang-II) induced hypertrophy. Methods: To
investigate loss-of-function mechanisms for Nrf2 on oxidative
stress induced cardiac hypertrophy, we administered Ang-II
(200 ng/gm bw/min, for 14 days) to (1) wild type (Wt), and (2)
Nrf2-/- (KO) mice (n=4–5/group) using osmotic mini-pumps.
Sham controls (Wt & KO) were implanted with saline pumps.
We assessed cardiac function (pre- and post-treatment
J.R. Chidester: None. A. Guggilam: None. C. Olson: None.
G. Khanderao: None. K. Ramachandran: None. C.J. Miller: None.
V. Rajasekaran: None. S. Kannan: None. S.E. Litwin: None.
R. Namakkal Soorappan: None.
P321Oxidative Stress Induces DNA Double-Strand Breaks
and Activates DNA Damage Responses in Vascular
Smooth Muscle Cells: A Possible Mechanism for
Atherosclerosis Progression
Takafumi Ishida, Mari Ishida, Satoshi Tashiro, Chiemi Sakai, Hitomi
Uchida, Hiroshima Univ, Hiroshima, Japan; Kiyoshi Miyagawa, Univ of
Tokyo, Tokyo, Japan; Masao Yoshizumi, Yasuki Kihara, Hiroshima Univ,
Hiroshima, Japan
Backgrounds: Oxidative stress is thought to be a pathogenic
mediator of atherosclerosis. Oxidative stress induces DNA
damage, and the unrepaired or improperly repaired DNA
damage increases genomic instability, which cause cell
death, senescence, or dysregulation of cellular functions.
Pathogenesis of both Hutchinson-Gilford Progeria
syndrome and Werner syndrome, which feature prominent
atherosclerotic disease at young age, involves impaired DNA
repair and the resultant genomic instability. The purpose of
this study is to determine whether oxidative stress causes
DNA damage in vascular smooth muscle cells (VSMC)
and to elucidate the role of DNA damage responses in
atherosclerosis and the fate of VSMC. Methods and Results:
Immunoreactivity against gamma-H2AX, a sensitive marker for
DNA double-strand breaks (DSBs), which is the most severe
form of DNA damage, was increased in human atherosclerotic
plaques, but not in the adjacent normal areas. gamma-H2AX
staining was observed in almost same regions where 8-oxodG immunoreactivity, an oxidative modification of DNA, was
observed. Apoptotic cells were abundant in atherosclerotic
lesions, but not in normal areas. In cultured human aortic
smooth muscle cells (HASM), 15 min incubation with H2O2
(100 microM) induced foci formation of gamma-H2AX in the
nuclei. H2O2 activated various signaling molecules involved
in DNA damage responses, including ATM, Chk2, DNA-PK
and p53 in HASM. Some H2O2-induced DSBs persisted after
24 hours, at which point apoptosis was induced in 7.1 ± 1.3
% of HASM, as detected by TUNEL method. Knockdown
experiments using siRNA revealed that ATM-, DNA-PK-, or
Chk2-deficient VSMC were more resistant to H2O2-induced
apoptosis. Conclusions: In summary, 1) DNA double-strand
breaks were accumulated in human atherosclerotic plaques, 2)
oxidative stress induced double-strand breaks and activation
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Poster Presentations (continued)
of DNA damage response in vascular smooth muscle cells,
and 3) impairment of DNA damage responses modulated
damage-induced cell fate such as apoptosis. Thus, DNA
damage itself or alteration in DNA damage responses may be
involved in the mechanisms for progression of atherosclerosis.
T. Ishida: None. M. Ishida: None. S. Tashiro: None. C. Sakai: None.
H. Uchida: None. K. Miyagawa: None. M. Yoshizumi: None.
Y. Kihara: None.
P322Chamber-Specific Differences in Oxidative Stress in
Newborn Versus Adult Rabbit Hearts Following
Ischemia-Reperfusion
E Bernadette Cabigas, Emory Univ Sch of Med, Atlanta, GA; Guoliang
Ding, Tao Chen, Talib Saafir, Mary B Wagner, Emory Children’s Ctr,
Atlanta, GA; Michael E Davis, Emory Univ Sch of Med, Atlanta, GA
. Cabigas: None. G. Ding: None. T. Chen: None. T. Saafir: None.
E
M.B. Wagner: None. M.E. Davis: None.
P323Waon Therapy, a Form of Thermal Therapy, Reduces
Oxidative Stress Systemically and Inhibits the Progression
of Cardiac Dysfunction in TO-2 Cardiomyopathic Hamsters
with Heart Failure
Yoshiyuki Ikeda, Masaaki Miyata, Yuichi Akasaki, Takahiro Miyauchi,
Yuko Furusho, Kanako Ide, Shuich Hamasaki, Chuwa Tei, Dept of
Cardiovascular Med, Kagoshima city, Japan
Background: Oxidative stress is one of the most crucial
factors that develop chronic heart failure (CHF), leading
to cardiac apoptosis and fibrosis and vascular endothelial
dysfunction. We have reported that Waon therapy, which is a
form of thermal therapy using a far infrared-ray dry sauna at 60
Y. Ikeda: None. M. Miyata: None. Y. Akasaki: None. T. Miyauchi: None.
Y. Furusho: None. K. Ide: None. S. Hamasaki: None. C. Tei: None.
P324Unhealthy Diet and Air Pollution Compromise Human
Cardiovascular Cell Functions: A First Mechanistic Analysis
Niloofar Ale-Agha, Nicole Buechner, Ulrich Sydlik, Klaus Unfried,
Joachim Altschmied, Judith Haendeler, IUF — Leibniz Inst for
Environmental Med, Duesseldorf, Germany
Diet and pollution are environmental factors known to
compromise “healthy cardiovascular aging”. The molecular
consequences of the permanent burden for aging of the
cardiovascular system are unknown, since they have never
been examined in primary, adult human cells. Therefore, this
study investigates the impact of unhealthy diet on agingrelated signaling pathways of human, primary cardiovascular
cells and of airborne particles on human endothelial cells,
as several studies demonstrated that ultrafine particles can
enter the circulation and thus may interact with endothelial
cells directly. Nutrition health reports have shown that the
diet in industrialized countries contains more than 100 mg/
dl low density lipoprotein (LDL) and a too high fraction of
monosaccharides, especially fructose, which is metabolized
insulin-independently. Both components have been shown
to increase the risk for cardiovascular diseases. To simulate
unhealthy diet we supplemented cell culture media of human,
primary endothelial cells (EC), smooth muscle cells (SMC)
and cardiomyocytes (CM) with 100 mg/dl LDL and replaced
1/3 of the glucose with fructose for one week. This treatment
did not induce cell death in any of the cell types. However,
we observed increased senescence, loss of endothelial nitric
oxide synthase and increased nuclear localization of Foxo3
in EC, increased proliferation in SMC and hypertrophy in CM.
With respect to pollution we have used ultrafine carbon black
particles (ufCB), one of the major constituents of industrial
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
ABSTRACTS
Each year, tens of thousands of children undergo
cardiopulmonary bypass (CPB) to correct congenital heart
defects. While necessary for surgery, CPB involves stopping
the heart and exposing it to ischemic conditions. While much
is known about adult injury, little is known about the effects of
global ischemia on newborn ventricles. The purpose of this
study was to determine age- and chamber-specific differences
in oxidative stress following global ischemia-reperfusion injury.
We studied newborn (2–4 days) and adult (>8 weeks) rabbit
hearts subjected to Langendorff ischemia-reperfusion (30
minutes ischemia, 60 minutes reperfusion). Catalase and
superoxide dismutase (SOD) activities were measured, as
well as real-time hydrogen peroxide (H2O2) and superoxide
(O2-) generation using confocal microscopy in isolated left and
right ventricular (LV and RV) myocytes exposed to hypoxia.
Our data demonstrate chamber and age-specific changes in
oxidative stress. During ischemia, H2O2 increased significantly
in the RV while remaining constant in the LV of newborn rat
rabbit myocytes. In contrast, there was a smaller, nonsignificant
increase in H2O2 in both both RV and LV myocytes of adults.
The increase seen in the RV of newborns was several fold
higher than that of adults (4.3-fold vs. 2.2 fold; p<0.05).
In whole heart tissue, catalase activity was significantly
increased following ischemia in both adult ventricles, while
no increase was seen in newborn hearts compared to sham
hearts. Additionally, levels in newborns were several fold lower
(p<0.05), indicating less scavenging potential. SOD activity
was increased from sham vs. ischemia in the LV of both adult
and newborn hearts, but only in the RV of the newborn heart
(0.80±0.09 to 3.0±0.43 U/mg; p<0.001). Intra-cardiac injection
of an H2O2 scavenger, Ebselen, loaded in nanoparticles,
improved recovery of developed pressure (49.53±16.25 to
80.0±11.31) in the RV of the newborn (p<0.05), suggesting the
local delivery of an exogenous antioxidant is cardioprotective
in newborn RVs. Our data demonstrate there are ventriclespecific differences in oxidative stress between newborn and
adult rabbit hearts. Local therapy is able to address some of
these differences and may limit damage during bypass surgery.
degrees centigrade, improves cardiac and vascular endothelial
functions and prognosis in patients with CHF. The aim of this
study is to investigate whether Waon therapy reduces oxidative
stress and prevents from developing cardiac dysfunction in
CHF. Methods: Thirty-week old male TO-2 cardiomyopathic
hamsters with CHF were divided into Waon therapy or control
group. Waon therapy group underwent Waon therapy daily
for 4 weeks. Control hamsters did not take any treatment. We
examined the amounts of reactive oxygen species of serum,
hearts and aortas using ELISA and immunohistochemistry.
We measured left ventricular % fractional shortening (LV%FS),
and performed TUNEL and Azan staining of hearts to assess
cardiac function, apoptosis and fibrosis, respectively. Antioxidants and apoptotic and angiogenetic factors were
assessed by Western blot. All examinations were performed
after 4 weeks of treatment. Results: Four-week Waon
therapy significantly decreased oxidative stress of serum,
hearts and aortas compared to those of controls. Waon
therapy significantly increased LV%FS and decreased cardiac
apoptosis and fibrosis (LV%FS, Waon therapy: 23.3±4.3 vs.
control: 16.5±4.2%, P<0.01, TUNEL positive nuclei, 22.0±2.6
vs. 49.3±7.2%, P<0.01, % fibrosis, 20.6±5.3 vs. 47.6±4.8%,
P<0.01). Waon therapy significantly increased the expressions
of manganese superoxide dismutase, heat shock protein 27
(HSP27) and HSP32 of hearts and aortas, which negatively
modulate oxidative stress, compared to those of controls.
Waon therapy significantly increased endothelial nitric oxide
synthase and decreased plasminogen activator inhibitor-1 of
aortas. In addition, Waon therapy significantly decreased Bax,
cleaved caspase 3 and cytochrome c and increased Bcl-2 and
hypoxia-inducible factor-1α of the failing hearts. Conclusions:
Waon therapy reduces oxidative stress systemically and
inhibits the progression of cardiac dysfuntion in TO-2
cardiomyopathic hamsters.
117
Poster Presentations (continued)
and exhaust emissions, in concentrations our vessels are
constantly exposed to. These concentrations of ufCB are
non-toxic and non-inflammatory for EC. Despite these missing
immediate effects, ufCB dramatically reduced the S-NO
content, a marker for NO-bioavailability in EC and increased
reactive oxgen species formation. As a consequence, ufCB
dramatically increased senescence of EC after two weeks.
Thus, unhealthy diet and a high burden of ultrafine carbon
black nanoparticles, to which we are exposed every day, seem
to induce a “cardiovascular aging” phenotype and can lead to
severe cardiovascular diseases.
N. Ale-Agha: None. N. Buechner: None. U. Sydlik: None. K. Unfried:
None. J. Altschmied: None. J. Haendeler: None.
P325Novel Insights in Transcriptome Remodeling Associated
with Heart Failure Revealed by Deep RNA Sequencing
ABSTRACTS
Chen Gao, Jae-Hyung Lee, Molecular Biology Inst, Univ of California,
Los Angeles, Los Angeles, CA; Shuxun Ren, Univ of California, Los
Angeles, Los Angeles, CA; Guangdun Peng, Molecular Biology Inst,
Univ of California, Los Angeles, Los Angeles, CA; Robb MacLellan,
Univ of California, Los Angeles, Los Angeles, CA; Jau-nian Chen,
Grace (Xinshu) Xiao, Yibin Wang, Molecular Biology Inst, Univ of
California, Los Angeles, Los Angeles, CA
The complexity of transcriptome and proteome is contributed
by alternative splicing of RNA. Altered RNA splicing is
also implicated in many human diseases including cancer.
However, little knowledge is available about the scope of
alternative splicing at whole genome level in heart diseases
and even less about the mechanisms underlying the regulation
of mRNA splicing in response to pathological injury in heart.
In order to investigate the key alternative splicing events
associated with cardiac pathogenesis, we used highthroughput RNA-Seq to profile total transcriptome in mouse
heart failure induced by pressure-overload. From preliminary
data, we identified >1000 novel exons that have not been
reported in any published database. Many of the novel exons
are detected in genes with potential importance in cardiac
gene regulation and signal transduction. In addition, we have
also identified a significant number of differentially expressed
exons between normal and diseased hearts, supporting the
notion that differential RNA splicing is associated with the
onset of heart failure. Interestingly, the differential splicing
events observed in failing hearts were also detected in
neonatal mouse hearts, suggesting that differential RNA
splicing is a part of the “fetal” gene expression program in
diseased hearts. Furthermore, we have also validated some
of these changes in human heart failure samples, supporting
the clinic relevance of these findings. In summary, this study
suggests that development of heart failure is associated with
alternative RNA splicing events at genome level. Further
studies on the functional consequences of differential RNA
splicing and the underlying regulatory mechanisms in heart will
advance our understanding to this important disease.
. Gao: None. J. Lee: None. S. Ren: None. G. Peng: None.
C
R. MacLellan: None. J. Chen: None. G. Xiao: None. Y. Wang: None.
P326An Innovative Peptide Spectral Library Search Engine for
Cardiovascular Proteomics
Haomin Li, Univ of California, Los Angeles, Los Angeles, CA; Ning
Deng, Zhejiang Univ, Hangzhou, China; Nobel Zong, Anna Zolyan,
Melissa Chan, Jenny Kim, Univ of California, Los Angeles, Los Angeles,
CA; Rolf Apweiler, European Bioinformatics Inst, Cambridge, United
Kingdom; Huilong Duan, Zhejiang Univ, Hangzhou, China; Peipei Ping,
Univ of California, Los Angeles, Los Angeles, CA
We developed a peptide spectral library search engine for the
cardiovascular community. Over 50,000,000 spectra obtained
with LTQ-Orbitrap instrument on cardiac mitochondria and
proteasome were analyzed, and 108,268 representative
118
spectra were included in this organelle-based library. An
improved dot product algorithm, slide dot product, was
coded to query user spectra against spectra in the library.
This procedure provides a solution to optimally balance the
speed and sensitivity in peptide identification. In addition,
an innovative noise decoy protocol was engineered to
distinguish spectra correlation attributed to noise signals of
the analytical instruments. The immediate advantages include
an effective improvement of both specificity and accuracy
in protein identification, overcoming a long-lasting concern
in experimental spectra library, the possibility of propagating
spectra with inaccuracy and error. With an independent test
dataset collected by LTQ-Orbitrap (~10,000 spectra), this novel
search engine identified 20% more spectra matches within
10% analytical time compared to other existing theoretical
database search route. Furthermore, the broad utility of this
search engine was also demonstrated in characterizing spectra
collected by LCQ and Q-TOF instrumentation. This peptide
spectral library search engine has been incorporated into the
Cardiac Organellar Peptide Atals Library (COPa Library) of
NHLBI Proteomics Center at UCLA and will be made accessible
at www.HeartProteome.org.
H. Li: None. N. Deng: None. N. Zong: None. A. Zolyan: None.
M. Chan: None. J. Kim: None. R. Apweiler: None. H. Duan: None.
P. Ping: None.
P327COPa Library: A Proteomic Knowledge Base for
Cardiovascular Biology and Medicine
Nobel Zong, Haomin Li, Univ of California, Los Angeles, Los Angeles,
CA; Ning Deng, Lingxuan Chen, Zhejiang Univ, Hangzhou, China; Yueju
Wang, The Scripps Res Inst, San Diego, CA; Caiyun Fang, Univ of
California, Los Angeles, Los Angeles, CA; Rafael Jimenez, European
Bioinformatics Inst, Cambridge, United Kingdom; Tao Xu, The Scripps
Res Inst, San Diego, CA; Henning Hermjakob, European Bioinformatics
Inst, Cambridge, United Kingdom; Huilong Duan, Zhejiang Univ,
Hangzhou, China; John Yates, The Scripps Res Inst, San Diego,
CA; Rolf Apweiler, European Bioinformatics Inst, Cambridge, United
Kingdom; Peipei Ping, Univ of California, Los Angeles, Los Angeles, CA
We constructed the Cardiac Organellar Peptide Atlas Library
(COPa library) as a targeted and interactive resource to the
cardiovascular community. Annotated peptide spectra are
hosted using a relational database in a modular fashion
based on species (e.g. human, mouse) and organelles (e.g.
mitochondria, proteasome). Within this release of COPa
library, a total of 108,268 spectra have been disseminated
via two avenues. A web portal was established to navigate
the library via parallel set of identifiers, such as protein
name, accession number, gene symbol, etc. In parallel, a
web-service cyber-infrastructure was engineered to aid the
annotation of mass spectra submitted via internet. The large
raw spectra files are dissected into small data packages at
the local PC before submission. This workflow surpasses the
limitation of network bandwidth, as well as enables parallel
data submission and search. A benchmark test with 897,327
ms/ms spectra showed the library searching covers 93.4% of
proteins identified via database searching, as well as additional
23.9% of proteins at the same level of statistical confidence.
In addition, a wiki-like web interface was embedded in
the library web portal in order to facilitate the synthesis of
consensus knowledge among the cardiovascular community
on innovations of functional proteomics. Overall, the COPa
library search supports targeted proteomic characterization,
which complements database search for exploratory survey.
The implementation of the COPa library-based proteomic
knowledgebase leverages state-of-the-art technology and
annotated datasets among the research community at large.
Its application bridges discovery-driven and hypothesis-driven
research while fostering translational medicine.
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Poster Presentations (continued)
mitochondria and energy generation. Because GPCR functions
are implicated in many vascular diseases, we confirmed the
differential expression of the arterial specific Regulator of
G-protein signaling — 5 (RGS5), and extended the analysis to
additional members of the RGS-R4 subfamily. RGS5 was the
most abundant RGS-R4 mRNA in the AA, and it was the most
differentially expressed family member across all vascular beds.
To analyze functional correlates to the differential expression of
RGS5, we performed aortic ring contraction assays from the
TA and AA. Consistent with the hypothesis that RGS5 blocks
angiotensin II (Ang II) type 1 receptor signaling via increased Gαmediated GTPase activity, Ang II-induced force generation was
significantly lower in AA relative to TA. Furthermore, whole-cell
voltage-gated K+ current (IKv) analysis revealed that IKv was
significantly greater in SMCs from the AA compared to cells
from TA. Kv1.5 mRNA was more highly expressed in the AA
relative to the TA. Conclusions: The differential gene expression
pattern between TA and AA suggests distinct differentiation
pathways may explain regional specificity of arterial diseases.
Specifically, the enrichment of GPCR-related genes in the AA
and mitochondrial genes in the TA may account for the diverse
physiological function of these two distinct vascular segments.
N. Zong: None. H. Li: None. N. Deng: None. L. Chen: None. Y. Wang:
None. C. Fang: None. R. Jimenez: None. T. Xu: None. H. Hermjakob:
None. H. Duan: None. J. Yates: None. R. Apweiler: None. P. Ping: None.
P328β-Blockers Can Enhance Short-Term Receptor Sensitivity
in Silico and in Vitro
Robert Amanfu, Jeffrey Saucerman, Univ of Virginia, Charlottesville, VA
A key feature of heart failure is the chronic elevation of
circulating catecholamines which desensitizes the β-adrenergic
receptor signaling pathway, rendering patients incapable
of increasing cardiac output in response to acute stress
(eg. exercise). β-adrenergic receptor blockers (β-blockers)
are commonly used to treat this condition, but precisely
how they work is still controversial and poorly understood.
Two opposing theories are that β-blockers work by 1)
blocking the harmful consequences of chronic signaling or
2) paradoxically increasing receptor sensitivity. To investigate
whether β-blockers function by increasing receptor sensitivity,
we extended a published computational model of the β1adrenergic receptor signaling pathway developed in our lab
by using a ternary complex receptor module. This receptor
model includes the spontaneous switching between the active
and inactive conformations of the receptor that is crucial for
accurate representation of β-blockers. Parameter values
were determined from literature to model 11 agonists and 10
β-blockers. The new receptor model was validated against
ligand binding studies and adenylyl cyclase assays from
literature. Chronic and acute stresses were modeled as low and
high (100X greater) concentrations of the agonist isoproterenol.
Simulations indicate that when the β-blocker propranolol is
present during chronic stress, cAMP levels are increased upon
application of an acute stress, elevating intracellular calcium
levels and contractility. This suggests that in addition to blocking
the harmful consequences of chronic signaling, β-blockers may
sensitize the response to acute stress. Preliminary data from
calcium imaging experiments in isolated cardiac ventricular cells
confirm this prediction. Simulations with a range of commonly
used β-blockers indicate that individual drugs differ significantly
in their ability to enhance receptor sensitivity. In addition,
simulation of the Gly389Arg polymorphism suggests that the
Arg389 variant has reduced sensitization to acute stress in
the presence of propranolol. These simulations are a first step
towards evaluating personalized β-blocker therapies with
computational models.
R. Amanfu: None. J. Saucerman: None.
P329The Thoracic and Abdominal Arterial Segments Are
Genetically and Functionally Different
Ick-Mo Chung, Ewha Womans Univ Sch of Med, Seoul, Korea,
Republic of; David K Pritchard, Luis F Santana, Williams M Mahoney Jr,
Stephen M Schwartz, Univ of Washington, Seattle, WA
Aims: Because arterial diseases are highly specific to different
segments and because smooth muscle cells (SMCs) of different
branches have distinct embryological origin, we undertook a
systematic effort to determine the extent of differences in gene
expression between the thoracic aorta (TA) and the abdominal
aorta (AA). We also correlated these expression differences
with physiologic function. Methods and Results: We utilized
whole genome gene expression microarrays to compare
expression patterns in the rat TA and AA. We identified 161
differentially expressed genes between these two segments.
Functional analysis of differences in expression by Gene Set
Enrichment Analysis identified different categories of genes
enriched in each section of the aorta. For example, GPCRrelated genes are overrepresented in the AA, while the TA
showed dramatic enrichment of gene categories linked to
I. Chung: None. D.K. Pritchard: None. L.F. Santana: None.
W.M. Mahoney: None. S.M. Schwartz: None.
P330Not published at presenter’s request.
P331Metabolic Dynamics and Phosphometabolomic
Fingerprinting Using 18O-Assisted 31P NMR
and Mass Spectrometry
Petras Dzeja, Emirhan Nemutlu, Song Zhang, Andre Terzic, Mayo
Clinic, Rochester, MN
Evaluation of disease phenotypes and drug effects requires
knowledge not only metabolite levels but also their turnover rates
from which metabolic fluxes and status of the whole energetic
system can be determined. Oxygen-18-assited 31P NMR and
mass spectrometric technique uniquely allow simultaneous
measurements of phosphorus-containing metabolite levels
and their turnover rates. This includes simultaneous recordings
of 18O-labeling rates reflecting ATP synthesis (β-ATP) and ATP
utilization (Pi), phosphotransfer fluxes through adenylate kinase
(β-ATP/ADP), creatine kinase (CrP) and glycolytic pathways
(G-6-P) as well as Krebs cycle associated mitochondrial nucleotide
turnover (γ/β-GTP), substrate shuttle (G-3-P) and glycogen
metabolism (G-1-P). Metabolomic and fluxomic profiling of hearts
and body fluids of phosphotransfer enzyme deficient transgenic
animals using 18O-assisted GC/MS, 1H and 18O-assisted 31P
NMR indicate selective metabolic perturbations and adaptations
in the whole energetic system. Adenylate kinase and creatine
kinase deficiencies were associated with altered metabolomic
profiles, increased mitochondrial capacities and redistribution of
phosphotransfer flux through glycolytic and guanine nucleotide
systems. These phosphometabolomic alterations were more
expressed when animals were subjected to treadmill exercise,
mitochondria-targeting drugs such as metformin and high-fat diet.
Metabolite turnover rates are correlated with heart performance
unveiling robustness and dynamics rearrangements in cell
bioenergetics infrastructure. Glycerophosphate, G-3-P, metabolic
dynamics is altered in transgenic animal models indicating
defects in substrate shuttle and supply of reducing equivalents
to mitochondria. This is of importance since G-3-P metabolic
abnormalities and metabolic arrest are linked to human diseases
such as sudden death syndrome. Thus, our study demonstrates
that phosphometabolomic and fluxomic profiling is a valuable
tool for metabolic phenotyping of transgenic animal models of
human diseases as well as for plasma biomarker identification and
monitoring of treatment efficacy and drug toxicity.
ABSTRACTS
This research has received full or partial funding support from the
American Heart Association, National Center.
P. Dzeja: None. E. Nemutlu: None. S. Zhang: None. A. Terzic: None.
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
119
Poster Presentations (continued)
P332 Effects of Intravenous Infusion of Polyunsaturated Fatty
Acids and Dextrose on Blood Pressure and Endothelial
Function in Obese Subjects
Aidar R Gosmanov, The Univ of Tennessee Health Science Ctr,
Memphis, TN; Dawn Smiley, Joselita Siquiera, Gonzalo Robalino, Limin
Peng, Guillermo E Umpierrez, Emory Univ Sch of Med, Atlanta, GA
ABSTRACTS
yperglycemia and elevated free fatty acids (FFAs) are implicated
H
in the development of hypertension and endothelial dysfunction.
We recently reported that 8-hour infusion of soy-bean oil
containing polyunsaturated fatty acids (Intralipid) results in the
elevation of blood pressure (BP) and endothelial dysfunction in
obese healthy subjects. However, the effects of dextrose infusion
or combination of dextrose and Intralipid on BP, endothelial
function and insulin action are not known. Accordingly, we
compared the effects of 8-hour infusion of normal saline at 40
ml/hr, Intralipid 20% at 40 mL/hr, dextrose 10% at 40 ml/hr and
combination of Intralipid and dextrose on BP, endothelial function
in 12 obese healthy subjects [ages:41±7 yrs, BMI:32±2 kg/
m2, BP:113/65 mmHg, HOMA-IR: 2.0±1.0]. Blood pressure,
brachial artery flow-mediated dilatation (FMD), and levels of
FFAs, glucose, and insulin were measured at 0, 4 and 8 h of
infusion. Intralipid infusion significantly increased BP, decreased
FMD, and increased plasma FFAs (Table). Unlike Intralipid alone,
the combination of Intralipid and dextrose did not increase
BP but resulted in FMD changes similar to Intralipid alone.
Levels of plasma glucose and insulin increased over time after
dextrose infusion alone or in combination with Intralipid but not
with lipid infusion alone. Compared with Intralipid, the addition
of dextrose to Intralipid led to restoration of FFAs to normal
level. In summary, Intralipid but not dextrose infusion alone or
in combination with Intralipid results in significant elevation in
blood pressure in obese healthy subjects. In contrast, dextrose
administration had no effect on Intralipid-induced endothelial
dysfunction. The mechanisms underlying differences in
vascular response after addition of dextrose to Intralipid are not
known, but these results indicate that dextrose-induced mild
hyperinsulinemia may regulate adverse hemodynamic effects of
fat administration in obese subjects.
0 hrs
4 hrs
8 hrs
SBP
(mm Hg)
SID
I+D
113±21
113±14
115±13
108±15
115±15
126±17†
117±13
110±22
113±13
125±16†
118±17
121±17
DBP
(mm Hg)
SID
I+D
65±12
68±10
68±12
68±12
64±11 74±14*
66±10 64±15
67±10 72±11*
67±17 71±12
P333 A Study of Miniature Catheters’ Spatial Electric Field
Heterogeneity During Murine Cardiac Catheterizations
Stelios Angeli, Richard Mean, Christakis Constantinides, Univ of
Cyprus, Nicosia, Cyprus
onductance catheters allow real-time quantification of
C
hemodynamics, allowing cardiac functional characterization,
an important predictor of long-term prognosis in cardiac
disease. The technique’s accuracy is, however, inherently
limited by the signal contribution from surrounding structures
(spatially and temporally varying). Despite prior attempts to
quantify this effect (known as parallel conductance) no prior
study assessed the spatial heterogeneity of the catheter’s
E-field. This study quantifies the E-field penetration pattern,
accounting for tissue properties and geometry. Ten C57BL/6J
mice were induced and maintained with 1.5% isoflurane mixed
in 100% O2. One C57BL/6J mouse underwent a right carotid
catheterization for placement of a 1.4 Fr Pressure-Volume
Millar catheter in the left ventricle (LV), followed by microCT
imaging (80kV/160mA/10ms exposure/240 projections/rotation
angle=1.5o). Multiphase MRI was performed using a 4D radial
spiral pulse sequence (TE=300μs/TR=2.4ms/BW=125kHz/
flip angle=45o/110μm3 resolution). Segmentation allowed
LV myocardial and blood region extractions from MRI and
construction of finite element End-Diastolic and End-Systolic
models. The catheter’s orientation in the LV was determined
from micro-CT image data renditions. Generated LV and
blood models were then imported in the software XFdtd and
electrical properties were assigned for all materials. Simulations
used a 20 μA, 20 kHz sinusoidal current, and ran for a lump
component equivalent and a constructed blood-myocardial
geometrical model. Specific absorption rate (SAR) maps
yielded total tissue deposited power. Simulations show that the
catheter’s E-field in lump component and geometrical models,
drops to 10% of its peak value at 0.4–0.6mm, and 1.1–2mm,
respectively, away from the excitation electrodes. SAR maps
yielded a <1% power leakage into surrounding structures at
two different myocardial permittivity values of εr=11844 and
38615. Results from this study map the spatial dependence
of the generated catheter E-field. Spatial E-field maps indicate
that the field is primarily confined within the ventricular chamber
with a relatively uniform spatial pattern, and with <1% of the
input power leaking in surrounding structures.
S. Angeli: None. R. Mean: None. C. Constantinides: None.
P334 Comparative Effectiveness of Left Ventricular Assist
Devices as a Bridge to Heart Transplantation
FMD (%)
SID
I+D
8.8±2.0
9.4±1.9
9.1±2.5
10.3±3.0
8.9±4.8
6.2±2.2†
8.2±2.3
7.9±2.9†
FFAs
(mmol/L)
SID
I+D
0.73±0.34
0.79±0.25
0.93±0.45
0.73±0.25
0.68±0.25
1.63±0.78†
0.80±0.50
0.63±0.39
0.75±0.50
1.82±1.1†
0.75±0.35
0.64±0.24
Glucose
(mg/dl)
SID
I+D
86.0±9.6
78.4±8.0
77.4±12.4
71.6±12.3
87.0±20.7
75.6±9.4
100.4±19.5*
101.2±16.6†
88.0±15.0
73.7±10.6
92.9±13.9
97.8±11.1†
Insulin
(µU/mL)
SID
I+D
8.4±4.9
9.1±5.5
8.4±9.6
10.4±9.6
11.3±6.4
11.3±7.7
25.1±10.2*
28.0±12.5†
10.1±6.6
9.3±7.5
33.4±21.0*
40.5±22.8†
Robert S Higgins, Sherri Wissman, Kelly MacBriar, Erin Bumgardner,
Emily Burke, Pallavi Solanki, Ayehsa Hasan, Juan Crestanello, Chittoor
Sai-Sudhakar, Gary Haas, William Abraham, Carl Leier, The Ohio State
Univ Medical Ctr, Columbus, OH
SBP- systolic BP, DBP- Diastolic BP, S- Saline, I- Intralipid,
D- Dextrose, I+D - Intralipid+Dextrose. Mean ± SD; *p
120
.R. Gosmanov: None. D. Smiley: None. J. Siquiera: None.
A
G. Robalino: None. L. Peng: None. G.E. Umpierrez: None.
eft ventricular assist devices (LVAD) have evolved to be more
L
frequently utilized in patients with advanced congestive heart
failure as a bridge to heart transplantation. In the era of limited
healthcare resources and diminishing funding for innovative
technologies, there is little data to evaluate the comparative
effectiveness of LVADs in comparison to more conventional
heart failure therapies leading to heart transplantation without
mechanical circulatory support. Objective. To evaluate the
clinical outcomes, complications, length of stay, financial
impact and satisfaction of patients with advanced heart failure
undergoing heart transplantation as part of a comprehensive
heart failure program with or without LVAD as a bridge to
transplant (BTT). Methods We retrospectively reviewed 25
patients undergoing heart transplantation from 2009–2010
(12 patients without LVAD, 13 with LVAD as BTT). Inpatient
Become an AHA/ASA Professional Member and save up to $300 off next year’s registration. (Promo code MBJ050ZZ)
Poster Presentations (continued)
hospital, financial and clinic records were reviewed to
evaluate length of stay, morbidity, mortality, DRG assignment,
insurance status, charges and cost, reimbursement and
patient satisfaction. All patients received surveys assessing
their satisfaction with the mechanical circulatory support and
transplant program. Results There were 14 males and 11
females (avg age 54). In the non-LVAD group, 9 patients fell
into DRG 1 ,3 were in DRG 2;in the LVAD group, 10 patients
fell in DRG 1, 3 patients were in DRG 2.Complications
occurring in the LVAD patients included pneumonia (2),CVA,
depression, rejection and bowel perforation; in the non-LVAD
patients pneumonia, pulmonary embolus, aspergillosis,
peri-rectal abscess and failure to thrive; Mortality at 1 year
occurred in 2 patients after LVAD; 1 patient after transplant
without LVAD. All patients responded that they were very
satisfied with the advanced heart failure program whether
they received LVAD prior to transplant or not Conclusions
Transplant recipients with advanced heart failure have
comparable clinical outcomes after transplant whether they
required LVAD implantation prior to transplant. Perioperative
care algorithms focused on reduction of common postoperative complications resulting in significant financial impact
(ie pulmonary), should be developed to address these issues. .
R.S. Higgins: None. S. Wissman: None. K. MacBriar: None.
E. Bumgardner: None. E. Burke: None. P. Solanki: None. A. Hasan:
None. J. Crestanello: None. C. Sai-Sudhakar: None. G. Haas: None.
W. Abraham: None. C. Leier: None.
P335Is GSK-3α a Regulator of Aging?
Jibin Zhou, Hind Lal, Xiying Shang, Ronald Vagnozzi, John Farber,
Morgan DeCaul, Thomas Jefferson Univ, Philadelphia, PA; James R
Woodgett, Samuel Lunenfeld Res Inst, Toronto, ON, Canada; Erhe
Gao, Thomas Force, Thomas Jefferson Univ, Philadelphia, PA
J. Zhou: None. H. Lal: None. X. Shang: None. R. Vagnozzi: None.
J. Farber: None. M. DeCaul: None. J.R. Woodgett: None. E. Gao:
None. T. Force: None.
P336Constitutive Activation of ROCK1 in Mouse Heart Leads
to Fibrotic Cardiomyopathy
Xiangsheng Yang Sr, Texas A&M Health Science Ctr, Houston, TX;
Qi Li, Hosp of Hainan Medical Coll, Haikou, China; Xi Lin, Texas A&M
Health Science Ctr, Houston, TX; Yanlin Ma, Hosp of Hainan Medical
Coll, Haikou, China; Xiaojing Yue, Zhengyin Tao, Jiang Chang, Texas
A&M Health Science Ctr, Houston, TX
Background: Our previous study demonstrated that genetic
deletion of ROCK1 inhibited stress-induced cardiac fibrosis
and ROCK1 was cleaved by caspase 3 into a constitutively
active Rho kinase isoform, ROCKΔ1 that led to increase in
Rho kinase activity in human failing hearts. However, the
function of truncated ROCK1 accumulated in human failing
myocardium and the molecular signaling linked between
ROCKΔ1 and cardiac remodeling remain obscure. Methods
and Results: To recapitulate this human pathophysiological
situation and define the pathogenic role of ROCKΔ1 in heart,
we generated transgenic mice expressing ROCKΔ1 in heart.
Significant increase in Rho kinase activity was achieved.
The mice developed fibrotic cardiomyopathy along with
upregulation of TGFβ1 and NF-κB signaling. Significant
increases in fibrotic factors were observed. Echocardiography
revealed diastolic dysfunction in the transgenic heart. The
transgenic mice were predisposed to angiotensin II treatment
that led to massive fibrotic cardiomyopathy. Rho kinase
inhibitor Fasudil treatment attenuated the fibrotic maladaption.
To elucidate the molecular mechanism underlying ROCKΔ1mediated fibrotic cardiomyopathy, wild-type cardiac fibroblasts
were co-cultured with transgenic cardiomyocytes. Large
amount of fibroblasts were activated and induced to express
α-SMA by the transgenic cardiomyocytes but not by the
wild-type control cardiomyocytes. Addition of Rho kinase,
TGFβR1 and NF-κB inhibitors to the culture media attenuated
the fibroblast activation. The results suggested that the
pro-fibrotic TGFβ and NF-κB signaling are responsible for
ROCKΔ1-mediated fibrotic cardiomyopathy. Finally, we
demonstrated TGFβ1 was a SRF (serum response factor) new
target gene. EMSA, luciferase and CHIP assay revealed SRF
binding to TGFβ1 promoter. Genetic deletion of SRF in mouse
heart resulted in marked decrease in TGFβ1 expression.
Conclusions: Our studies demonstrated the pro-fibrotic role
of truncated ROCK1 in vivo and provided an animal model
that recapitulated human heart failure. The results elucidated
that activation of NF-κB and TGFβ1 signaling contributed to
Rho kinase-mediated fibrotic cardiomyopathy, the later was
regulated by SRF.
ABSTRACTS
The glycogen synthase kinase-3 (GSK-3) family of protein
kinases consists of two highly related isoforms, α and β. The
GSK-3 family has been reported to regulate an astonishing
variety of physiological and pathophysiological processes,
whereas virtually nothing is known as for whether they
regulate aging. Aging, defined as the progressive loss of
function accompanied by decreasing fertility and increasing
mortality with advancing age, is a complex biological
process, controlled by multiple genetic, epigenetic and
environmental factors. We now present the first studies
defining the role of GSK-3α in the aging process using
germline GSK-3α knockout (KO) mice at age of 3, 6, 12,
and 24 mos. Age-matched wildtype (WT) mice served as
control. Kaplan-Meier analysis shows that percent survivals
(WT vs. KO) was 73.33 vs. 42.11‡ at 24 mos of age. In the
heart we find cardiac hypertrophy, marked cell loss with
replacement fibrosis, contractile dysfunction with profound
abnormalities of mitochondrial structure, and impaired
autophagy (assessed by Beclin1/ATG6), all becoming evident
after 6 mos of age. Echocardiographic LV mass (in mg)
is as follows (4 time points): 124.0±3.15 vs. 132.4±3.93,
136.2±4.75 vs. 171.5±7.57‡, 169.2±8.53 vs. 203.5±8.18‡,
195.4±9.82 vs. 257.4±7.24‡; ejection factor: 60.6±1.08
vs. 62.5±1.52, 64.3±1.10 vs. 60.7±1.61*, 58.5±1.74 vs.
52.2±1.41‡, 55.5±0.96 vs. 49.4±1.02‡; invasive hemodynamic
dP/dt+: 6863.8±673.8 vs. 6999.4±473.2, 7115.4±392.9
vs. 6012.6±407.3*, 7509.6±419.6 vs. 4291.6±473.5*,
6889.0±460.9 vs. 3955.5±306.9‡. We also observed
increased BrdU positive nuclei in isolated cardiomyocytes
from the KO at 6 mos of age, and increased stem cells at
every time-point, compared with age-matched WT. In the
small intestine, we find marked senescence-associated
β-galactosidase (SA-β-GAL) activity associated with thinning
of the crypts, consistent with reduced cell cycling in this
tissue. The results imply accelerated development of agerelated pathologies in the KO heart and small bowel. These
phenotypes are associated with a reduced lifespan in the
mouse. Thus GSK-3α is a novel regulator of aging which
retards age-related pathologies and prolongs lifespan of the
organism. (*, P<0.05; ‡, P<0.01 KO vs. WT).
X. Yang: None. Q. Li: None. X. Lin: None. Y. Ma: None. X. Yue: None.
Z. Tao: None. J. Chang: None.
This research has received full or partial funding support from the
American Heart Association, South Central Affiliate (Arkansas,
New Mexico, Oklahoma & Texas).
P337Not published at presenter’s request.
P338 Withdrawn
Next year’s conference: July 23–26, 2012. Visit my.americanheart.org/bcvssessions for more information.
121
Poster Presentations (continued)
P339The Mechanistic Relationship Between GRK2 and eNOS
During Cardiac Ischemia/Reperfusion Injury
Zheng Maggie Huang, Erhe Gao, Xiying Shang, Xufan Tian, Gang
Qiu, J Kurt Chuprun, Thomas Jefferson Univ, Philadelphia, PA; David J
Lefer, Emory Univ, Atlanta, GA; Walter J Koch, Thomas Jefferson Univ,
Philadelphia, PA
Background: Previous studies have shown that both cardiacspecific GRK2 transgenic (TG) (αMHC-GRK2) mice and eNOS
knockout (KO) mice have larger infarcts after I/R compared
to control mice. In contrast, cardiac-targeted eNOS TG mice
and cardiac specific GRK2 KO mice show cardioprotection
after I/R, suggesting a dynamic interaction between the two
proteins. Mechanistically, GRK2 can be inhibited by cellular
NO through S-nitrosylation with Cys340 being the major
site; meanwhile GRK2 has been shown to directly bind to
and inhibit Akt, which is a strong activator of eNOS. Aim: to
investigate the potential novel interaction between GRK2 and
eNOS, and the consequent functional impact on the protein
activity and cardiac phenotype after I/R injury. Methods:
αMHC-GRK2 mice were crossed with either eNOS TG or
eNOS KO mice. All mice were subjected to sham or 30min
myocardial ischemia via coronary artery ligation followed
by 24hrs of reperfusion. Infarct size, cardiac function, and
tissue apoptosis were examined. Co-IP was used to test the
interaction between GRK2 and eNOS, and phosphorylation
of eNOS was studied in neonatal myocytes. Results: 1).
αMHC-GRK2/eNOS TG hybrid mice showed a significantly
reduced infarct size after I/R compared to αMHC-GRK2 mice,
accompanied by improved cardiac function measured by
echocardiography and hemodynamics, and significantly less
apoptosis tested by TUNEL assay, implying a rescue effect by
eNOS. 2). αMHC-GRK2/eNOS KO mice exhibited a bigger
infarct size compared to either αMHC-GRK2 mice or eNOS
KO mice. 3). CO-IP confirmed the interaction between GRK2
and eNOS in cardiac tissue, which was increased upon β-AR
agonist treatment. 4). In neonatal ventricular myocytes, GRK2
overexpression significantly decreased eNOS phosphorylation
at Ser1177 after exposure to H2O2, while GRK2 knockdown
by siRNA led to slight increase in pSer1177. Conclusions:
eNOS interacts with and may be a downstream target of
GRK2 in the heart. Decreased activation of eNOS may mediate
the deleterious effect of GRK2 overexpression during cardiac
I/R injury.
ABSTRACTS
.M. Huang: None. E. Gao: None. X. Shang: None. X. Tian: None.
Z
G. Qiu: None. J. Chuprun: None. D.J. Lefer: None. W.J. Koch: None.
This research has received full or partial funding support from the
American Heart Association, Great Rivers Affiliate (Delaware, Kentucky,
Ohio, Pennsylvania & West Virginia).
P340The Role of Osteoclast-like Cells in Abdominal
Aortic Aneurysm
Dai Yamanouchi, Colin Stair, Stephanie Morgan, K Craig Kent, Univ of
Wisconsin Sch of Med and Public Health, Madison, WI
Increasing evidence suggests that arterial calcification is the
result of highly organized processes resembling those seen in
bone which relies upon a delicate balance between mineral
deposition and resorption by osteoblasts and osteoclasts,
respectively. Osteoclast-like cells (OLCs) are derived from the
monocyte/macrophage lineage and share osteoclast features
such as the ability to dissolve extracellular matrix. Although
osteoclastogenesis, the development of OLCs, has been
reported to occur in calcified arteries, its potential role in the
development of abdominal aortic aneurysm (AAA) has yet to
be explored. Firstly, we obtained aortic tissues from patients
undergoing surgical repair for AAA (N = 5) and aortic occlusive
disease (N = 5). Alizarin-Red staining showed calcification
in both aneurysmal and occlusive aorta. In contrast, OLCs,
122
recognized as multi-nucleated cells positive for enzymatic
TRAP staining, were identified only in aneurysmal aorta (100%,
5 of 5). A time course experiment in mouse calcium chlorideinduced AAA (CaCl2 AAA) showed similarities in both spatial
and temporal patterns of calcium deposition and monocyte/
macrophage accumulation starting 48 hours followed by
OLCs formation peaked at 7 days after CaCl2 injury. Secondly,
we administered bisphosphonate (pamidronate disodium, 1.25
mg/kg, weekly), an inhibitor of osteoclasts, or normal saline
as a control, intravenously to mice after CaCl2 injury (N = 5).
Bisphosphonate-treated mice showed a significant reduction
in aortic dilation compared to control mice after 7 and 42 days
with mean fold change of 1.16 ± 0.04 (vs. 1.65 ± 0.05, P <
0.01) and 1.37 ± 0.12 (vs. 2.17 ± 0.08, P<0.01) respectively.
We also studied the effect of bisphosphonate on “developed
aneurysm” by delaying the injection of bisphosphonate
until one week after the injury to allow the aneurysm to
develop prior to injection (N = 5). The mice with delayed
bisphosphonate injection also showed significant inhibition
compared to the saline control at 42 days (1.56 ± 0.13 vs.
2.42 ± 0.06, P < 0.01). In conclusion, we have demonstrated
the existence of OLCs in AAA. We also have demonstrated
the inhibitory effect of bisphosphonate on aneurysm. These
results indicate a novel approach for the pathogenesis and
therapy of AAA.
D. Yamanouchi: None. C. Stair: None. S. Morgan: None.
K.C. Kent: None.
P341MRTF-A, a Rho-Dependent Coactivator of SRF, Plays a
Critical Role in Vascular Remodeling
Takeya Minami, Koichiro Kuwahara, Yasuaki Nakagawa, Yoshihiro
Kuwabara, Kazuhiro Nakao, Hideyuki Kinohita, Satoru Usami, Chinatsu
Yamada, Junko Shibata, Toshio Nishikimi, Kyoto Univ Graduate Sch
of Med, Kyoto, Japan; Minoru Takaoka, Graduate Sch of Med, The
Univ of Tokyo, Tokyo, Japan; Masataka Sata, The Univ of Tokushima
Graduate Sch, Tokushima, Japan; Ryozo Nagai, Graduate Sch of Med,
The Univ of Tokyo, Tokyo, Japan; Kazuwa Nakao, Kyoto Univ Graduate
Sch of Med, Kyoto, Japan
Objective: Rho-ROCK pathway is involved in the progression
of pathological vascular remodeling through the regulation
of cell proliferation and migration by controlling gene
transcription. While a transcriptional co-activator Myocardinrelated transcription factor(MRTF)-A transduces Rho-actin
signaling to SRF activation in the nucleus, roles of MRTF-A in
the process underlying vascular diseases remain unknown.
Methods and Results: We found that MRTF-A mRNA and
protein expression in femoral arteries 2weeks after wire injury
was significantly higher than that in sham-operated arteries of
mice. In contrast, the expression of myocardin or MRTF-B was
significantly decreased in injured arteries. We then evaluated
the role played by MRTF-A in pathological vascular remodeling
by creating several vascular disease models of mice lacking
MRTF-A (MRTF-A-/-). Neointima formation induced by ligation
in carotid arteries of MRTF-A-/- was significantly smaller than
that of MRTF-A+/-. Neointima formation induced by wire
injury in femoral arteries of MRTFA-/- was significantly smaller
than that of MRTF-A+/+. Atherosclerotic lesions of MRTF-A/;ApoE-/- was also significantly smaller than those of MRTFA+/+;ApoE-/. The expression of SRF-target genes involved in
cellular migration, such as MMP9 and vinculin,within injured
arteries of MRTF-A-/- was significantly attenuated compared
to that of MRTF-A+/+. In cultured, de-differentiated rat aortic
vascular smooth muscle cells (RAVSMC), the expression of
these genes was decreased by knocking down MRTF-A.
Indeed, the promoter activity of vinculin gene was controlled
by MRTF-A in RAVSMC. In addition, knocking down MRTF-A
in RAVSMC resulted in a significant impairment in migration
capacity. Furthermore, treatment with a small molecule
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Poster Presentations (continued)
CCG1423 inhibiting MRTF-A significantly reduced neointima
formation induced by wire injury in femoral arteries of mice.
Conclusions: Our study revealed that in de-differentiated
vascular smooth muscle cells, in which myocardin and
MRTTF-B expression is decreased, increased expression of
MRTF-A contributes to the acquisition of migration capacity by
maintaining SRF-target genes transcription, thereby promoting
stress-induced vascular remodeling.
T. Minami: None. K. Kuwahara: B. Research Grant; Modest; Grantsin-Aid for Scientific Research from the Japan Society for the Promotion
of Science. Y. Nakagawa: None. Y. Kuwabara: None. K. Nakao: None.
H. Kinohita: None. S. Usami: None. C. Yamada: None. J. Shibata:
None. T. Nishikimi: None. M. Takaoka: None. M. Sata: None. R. Nagai:
None. K. Nakao: B. Research Grant; Modest; Grants-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science.
P342Mitochondria-Targeted Antioxidant Therapy Prevents
Angiotensin II-Mediated Connexin43 Remodeling and
Sudden Arrhythmic Death
Ali A Sovari, Shadi Zandieh, Univ of Illinois at Chicago, Chicago, IL;
Shahriar Iravanian, Emory Univ, Atlanta, GA; Lianzhi Gu, Euy-Myong
Jeong, Marcelo G Bonini, Samuel C Dudley, Jr, Univ of Illinois at
Chicago, Chicago, IL
A.A. Sovari: None. S. Zandieh: None. S. Iravanian: None. L. Gu:
None. E. Jeong: None. M.G. Bonini: None. S.C. Dudley, Jr: None.
This research has received full or partial funding support from the
American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa,
Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota,
South Dakota & Wisconsin).
P344A Natural p300-Specific Histone Acetyltransferase
Inhibitor, Curcumin, Prevents the Development of Heart
Failure in Addition to ACE Inhibitor After Myocardial
Infarction in Rats
Tatsuya Morimoto, Univ of Shizuoka, Shizuoka, Japan; Yoichi
Sunagawa, Graduate Sch of Med, Kyoto Univ, Kyoto, Japan; Hiromichi
Wada, Kyoto Medical Ctr, Kyoto, Japan; Yasufumi Katanasaka, Univ of
Shizuoka, Shizuoka, Japan; Tomohide Takaya, Graduate Sch of Med,
Kyoto Univ, Kyoto, Japan; Teruhisa Kawamura, Kyoto Medical Ctr, Kyoto,
Japan; Shigeki Yanagi, Akira Marui, Ryuzo Sakata, Akira Shimatsu,
Takeshi Kimura, Masatoshi Fujita, Graduate Sch of Med, Kyoto Univ,
Kyoto, Japan; Koji Hasegawa, Kyoto Medical Ctr, Kyoto, Japan
Background: We found that curcumin, a p300 histone
acetyltransferase (HAT) inhibitor, prevents deterioration of
the systolic function in rat heart failure models in vivo. To
clinically apply this novel therapy to humans, it should be
clarified whether or not curcumin has additional effects on
conventional heart failure therapy comprising angiotensinconverting enzyme inhibitors (ACEI). Methods and Results:
Rats were subjected to a sham operation or myocardial
infarction (MI). One week later, 32 rats were randomly
assigned to solvents (control), enalapril (ACEI, 10 mg/kg/
day) alone, curcumin (50 mg/kg/day) alone, or curcumin plus
enalapril for 6 weeks. ACEI, but not curcumin treatment,
decreased the blood pressure in post-MI rats. After treatment,
LVFS (fractional shortening) was significantly (p<0.05) higher
in the ACEI (29%) and curcumin (29%) groups than in the
vehicle group (22%). Notably, LVFS significantly (p <0.05)
increased on ACEI/ curcumin combination therapy (35%)
compared with therapy comprising either ACEI or curcumin
alone. The LV wall thickness and cardiomyocyte diameter
were significantly smaller in the ACEI/curcumin than the
ACEI group. Moreover, perivascular fibrosis was significantly
reduced in the ACEI and curcumin groups compared with the
vehicle group. This reduction was further augmented by the
ACEI/ curcumin combination therapy. Conclusion: Curcumin,
restores the post-MI LV systolic function in rats without
affecting the blood pressure. This natural non-toxic dietary
compound in addition to ACEI has beneficial effects on LV
systolic function.
. Morimoto: None. Y. Sunagawa: None. H. Wada: None.
T
Y. Katanasaka: None. T. Takaya: None. T. Kawamura: None.
S. Yanagi: None. A. Marui: None. R. Sakata: None. A. Shimatsu:
None. T. Kimura: None. M. Fujita: None. K. Hasegawa: None.
P345The Neuregulin Glial Growth Factor 2 (GGF2) Produces
Sustained Improvement in Left Ventricular Function in
Rats with Both Early and Established Heart Failure
Anindita Ganguly, Erika Troy, Maya Srinivas, Andrea Vecchione,
Patrick Sarmiere, Tao Hu, Jennifer Iaci, Craig Hackett, Donald Button,
Anthony Caggiano, Tom J Parry, Acorda Therapeutics, Hawthorne, NY
Neuregulin-1β is essential for fetal cardiac development
and adult cardiac function. Previous reports indicate that
neuregulins improve left ventricular function in heart failure
models, however the duration of the functional improvements
with early or late initiation of neuregulin treatment has not been
characterized. The present studies examine the effects of early
and delayed initiation of intravenous GGF2 treatment on left
ventricular (LV) function in rats with myocardial infarction (MI).
Rats underwent surgically-induced MI by left anterior coronary
artery ligation. Treatment with vehicle or GGF2 (2.6 mg/kg)
was initiated at 2 or 16 w post-MI and continued once or twice
weekly or once every two weeks for the in-life duration of the
study (approximately 40 weeks). LV function was assessed
echocardiographically up to once weekly for the duration
of the study. Early and delayed initiation of GGF2 treatment
caused sustained and significant improvement (p < 0.05) in
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ABSTRACTS
Introduction: Angiotensin II activation and associated elevation
in ROS have been implicated in pathogenesis of arrhythmia.
Nevertheless commonly used antioxidants have been ineffective
in clinical trials. We created a transgenic mouse model of
cardiac restricted overexpression of ACE (ACE8/8). These mice
show spontaneous VT/ VF, SCD, and a reduction in Cx43 level,
which impairs conduction and predisposes to arrhythmia. We
sought to determine the role and the major source of ROS
by angiotensin II in VT/ VF and Cx43 remodeling. Method:
Wild type and ACE8/8 mice with and without 2 weeks of
treatment with a NOS inhibitor (L-NIO, 25mg/Kg IP injections
daily), a mitochondria-targeted antioxidant (Mito-TEMPO,
0.7mg/Kg IP injections daily), a NADPH oxidase inhibitor
(Apocynin 80mg/L in drinking water), and ACE8/8 crossed
with P67DN were studied. Western blotting (with derivatization
to dinitrophenylhydrozone to detect oxidized protein levels),
detection of superoxide production in mitochondria by red
mitochondrial superoxide indicator and immunohistochemistry
staining for Cx43 were performed. EP study was performed
by a 1.1F octapolar catheter through pacing the right ventricle
using a burst pacing protocol. Results: Proteins were more
oxidized (increased protein-carbonyl detection), and Cx43 was
reduced in ACE8/8 to 33% of control. Treatment with MitoTEMPO prevented SCD and improved survival in ACE8/8 mice
(p=0.0005, hazard ratio 4.76 with 95% CI of 1.96 to 11.53).
Inducibility of VT/VF was higher in ACE8/8 mice compare to WT
(87.5% vs. 2.3%) and VT inducibility was reduced with MitoTEMPO treatment (50% in treatment group). Cx43 level was
increased by 1.7 fold with Mito-TEMPO treatment. Treatments
with L-NIO, Apocynin and crossing with P67DN mice did
not prevent VT/VF and SCD in ACE8/8 mice. Conclusion:
In a model of angiotensin II activation mitochondria-targeted
antioxidant, prevents VT/VF/SCD and Cx43 remodeling.
Suppression of NADPH oxidase activity by Apocynin and
crossing the ACE8/8 mice with P67DN or inhibition of NOS by
L-NIO did not prevent the arrhythmic deaths in ACE8/8 mice.
This result suggests that mitochondria are the major source of
ROS by angiotensin II and mitochondria-targeted antioxidants
may be effective antiarrhythmic drugs.
P343 Not published at presenter’s request.
123
Poster Presentations (continued)
both ejection fraction (EF) and fractional shortening (FS) in all
regimens tested. The greatest improvements were seen with
the once weekly dosing paradigm after early initiation (average
EF (%) at 40 weeks post initiation of dosing: vehicle = 44.4 ±
6.0, n = 8 rats, vs. GGF2 = 64.7±6.1. n = 9 rats) and twice
weekly dosing paradigm after delayed initiation (average EF
(%) at 4 weeks post initiation of dosing: vehicle = 34.18±1.6,
n = 7 rats, vs. GGF2 = 50.69±4.68, n = 7 rats). In addition, LV
function improved when rats were re-challenged with GGF2
following an extended wash out period. This observation
indicates potential efficacy for treatment paradigms that
utilize intermittent dosing. These findings suggest that GGF2
produces sustained improvement in LV function after early or
delayed initiation of treatment following MI in rats.
A. Ganguly: A. Employment; Significant; 100. F. Ownership Interest;
Modest; 1. E. Troy: A. Employment; Significant; 100. F. Ownership
Interest; Modest; 1. M. Srinivas: A. Employment; Significant; 100.
F. Ownership Interest; Modest; 1. A. Vecchione: A. Employment;
Significant; 100. F. Ownership Interest; Modest; 1. P. Sarmiere: A.
Employment; Significant; 100. F. Ownership Interest; Modest; 1. T. Hu:
A. Employment; Significant; 100. F. Ownership Interest; Modest; 1.
J. Iaci: A. Employment; Significant; 100. F. Ownership Interest; Modest;
1. C. Hackett: A. Employment; Significant; 100. F. Ownership Interest;
Modest; 1. D. Button: A. Employment; Significant; 100. F. Ownership
Interest; Modest; 1. A. Caggiano: A. Employment; Significant; 100. F.
Ownership Interest; Modest; 1. T.J. Parry: A. Employment; Significant;
100. F. Ownership Interest; Modest; 1.
P346Regulation of GRK5 in Myocytes Is Mediated by Nuclear
Transcription Factor [κ]B: A New Clue in Heart Disease
ABSTRACTS
Kazi N Islam, Walter J Koch, Thomas Jefferson Univ, Philadelphia, PA
G protein coupled receptor kinase-5 (GRK5) plays a key role
in the heart and it’s expression is increased in heart failure.
Moreover, a human mutation within its amino-terminus
appears to play a role in therapeutic responses to β-blockers
in heart failure patients. Nuclear transcription factor kappa B
(NF-κB), a ubiquitous transcription factor, is involved in the
regulation of numerous genes in various tissues and activation
of NF-κB has also been shown to be associated with heart
disease. Here, we investigated the role of NF-κB signaling
in the regulation of the GRK5 gene and GRK5 expression in
neonatal rat ventricular myocytes (NRVMs). In analyzing the
5’-flanking DNA of GRK5, the presence of a potential NF-κB
binding site was observed in the promoter. Phorbol myristate
acetate (PMA), a known stimulator of NF-κB, increased the
levels of GRK5 in NRVMs. Conversely, treatment of NRVMs
with N-acetyl cysteine (NAC), a known inhibitor of NF-κB,
decreased the levels of GRK5 compared to control cells.
Utilizing electrophoretic mobility shift assay (EMSA) with
nuclear extracts from NRVMs treated with or without PMA,
it was found that proteins bound to the GRK5 promoter
containing the putative NF-κB binding site. When an antibody
specific for NF-κB was utilized, a super-shift of the proteins
binding to the putative NF-κB binding site was observed.
Furthermore, interaction of NF-κB with GRK5 was confirmed
by shift Western blot. Chromatin immunoprecipitation (ChIP)
showed dynamic recruitment of both p50 and p65 to the
NF-κB site of GRK5 promoter after treatment of NRVMs with
PMA. Treatment of myocytes with siRNA and subsequent
knock-down of NF-κB p65 decreased the levels of GRK5
as judged by real time quantitative PCR. Finally, adenovirus
mediated overexpression of a dominant negative IκB-α in
NRVMs inhibited the levels of GRK5. Taken together, our
present studies suggest that NF-κB plays a potential role in
the regulation of GRK5 transcription in myocytes and this may
translate to key expressional changes seen in heart disease.
K.N. Islam: None. W.J. Koch: None.
P347 Withdrawn
124
P348High Glucose Induces Smad Activation via the
Transcriptional Coregulator P300 and Contributes to
Cardiac Fibrosis and Hypertrophy
Kim A Connelly, Esq, Maral Ouzounian, Andrew Advani, Suzanne
Advani, Kerri Thai, Peter P Liu, Richard E Gilbert, St Michael’s Hosp,
Toronto, ON, Canada
Background: Despite advances in the treatment of heart
failure (HF), the mortality remains high, particularly in those
individuals with diabetes mellitus. Activated transforming
growth factor beta (TGF-ß) contributes to the pathogenesis
of diabetic cardiomyopathy. We hypothesized that the
transcriptional co-activator p300 regulates glucose induced
activation of TGF-ß via acetylation of a specific Lysine residue
(Lys19) in the Mad homology 1 domain of Smad 2, and that
by inhibiting p300, TGF-ß activity will be reduced and heart
failure ameliorated/prevented. Methods: p300 activity and
Smad acetylation in normal glucose (5.6 mmol/L - NG) and
high glucose (25 mmol/L - HG) media were assessed in H9c2
rat cardiomyoblasts. [H]3 proline incorporation was assessed
in cardiac fibroblasts as a marker of collagen synthesis. The
role of increased p300 activity was assessed in vitro by using
a known p300 inhibitor, curcumin or siRNA directed at p300
and in vivo in a hemodynamically validated model of diabetic
cardiomyopathy, the (mRen)2–27 transgenic rat. Results:
H9c2 cells exposed to HG demonstrated increased p300
activity c/w NG media, that was reduced by p300 inhibition
using curcumin or p300 siRNA (all p<0.01). Increased p300
activity in HG media increased [H]3 proline incorporation
(p<0.05). This effect was attenuated by treatment with
curcumin/p300 siRNA (p<0.01). Finally, H9c2 cells were
stimulated, extracted protein was immunoprecipitated with
Smad2, and lys19 acetylation assessed. Acetylation of the
Lys19 was reduced in cells pre-incubated with the p300
inhibitor (p<0.05). To determine the functional significance
of p300 inhibition, diabetic Ren-2 rats were randomised
to receive either curcumin/vehicle for 6 weeks. Curcumin
treated diabetic rats had reduced cardiac hypertrophy and
improved chamber compliance when c/w untreated diabetic
counterparts (all p<0.01). Conclusions: These findings
demonstrate that high glucose increases activity of the
transcriptional coregulator p300, acetylating Smad2 and
promoting cardiac fibrosis and hypertrophy. Inhibition of p300
reduces cardiac hypertrophy and results in improved diastolic
function. Modulation of the p300 may be a novel strategy to
treat diabetes induced heart failure.
K.A. Connelly: None. M. Ouzounian: None. A. Advani: None.
S. Advani: None. K. Thai: None. P.P. Liu: None. R.E. Gilbert: None.
P349Ambient Air Pollution Compromises Nitric Oxide
Bioavailability, Leading to Accelerated Endothelial
Cell Senescence
Nicole Buechner, Ulrich Sydlik, Klaus Unfried, Joachim Altschmied,
Judith Haendeler, IUF, Duesseldorf, Germany
Particulate matter (PM) pollution is a burden to public health.
Although PM causes a health risk to the lung, the overall
evidence indicates that the majority of the PM effects are upon
the cardiovascular system. Several studies demonstrated
that ultrafine particles can directly enter the circulation and
thus may interact with endothelial cells. However, intracellular
mechanisms affected by non-cytotoxic, in vivo relevant
concentrations of particles have not been investigated in
endothelial cells in vitro and in vivo. Therefore, this study aims
for the first time to analyze the effects of ultrafine and fine
particles of different materials in non-toxic concentrations on
human endothelial cells (EC) and in animals. We incubated EC
with ultrafine and fine Carbon Black (ufCB and fCB) particles
as well as Titaniumdioxide (ufTiO2 and fTiO2) and determined
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Poster Presentations (continued)
the non-toxic concentrations. MTT measurements revealed
that 0.1 and 1 µg/cm2 did not reduce endothelial cell viability.
To test whether these concentrations influence endothelial cell
function, we measured nitric oxide (NO) bioavailability, which is
important for vessel function. Only ultrafine particles reduced
the S-NO content of EC, whereas fine particles had no effect
at the same concentrations. Interestingly, the effects observed
with ufCB and ufTiO2 were more pronounced than with the
known reducer of NO bioavailability, H2O2. We previously
demonstrated that NO increases activation of Telomerase
Reverse Transcriptase (TERT), an enzyme essential for
telomere maintenance. TERT activation is required to
protect EC from apoptosis and the onset of senescence
and TERT is inactivated by the Src kinase under conditions
of oxidative stress. Therefore, we investigated the effects
of ufCB and ufTiO2 on TERT and Src activation. ufCB and
ufTiO2 significantly reduced TERT and increased Src kinase
activation. To investigate whether ufCB show also effects in
vivo, we instilled ufCB into rats and determined eNOS and
TERT expression in the aorta. ufCB reduced eNOS and TERT
expression in the abdominal aorta of animals treated with
ufCB. Thus, ultrafine nanoparticles, which we inhale every
day, seem to reduce endothelial function and thus should be
considered a risk factor for cardiovascular disease.
N. Buechner: None. U. Sydlik: None. K. Unfried: None. J. Altschmied:
None. J. Haendeler: None.
P350Interstitial Volume Regulation in the Heart: Mechanistic
Insights from ex vivo Fluid Dynamics in Pig Myocardia
Maria P McGee, Michael Morykwas, Jim Jordan, Louis Argenta, Wake
Forest Sch of Med, Winston-Salem, NC
P351Statins Prevent Expansive Remodeling in Arterialized
Vein Grafts
Beiping Qiang, Jonathan Toma, Schulich Heart Program, Sunnybrook
Res Inst, Toronto, ON, Canada; Hiroko Fujii, Terrence Donnelly Heart
Ctr, St Michael’s Hosp, Toronto, ON, Canada; Azriel Osherov, Nafiseh
Nili, Paul Fefer, Michelle Samuel, Schulich Heart Program, Sunnybrook
Res Inst, Toronto, ON, Canada; Jagdish Butany, Toronto General
Hosp, Univ Health Network, Toronto, ON, Canada; Howard Leong-Poi,
Terrence Donnelly Heart Ctr, St Michael’s Hosp, Toronto, ON, Canada;
Bradley H Strauss, Schulich Heart Program, Sunnybrook Res Inst,
Toronto, ON, Canada
Vein grafts (VG) account for more than 70% of conduits used
in aortocoronary bypass surgery, but have high failure rates
by 10 years. We have previously shown in an experimental
model that VG undergo marked expansion by 4 weeks, which
was followed by increased total LDL retention at 12 weeks.
The objective of this study was to determine whether statin
therapy would prevent these expansive remodelling changes.
Methods and Results Reversed jugular vein-to-common
carotid artery interposition graft were constructed in 24
cholesterol-fed (0.5%) rabbits. Rabbits were randomized to
either control or atorvastatin (5 mg/kg/day) groups, starting
two weeks prior to vein graft implantation and continuing
until sacrifice at 1 or 12 weeks post-surgery. Ultrasound
measurements of arterial luminal cross-sectional area (LCSA)
were done at day 3 and at 4, 8 and 12 weeks post-surgery.
Histomorphometric measurements were performed following
sacrifice at 12 weeks. Atorvastatin treatment significantly
decreased total plasma cholesterol levels at 12 weeks
(6.70±4.19mmol/L vs placebo 38.65±10.55mmol/L, p<0.001).
Atorvastatin significantly reduced expansive remodelling
at all time points, including at 12 weeks (44.6±6.62mm2
vs placebo 77.6±10.68mm2, p<0.001). Intimal CSA by
histomorphometry was significantly reduced by atorvastatin
at 12 weeks (5.59±2.19mm2 vs placebo 9.57±2.43mm2,
p<0.05). At 1 week, macrophage infiltration was significantly
reduced (Ram11 positive intimal staining 0.018±0.019 vs
0.189±0.049, p<0.05). Atorvastatin treated samples at 1 week
showed reduction in both MMP 2 activity (gelatin zymography)
and metalloelastase activity (casein zymography). Conclusion:
Atorvastatin prevents expansive remodelling in arterialized
VG, likely through inhibition of macrophage infiltration and
reduction of tissue proteolytic activity. This may be an
important mechanism preventing VG atherosclerosis and late
VG failure.
. Qiang: None. J. Toma: None. H. Fujii: None. A. Osherov: None.
B
N. Nili: None. P. Fefer: None. M. Samuel: None. J. Butany: None.
H. Leong-Poi: None. B. Strauss: None.
P352The Anticancer mTOR-Inhibitor Temsirolimus Produces
Left Ventricular Dysfunction in Murine Hearts
Carlo G Tocchetti, Carmela Coppola, Natl Cancer Inst, Sen Pascale
Fndn, Naples, Italy; Cristina Quintavalle, Federico II Univ, Naples,
Italy; Antonio Barbieri, Domenica Rea, Giuseppe Palma, Marianna
Gala, Antonio Luciano, Aldo Giudice, Natl Cancer Inst, Sen Pascale
Fndn, Naples, Italy; Nazareno Paolocci, Johns Hopkins Medical Insts,
Baltimore, MD; Claudio Arra, Rosario V Iaffaioli, Natl Cancer Inst, Sen
Pascale Fndn, Naples, Italy; Gerolama Condorelli, Federico II Univ,
Naples, Italy; Nicola Maurea, Natl Cancer Inst, Sen Pascale Fndn,
Naples, Italy
ABSTRACTS
Developing and testing specific therapies for myocardial edema
require understanding their mode and site of action. However,
parsing determinants of interstitial volume/pressure relationships
in vivo is difficult, particularly in the myocardium, where
rhythmic contractions add to the confounding components
of fluid-transfer driving pressures. Here, we describe a novel
ex-vivo model system based on osmotic stress techniques
and illustrate its application to analyses of local myocardial fluid
dynamics that exclude systemic influences and systole/diastole
compressive cycles. Freshly harvested ventricular explants were
equilibrated in physiologic media at colloidosmotic pressures
ranging from 3 to 219 mmHg, and fluid transfer in/out was
measured gravimetrically as a function of time and pressure.
The relationship between the change in explant volume and
the imposed colloidosmotic pressure of the media was linear.
The hydration potential, defined empirically as the pressure at
which explant volume did not change, was calculated from the
abscissa intercept at volume change = 0, and the compliance
from the slope of volume/pressure regression lines. Fluid-transfer
rates and energies were derived from volume versus time
trajectories measured at 4 and 37 °C. Hydration potential was
71.3 ± 10.6 and 23.0 ± 15.2 mmHg at 4 and 37°C (significant,
P = 0.002), while compliance was 1.09 ± 0.3 and 1.22 ± 0.2
µl/g/ mmHg (not significant, P = 0.2, n = 5). Temperaturedependent differences between in/out flow rates were also
significant, giving experimental activation energies of -5.9 ±
2.2 and 1.4 ± 0.7 kcal/mol for inflow and outflow, respectively.
Results show that at physiologic temperatures, even without
vascular hydrostatic pressure gradients and lymphatic drainage,
the myocardial bias toward interstitial fluid efflux persists. These
findings are consistent with local fluid-control mechanisms
actuated by colloidosmotic and tensional forces rather than
passive changes in flow resistance. This new approach allows
quantitative evaluation of interstitial components of Starling’s
forces and should help in mechanistic preclinical characterization
of treatments to correct interstitial myocardial edema.
M.P. McGee: None. M. Morykwas: None. J. Jordan: None.
L. Argenta: None.
Background: Cardiotoxicity is a major drawback and
social problem linked to many anticancer treatments. Early
identification of signs of this adversity would certainly benefit
the management of oncologic patients. The mTOR-inhibitor
temsirolimus is currently being evaluated for anticancer
efficacy in hundreds of clinical trials and is approved for
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125
Poster Presentations (continued)
Tracking.echocardiography.we.found.that.in.the.temsirolimus.
group.radial.strain.was.already.decreased.at.7.days:.42±5%,.
p=.01 vs sham.(59±1%)..Conclusions:.The.antineoplastic.
mTOR-inhibitor.temsirolimus.induces.LV.dysfunction.in.
mice..Such.dysfunction.occurs.later.than.the.one.observed.
with.Doxo,.but.speckle.tracking.echocardiography.is.
more.sensitive.than.conventional.echocardiography.and.
can.detect.early.signs.of.myocardial.alteration.that.may.
prelude.to.overt.LV.dysfunction..The.clear.mechanisms.of.
temsirolimus.cardiotoxicity.are.to.be.elucidated.in.further.
experimental.studies..We.also.plan.to.apply.speckle.tracking.
echocardiography.to.clinical.studies,.in.order.to.evaluate.the.
impact.of.early.identifi.cation.of.temsirolimus.cardiotoxicity.in.
the.treatment.of.renal.cell.carcinoma.
C.G. Tocchetti: None. C. Coppola: None. C. Quintavalle: None.
A. Barbieri: None. D. Rea: None. G. Palma: None. M. Gala: None.
A. Luciano: None. A. Giudice: None. N. Paolocci: None. C. Arra:
None. R.V. Iaffaioli: None. G. Condorelli: None. N. Maurea: B.
Research Grant; Significant; Pascale Foundation Institutional Grant.
ABSTRACTS
treatment.of.advanced.renal.cell.carcinoma..However,.
the.PI3K/Akt.pathway.converges.on.mTOR,.which.is.a.
central.regulator.of.cell.growth,.including.cardiomyocyte.
growth..Here,.we.aim.at.evaluating.the.cardiac.effects.of.the.
anticancer.mTOR-inhibitor.temsirolimus.in.a.mouse.model.
in vivo..Methods:.Left.Ventricular.(LV).fractional.shortening.
(FS).was.assessed.by.M-mode.echocardiography.in.sedated.
C57BL/6.mice.(2–4.mo..old).at.day.0,.and.after.2,.7,.14,.
21.days.from.a.single.i.p..injection.of.temsirolimus.(0.1mg/
kg,.a.dose.comparable.to.the.one.used.to.treat.cancer.in.
humans).or.vehicle..Doxorubicin.(Doxo,.2.17.mg/kg/day.for.7.
days).was.used.as.a.positive.control..With.Speckle.Tracking.
echocardiography.(ST).we.also.evaluated.radial.myocardial.
strain.(%),.a.very.sensitive.parameter.which.can.detect.
subtle.changes.in.cardiac.function..Results:.After.2.days,.
there.was.no.change.in.FS.with.temsirolimus,.but.FS.was.
already.reduced.with.Doxo:.52±0.2%,.p=.0000001 vs sham.
(60±0.4%)..With.temsirolimus,.FS.was.reduced.only.after.21.
days:.50±3%,.p=.009 vs sham..Interestingly,.with.Speckle.
126
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