1. No category

MODUIATION OF Ca,* TRANSPORT CHARACTERISTICS VITH LIPIDS IN
ISOIATED SARCOPLASMIC RETICULUM VESICLES
BY
PENELOPE P. RAMPERSAD
A Thesis
Submined to the Facuþ of Graduate Studies
in Panial Fulfill¡nent of rhe Requirements
for the Degree of
MASTEROFSCIENCE
Depanment of Physiology, and
The Division of Stroke a¡d Vascular Disease
University of Manitoba
\Mnnipeg, Manitoba
@
December
2OO3
THE UNIVERSITY OF MANITOBA
FACULTY OF GRADUATE STUDIES
COPI'RIGHT PERMISSION PAGE
Modulation of Ca2* Transport Châracteristics with Lipids in
Isolated Sarcoplasmic Reticulum Vesicles
BY
Penelope P. Rampersad
A ThesisÆracticum submitted to the Faculty of Graduate Studies of The University
of Manitoba in partial fulfillment of the requirements of the degree
of
MASTER OF SCIENCE
PENELOPE P. RAMPERSAD @2003
Permission has been grânted to the Library ofrhe university of Manitoba to lend or sell copies
of this thesis/practicum' to the National Library ofCânada to microfilm this thesis and to lend
or sell copies of the
thesis/practicum.
film, and to university Microfilm Inc. to publish an abstract of this
The author reserves other publication rights, and neither this thesis/practicum nor extensive
extrâcts from
permission.
it
may be printed or otherwise reproduced without the author's written
TABLE OF CONTENTS
ABSTRACT
ACKNO\øLEDGEMENTS
III
DIPIçATIAN
TV
LIST OF FIGIJRES
LISTOFTABLES
LIST
I.
\1I
OFABBRE\'IATIONS
RE\IIE\øOF THE
\TII
LITERATTJIRE
1
THE ROLE OF THE SARCOPI.A.SMIC RETICULL'tr{ IN STRIATED MUSCLE
EXCIT,A.TION CONTRACTION COIJPLING
CARDIACMUSCLE
SKELETATMUSCLE
I
2
4
SARCOPIASMIC RETICULI.IM CONSTITUENTS
5
CATSEQUESTRIN
6
INOSITOL TRIPHOSPIIATE RECEPTOR
RYANODINE RECEPTOR
7
SARCOPLASMIC
,/
ENDOPLASMIC RETICIJLUM
CATCILM
TRIPHOSPATASE
8
ADENOSINE
t6
INVOLVEMENT OF THE SARCOPIASMIC RETICULUÀ{ IN
ARRHYTHMOGENESIS
2I
EVIDENCE FOR CARDIOPROTECTION BY OMEGA-3 POL\-LINSATI-IRATED
FATTYACIDS
EPIDEMIOLOGY AND CONTROLLED TR]ALS
22
DIETARYMECTIANISMS
FREE FATTY ACID MECHANISM OF ACTION
2+
26
29
II.
,,
EXPERIMENTAL
PROCEDI.JBE'
MATERIAIS
32
METHODS
32
ACUTE STLJDY
FEEDING STTIDY
36
III.
RESULTS
EFFECTS OF EXOGENOUSLYAPPLIED PUFAS ON ISOLq.TED SKELETAL HSR
\aESICLES
44
EFFECTS OF CHOLESTEROLAND FIAXSEED FEEDING ON CARDIACSR
FUNCTION
DIETARYANAIYSIS
CHARACTERISATIONOFRABBITS
CI{ARACTERISATIONOFCSR
TV.
V.
DISCUSSION
LISTOFREFERENCES
62
65
66
78
90
ABSTRACT
Omega-3 poly'unsarurated fatty acids (n-3 PUFA) reduce the anhlthmogenic
potential
of the myocardium during ischaemia.
Since disrurba¡lces
in intracellular Ca'?*
regulation are arrhlthmyogenic, we examined n-3 PUFA effects on sarcoplasmic rericulum
(SR)
Cl.
handling. Concentration-dependent responses of n-3 PUFAs on SR
Ca'?* uptake
were measured using stopped flow rapid kinetic fluorimetrìc assay of
sarcoplasmic/endoplasmic rericulum Ca2*-ATPase (SERCA) cataly-ric acriviry and
þ)
(")
Ca,.
sequestration. Under conditions favouring Ca2*-dependent ryanodine receptor @yR)
activation, n-3 PUFAs significantly improved functional coupling
of net
Ca2* rranspon.
Subsequent studies showed improvements
in functional coupling were due to effects upon
In
ionomycin-permeabilsed vesicles, n-3 PfJFAs
both SERCA and RyR proteins.
sdmulated Ca'?*-dependent activation
of
SERC,A caral).dc acriviry. Howeve¡
in the
same
concenüation range, n-3 PUFAs reduced [3F!-ryanodine binding significantly. In addition,
the n-3 PUFA, docosahexaenoic acid, markedþ diminished Ca2*-induced Ca2* release in
active Câ2*-rranspon studies. Overall these observations indicate that n-3
Ca2* uptake mechanisms
PllFAs promote
panþ by improvemenrs in catal1'tic activþ and panþ by inhibitìng
Ca2* release.
Since impùed Ca'?. hârìdling by cardiac sarcoplasmic reticulum (CSR) is often
implicated in ischaemia-induced arrhlthmias, we hlpothesised that RyR-mediated Ca'?* leaks
from CSR in a model of arrhlthmia s¡ould be improved by feeding
fla-rseed, an enriched
source of the n-3 PtrFA alpha linolenic acid. Male NZ\Ø rabbits were fed one of four diets
for 8 weeks: regular chow, or regular chow
cholesterol,
or
10olo
supplemented
with
1oolo fla-rseed,
or
0.5%
flaxseed and 0.5% cholesterol. Flearrs were excised rapidþ frozen or
subjected
assayed
to an ischaemia-reperfusion protocol then frozen. cSR
for
vesicles were isolated and
SERCA activity, Ca2* trarspon, and membrane lipid compostition. Gas
chromatography revealed an increase in n-3 PLtrA conrent of CSR in both flax-fed groups
(AIA, p<0.001). Fura-2 fluorescenr measuremenrs of CSR Ca'?* transpor!
of diet in pre-ischaemìc controls. llowever, after
ischaemia, CSR
showed no effect
from flax-fed animals
transponed Ca2* at higher rares than CSR from all other groups (p<O.OO1). These data
suggest increased
n-3 PUFA content of csR membrane confers protection
ischaemia-induced impairment
of SERCA-mediated
against
ca2n rransporr. This protection may
accoun! for some of rhe anti-arrhythmic effects observed from n-3 PLIFA enriched diets.
ACKNO\IT-EDGEMENTS
The author wishes to tha¡k and acknowredge the commitment of her colleagues to
this study: Ms. Andrea Edel, Mr. Brad A¡der, Ms. Melanie Kopilas, and Ms. Anna \ffeber.
She extends special thanks m
Mr. Alejandro Austria, Dr. Elena Dibrov, the R.o. Burrell
Animal Handling Facilìry, the Dìvision of Neurovirolog, and Neurodegenerarive Diseases,
and the members
of the Division of Stroke
a¡rd vascular Disease at St. Boniface Research
cenre for their technical advice and suppon. Felicitations are extended to
trrlr. Tom
cook
and Mr. Bemard Abrenica for their expenise, humour and camaraderie trroughour
this
process. she would also like ro recognise the guidance and friendship of her supervisor Dr.
James Gilchrist and her commitree Members:
Ranjana
Bird
Dr. Grant pierce, Dr. Anton Lukas. and Dr.
DEDICATION
This manuscript is dedicated to my famiþ with love.
LIST OF FIGURES
1.
Synchronous Fluorimetry of SR Ca2* transporr
2.
The Effects of Alpha-Linolenic Acid on A. Ertraluminal Ca2* Transpon and B.
SERC.A ATP
3.
activþ
and ATP hydroþsis
Hydroþis by HSR Membranes
The Effects of Eicosapentaenoic Acid on A. Extraluminal Ca2* Trznsport and B.
SERC,A ATP Hydroþsis by HSR Membranes
4.
The Effects of Docosahexaenoic Acid on A. Extraluminal Ca'?* Transpon and B.
SERC,A ATP
5.
Hydroþis by HSR Membranes
The Effects of Linoleic Acid on A. Extraluminal Ca2* Transpon and B. SERCA
ATP Hydroþsis by HSR Membra¡res
6.
Ef[ect of Alpha-Linolenic Acid on SERCA1 ATP Hydroþsis
7.
Effect of Eicosapentaenoic Acid on SERC,AI ATP Hydroþis
8.
Effect of Docosahexaenoic Acid on SERCA1 ATP Hydroþis
9.
Effect of Linoleic Acid on SERCA1 ATP Hydroþsis
10. Effect of Alpha-Linolenic Acid
[fi-ryanodine Binding
11. Effect of Eicosapentaenoic Acid on []Fll-ryanodine Binding
12. Effect of Docosahexaenoic Acid
13. Effect of Linoleic Acid on
14.
on []Fll-ryanodine Binding
[fi-ryanodine Binding
Effecr of Docosahexaenoic Acid
on Car* PulseJoading in HSRMembranes
15. Effect of Ruthenium Red Ca'?* pulseJoading
16. SDS-PAGE of SR response to PUFAs
12. Mear Rabbìt lVeights of Feeding Groups at 8 lWeels
18. Toml Plasma Fatty Acid Concenûarions After 8 \Veeks of Feeding
19. Total Plasma cholesterol and rriglyceride concenrrarions after
g rù/eeks
Feeding
20. Comparison of CSR and HSR by SDS-PAGE Gel
21. Characterisation
of
Extraluminal Ca'?* Trznspon by Isolated CSR Vesicles
22. Yield of CSR from Feeding Groups
23. Cholesterol content of CSR isolated from rabbits fed a fla_x, cholesterol, or
cholesterol
/
flax supplemented diet from both non-ischaemic and ischaemic
reperfused heans
24. Extralurninal ca'?* uptake by cSR isolated from rabbits fed a flax, cholesterol, or
cholesterol
/
fla-x supplemented diet
from both non-ischaemic and ischaemic-
reperfused heans.
25. comparison of SERCA cataþic acriviry between cSR isolated from rabbits fed a
flax, cholesterol, or cholesterol
/
and ischaemic-reperfu sed heans.
flax supplemented diet from both non-ischaemic
LIST OF TABLES
1.
Fatry Acid Composition of Rabbit Diets
2.
Effects of Feeding on Plasma Fatty Acid profiles ar g \X/eeks
3.
Effect of Feeding on Brain Fatty Acid profile ar g \X/eeks
4.
Eff ect of Feeding on Kidney Fatry Acid profile at 8 lX/eeks
5.
Effect of Feeding on Liver Fatty Acid profile at 8
6.
Effects of Feeding on Fatty Acid profile of Non-ischaemic Fleans at g \ffeeks
7
EÍfects of Feeding on Fatty Acid profile of Ischaemic-Reperfused Hearts ar g rveeks
8-
Effecrs of Feeding on Fary Acid profiles of csR from Non-ischaemic Hearts
at
lùØeek
vll
Süeeks
g
LIST OF ABBREVIATIONS
CaÀ4 Calmodulin
CaMKII Calmodulin Kinase Tv¡o
CF
0.5 % Cholesterol / 1Oo/o Fla-x Dier
CPA
Cyclopiazonic acid
CSR
Cardiac Sarcoplasmic Reticulum
DFIPR Dihydroppidine Receptor,/ L-type Calcium Channel
ECC
Excitation Contracrion Coupling
FX
10
Flax Diet
HSR
Heary Sarcoplasmic Reticulum
i,/R
Ischaemia-Reperfusion
IP
Ischaemic-Preconditioning
FrR
Inositol Triphosphate Receptor
LCC
L-qpe Calcium Channel / Dihydroppidine Receptor
MgATP MagnesiumAdenosineTriphosphate
n-3 PLIFA Omega-3 Pollunsaturated Fatty Acid
o/o
NC)(
OL
PC
PE
PL
RG
RyR
Sodìum Calcium Exchanger
0.5% Cholesterol Dier
Phosphatidylcholine
Phosphatidylethanolamine
Phospholipid
Regular Rabbit Chow
Ryanodine Receptor Ca.lcium Release Channel
viii
SERCA
SL
SR
Tg
TM
VGSC
Xc
Sarco-endoplasmicreticulumCalciumATPase
Sarcolemma
Sarcoplasmic Reticulum
Thapsigargin
Transmembrare
Voltage-gated sodium cha¡mel
Xestospongin
I.
REVIE\øOF THE LITERATTIRE
THE ROLE OF THE SARCOPLASMIC RETICULUM IN STRIATED
M USCLE EXCITATIO N CONTRACTIO N COUPLI NG
-"Of the ions invoh¡ed in the intricate worhings of the heørt, cabium is
considered perbaps the most important It is crucial to"tbe øery proce ss' tbit ,iàøt",
tbe chambers of- the heart to contract and rerax, o prorótí called excìtationcontractio.n coupli_ng. It is important to understand in quantiøtiae detail exactþ hou
calcium is moaed around the oarious
es of tbe myocyte in order to'bring
about excitation-contraction coupllryt if.organe
tie qre to ,idrrstaid tire basic physiology ;f
heart function. Fwrtbermore, spàüal-microdomains tpithin the cell orr'iríporøit i,
localizing the molecular playeri that orchestrate cørdiac
fanction.,'
hnaUM.
Preliminary mrdies of muscle conrraction by Sidney Ringer
in
B IU
1gg3 revealed that
calcium s¡as required for contraction of an isolated frog hean. The significance
of
this
observation remained relativeþ obscure until Heilbrunn (t940) proposed an essential role
for calcium in conrracrion. He showed that calcium diffusing inro cut ends of muscle fibres
caused them
ro conrracr: Kamada et al (1943) rhen confirmed
same time Batley (t0+z) began
myosin
ATPase' By
to
deduce
these observations.
At
the
that calcium regulated contraction through the
1950 Sandow coined the term Exaøtior-c-ottranírrn ewpling the
hlpothesis that calcium released from terminal regions of muscle fibres activates the myosin
ATPase. Emesto carafoli provides an account of the history and imponance of calcium ìn
muscle contraction in a recent review [2].
The sarcoplasmic reticulum (SR) is a speci¡lizsd intracellula¡ calcium store that is
integral to the reguladon of muscle contracdon in eukaryotes. Analagous to the endoplasmic
reticulum,
it
fonns an intramembra¡reous structure that associates with both the plasma
membrane and myofilaments. The SR acts as both a source and sink of car* in the cell.
ca2"
is released and sequestered respectiveþ by
ryanodine recepror ca2*-release channels (RyRs)
and sarco,/endoplasmic reticulum ca2*-ATpases (SERCA) rocated in the SR membrane
bilayer. The movemenr of ca'?* is Ð,nchronised in the myoc¡e so that an electrical signai
coordinates its release into the cJtosol from the SR resulting
in
subsequent removal by the SR causing relaxation. The following
contraction and its
is an overviev¡ of
excitation-contraction coupling in both cardiac a¡rd skeleral muscle.
CARDIACMUSCLE
The purpose
trânsduce
of
excitation-conrracrion coupling (ECC)
in
cardiac muscle
an electrical or chemical signal into physical muscle tensìon.
is to
Electrical
coordination of myoct'tes pumps blood out of the heart in a sFrch¡onous ma¡ner. calciuminduced calcium release (GICR) is the mosr wideþ held postulate on hov¡ ECC occurs in the
hean. Publications by Endo, Ford and Podolsþ and Fabiato ard Fabiato in rhe earþ
gave insìght
1970s
into the mecha¡ism of CICR 13-51. CICR begins at the level of the myoq.te,s
transverse rubule
(ttubule). These invaginations of the sarcolemma (SL) propagate
an
action potendal that is usually initiated at the sinoatrial node. Activation of DHpRs by
depolarization of the SL initiates calcium current
(Q
that is functionaþ coupled to RyRs in
the SR. ,I." is often labelled "trigger calcium" because it potentiates its own release from the
SR by activating RyRs; hence the
increase ìn c¡toso.lic
Ca2n
[7].
calcium-induced calcium release. The result is a¡
from 8 x 10{ M to 1-3 x 10, M [6]. In fact
needed to increase q4osolic ca2*
during contraction
tem
from
1oo
5O-1OO ¡.rM Car* is
nM to 1 pM, since buffering occun in the cJtosol
Elevated cltosolic calcium then binds troponin
c
allowing the
contractile filaments to interact ard contract the cell.
Involvement of the SR in CICR begins with RyR activation in the j'nctional cleft.
RyRs are located in the terminal cisrernae of the SR and ertend into the diadic cleft between
the SR and t-tubules [6]. Electron-microscopy reveals that j'nctional complexes present in
the cleft are approximately
1OO-2OO
[6-8]. These complexes are
nm in diameter, 10-15 nm high and
composed
of up to
1OO
accessory proteins junctin, triadin, a¡rd calsequestrin
3OO-2OOO
nm apan
RyRs in associarion wirh the SR
[9]. As well, a number of regulato¡y
proteins can be associated with the RyRCa2* release
from the SR is by couplon acrivation. A couplon consisrs of
10-25
DHPRs and 100 RyRs [1]. Thus four to nine RyRs are coupled to a single DHpR depending
on the species [8]. The high ratio of RyRs to D]IPRs ensures sufficient ca2* release ar rhe
junction and ca'?* propagation in response to ca2* enrry via DHpRs
mechanisms of RyR activation by ca'?* include the
Na*/caa
[1]. other
porenria-l
exchanger (l.JOÇ and inositol
trþhosphate receptor (IPrR).
SR ca2* release is trìggered in
¡¿M ca'?.
tlie firsr
10 ms
of an action potential and peaks at 5-10
q¡ithin 2-5 ms of the acrion potenrial upstroke
[2, 1o]. The ca2* transienr or witch
is comprised of approximateþ
io ooo concened
from the SR [7, 11]. A spark is the
summatio
sparks, the fundamental release unit of ca2*
n oî
6-20 RyRs opening
[6, 1, 1.2-16] and
results in a large increase of q,tosolic ca':* in a concenrrated area [2]. They appear close to Z
lines v¡here diads or couplons are expected
[6]. In every junction
1 spark produces 2-3 x1o'le
mol Ca2*, which is equivalent to 12 OOO-18 OOO Car ions/ spark [Z].
CaZ* sparks can
several RyRs
function
as
local events, since a spark is the simultaneous opening of
from one DFIPR The theory of local control [17, 18] states
mediated by the intensig' of
I.^
SR caz* release is
1191. Spontaneous distinct local evenrs occur
onþ witfrin
a
given j,nction usualþ, except ìn ca2* overload where propagation of the spark results in
Cat*waves
[1].
Sparks have a low probabilty, less than
increase zubstantialþ during FCC
additional cha¡inel activation [20].
t7l.
1O'a
in the resting cardiomyoq.,te, but
Sparks also amplif' Ca,. signals and spread by
The process of GICR is terminated when calcium is re-sequestered reading to
relaxation. To maintain a homeostatic state in the cell total ca2* content musr remain
con$ant and be retumed
to its
respective stores. There are four known methods of
ertruding calcium from the sarcoplasm after contraction [z]. In rabbit and huma¡ the sarco,/endoplasmic retjculum ca'?*-ATPase (SERCA) accounrs for approximate
ly
7ao/o
of c**
efflrx while the SL sodium/caicium exchanger 0.JOq accounrs for 2g
"/o [1, /]. The plasma
membrane ca'?*-ATPase (PMC"{) and mitochondrial caz* uniporter each represenr abour
1"/o
of lotal
ca2* removed
activity in rar and mouse
[1]. In comparison
SERCA represenrs
l7l. The SR sequesters most of the ca2*
due
9oo/o
oî
c].
removal
to the large number of
SERCAs in its tubular region in all species [6].
SKELETALMUSCLE
skeletal muscle is similar to cardiac muscle in thar a process of ca2* release from
RyRs activates conrracdon of the myofilaments. However, a process terrned depolarization-
induced ca2* release (DIC$, rather than CICR, primanly regulates ECC in skeletal muscle.
DICR is initiated in skeletal myofibrils by a motor neuron-induced depolarisation of ttubules. DFIPRs act as voltage-sensors and'ndergo a physical change in response to the
action potential. This confornational change is mechanicalþ transferred to RyRs by an
unknown mechanism[6], so RyR activation is independent of 1." [21].
As compared ro cardiac muscle, skeletal RyRs differ in their arrangement. A triad
like the cardiac diad, consisting of the t-tubule and two j'rtaposed terminal cistemae is
formed in skeletal muscle. Every other RyR is linked ro a retrad of DHpRs allowing for
tighter regulation of RyR activation than in cardiac muscle [6, 22]. In skeletal muscle ECC is
coupled between RyRl and DFIPRI23I.
It
has been deduced thar loop
II-III of DHpR
acrivares
RyRl [2, 23]. RyR1, like RyR2, is also regulated by cat* and shows ca2*-dependent
actìvation in siw and in aino. snce all RyRls are not coupled to DHpRs, it is hlpothesised
that the function of CICR rn skeleral muscle is to amplify the DICR response
[23].
The mechanism by which ca2* release is activated also differs between cardiac and
skeletal muscle
in some respects. During exciration-conrraction coupling in cardiac muscle
the RyR2 is generally thought to interact with the L-t¡pe ca'?* channel to initiate
CICR In
contrast RyRl in skeleral venebrates interacts with a DHpR that acts as a voltage sensor
and
not a cat* conducting channel. RyRs exist
[21]' coupling of DFIPRs to RyRs
as
RyRl and RyR3 isoforms in skeletal muscle
is thought
to occur through RyRl whìre it is suggested
that RyR3 accounrs for the CICR component in skeletal muscle
[21].
Although GICR is one pathway that can induce ca2* release from the SR in skeletal
muscle,
a role
DICR is the dominant mechanism. \x/hile both GICR
a¡rd
DICR are thought to play
in both cardiac and skeleral muscle they are thought to contribure to the process of
muscle rwitch to differing extents [6].
SARCOPLASMIC RETICULUM CONSTITUENTS
The sarcoplasmic reticulum is organised into longitudinal a¡rd terminal
cisremae
regìons. Longitudinal sR is enriched in ca2*-ATpase pumps and encases myofilamenm.
Terminal cìstemae are the juncdonal regions of sR, so they contain the majority of car*
release
channels. Two families of ca2t release channels, RyRs and IprRs, are known ro
function in the SR as well as an ATP-dependent ca2* pump.
coordinate the effons
of
A
variety of proteins
these main affectors including calsequestrin, FKBp 12, and
phospholamban all to be discussed in this section.
CALSEQUESTRIN
calsequestrin
(csQ is a soluble low affinþ high
capacity ca'?* binding protein
found in the lumen of the SR in skeletal, cardiac and smooth muscres
[24].
cha¡acterised by David Maclennan as calsequestdn
as an acidic 44
in
1921,
it was described
Initially
kDa protein, the major ca'?* storage protein in the lumen of the SR
[24]. It comprises
SR protein and
it is a monomer in
carries a net negarive charge
aqueous solution
[24]. cse selectiveþ binds car.
.5,
KD: 4 x
of
and
[25]. Approximately 3z% of cse amino acid residues are
glutamate or asparrate [24]. These acidic amino acids bind 43 mol ca2*
7
zolo
/
mol protein at pH
cSQ undergoes conforrnational changes in response to ca2" binding
70'5 L241.
due to increased alpha-helical conrenr and decreased surface hydrophobicþ
[25].
In
the
absence of ca2*, electrosmtic repulsion berween carboxyl groups prevenrs its foldìng
[25].
CSQ hydrophobicalþ binds the intemal face of the SR junction
pa,
26,
271. CSe is
linked to RyR [28] through the SR proteins triadin, 95 kDa,lz9,3Ol and junctin, a 26 kDa
protein that shares some homolog' with triadin [31]. Triadin and junctin both have single
tra¡rsmembrane domains in the SR lipid bilayer that bind
cse to their basic
regions [2].
one physiological function of cSQ may be to interact with RyRs in the terminal cistemae
[26]. Structural
changes
in cSQ due to pH- and ca'?*-dependent conformarions rnay affecr
RyR P" [32-3a]' In addition it may complex with the SR protein riadin to produce RyR
effects [9].
Anorher mejor
fi.rncr
ion of CSQ is ro m¿intain Ca2' homeosrasis
buffer up to 20 mM cat* in the sR t241.
cse
reduces free luminar
[2]. CSe ma¡
ca,* concenrarions ro
prevent reverse-mode sERCA activþ and formation of ca'?* precþtates
[2].
compared to
other binding proteins cSQ is larger, contains more binding sites, has a larger dissociation
constant a¡d is bound ro rhe membrane instead of {ree 1241. A_ll of these properdes may
contribute to its unique function.
INOSITOL TRIPHOSPI{ATE RECEPTOR
There are two families
of
calcium release channels that can be found
sarcoplasmic reticulum, IPrR and RyR [2,
muscle whìle IPrR are found
23]. RyRs are found predominantþ in
in mosr orher
as
the
skeletal
tissues including smoorh muscle cells, plasma
membrane, nucleus, secretory vesicles, and golgi apparatus
present in striated muscle
in
[2].
However, IprR are also
well [35].
Some similarities are shared between the IPrR and the RyR, the largest ion-cha¡nels
known [35]. Borh channels are retrârners composed of large subunits of 290 kDa and 550
kDa for IP,R and RyR respectiveþ [2, 35]. They exhibit homologz near the carboxyterminus that forms the transmembrane channel region of each receptor [2]. These channels
carry large conductances of up to
1OO
pS in compadson
to
10 pS
in
a
DFIpR [2]. The iprR
cha¡rnel exists in at least 3 isoforrns. IP,R's sr.'rnmetrical central pore shares homologr with
the RyR 17, 23, 36,321.
ln
contrast, RyRs are activated by voltage mediated changes,
calciurr¡ and nicotinamide adenine dinucleotide [38, 39] while IP,Rs are primarþ activated
by the second messenger inositol 1,4,5 triphosphate, IPr. Cah tra¡lsients arising from RyRs
are termed sparks whereas IPrRs produce puffs, large increases
concentrated area[2,
4Q.
â.n
q,,tosolic Ca.*
in
a
Recent cryo-electron microscopic images have shown that lprRs
and RyRs are stmcturaþ different despite their molecular similarity
described as
in
t+1]. The IprR has been
uneven dumbbell reaching iZO,Ä, and spanning 1OO,Â. a¡rd
150,[ lateralþ
at
either end [41]. "The distinguishing frrnctional attributes of each RyR or Ip,R channels likeþ
underlie the spatiotemporal complexiry of intracellular Ca':* signaling in cells"[23].
A number of modulators of IP,R lirnction have been identified
is formed in addition to
diarylgþerol
as a by-product
[2].
The ligand IP,
of phosphotidyl inositol bisphosphate
hydrolysis by phospholipase C in the inner leaf of the plasma membrane. Phospholipase C
is
activated
by a number of primary
vasopression, thrombin,
messengers including acetylcholine, adrenaline,
AIP, platelet-derived gronth factor and endothelial gronth factor
acting through G-proteins and tyrosine kinases. Other modulators of the cha¡rnel include
both luminal and qnosolìc calcium, calmodulin, ATP, protein kinase A, cGMP-dependent
protein kinase, Ca2*/CaM-dependent protein kinase (CaMKII), prorein kinase C, ty,rosine
kinases,
NADH, calcineurin, FKBP12, heparin, and xestospongin
[2]. It
has been
hypothesìsed that IPrRs are biphasicaþ regulated by Ca2**ensitive intrinsic activatory and
inhibitory sites, which indicates that IPrRs are responsive to changes in q.tosolic
Cí-
142).
Similar mechanisms of modulation are proposed for the RyR below.
RYANODINE RECEPTOR
Structure and Function
The RyR functions as a Ca2*-release channel during ECC in both skeletal and cardiac
SR. It also has a role to pÌay in non-muscle cells. First cited by Fleischer
et. at
n
1985,
ryanodine's effects on Ca2* accumulation in crude preparârions of both longitudinal SR a¡rd
terninal cistemae were examined [a3]. Since those preliminary studies [44-48] detailed
understanding
of RyR
structure/function has been hampered (or hindered)
by
its
intracellular location and size. This has made it difficult ro crysrallize for x-ray analysis and
study in rwn. Nevenheless, this protein has attracted an enoÍnous interest and much is now
known from controlled studies zz siu
nd
in aiho.
A
relatively brief overview of RyR
isoforms, morphologr and ârangement, a¡ld conducta¡ce propenies is presented here.
RyRs exists in at least 3 isoforms in mammals. These three genes are located on separate
chromosomes and can differ by splice variation as well [49-521.
muscle
In mamma]ia¡
striated
RyRl is predominantþ found in skeletal muscle and brain, RyR2 in cardiac muscle
and brain, and RyR3 is at relativeþ low levels in brain, diaphragm and jurkat T-þnnphoc¡tes
[2, 23]. RyR2 and RyRj respectiveþ share 66Vo and
The RyR cha¡nel is a 2.3 lrIDa holoreceptor
tetrarners, so rhe size
7Ao/o
p5l.
identity with RyRl [50, 53].
RyRs are q..mmetricaly shaped as
of each monomer is about 550 kDa [35]. The qtoplæmic domain
approximateþ 29 nm x 29 nm
x
12 nm while the transmembrane
approximateþ z nm into the SR lipid bilayer [2,
35].
(rM) domain
Approximateþ 4 /
q.'toplasmic with the N-terminus oriented in thar direction
5'h
is
extends
of the chan¡el
is
[35]. There are ar least ten
distinct domains within the looseþ packed qtoplæmic portion of the channel that contains
2-4 ca2* binding sites 12,7,35,
541.
The remaining 1/5'h of the cha¡rnel creates the TM
domain with its c-terrninus [35]. The number of
disputed. A¡fvhere from 4
to
12
rM
spanning segmenrs in RyRs is activeþ
TM domains have been proposed t5O, 521. The TM
domain forrns a 2 nm central hole between rhe monomers thereby creating the ca2*sensitive
gate
[55]. The width of the RyR2 channel pore is approximately
3l
v¡hile the length of the
voltage drop is 10.4 ? , a relativeþ shon permeation pathway consisrent with its rapid ionic
conduction [23, 56].
The arrangement of RyBs differs according to isoform a¡rd tissue. In skeretal systems
the gtosolic domains of RyRls closeþ align themselves with DFIpRs filling the junctional
cleft. Fast-twitch
skeletal muscle is configured so that every other RyR corresponds
DHPR, for every 2 DFIPR there is
1 RyR
to
a
[52]. The direct interaction berween the DFIpR
loop and RyRl in skeleral muscle allows for rapid signal transduction, since the action
potential is onþ 2 ms [23]. In conrrasr, the order of arrangement in cardiac muscle is 5-10
RyRs for every DFIPR
(-
[52]. This lax organization is functional
because the acrion porential
100 ms in rat) is signifìcantly slower than skeletal muscle [23].
All RyR isoforms exhibit high ,nitary conductance of up to 5oo ps in
monova.renr
cation solutions and lo0 pS in diva.lent solutions t58, 591, ìn comparìson to DFIpRs (20 ps)
or ma-xi-Ç" (250 pS) channels [60, 61]. The ion selectivity of the RyR is relativeþ poor. It is
predominantly a calcium chamel but it also conducts K., Mgr. and Nar. [23]. RyRs select
for divalent over monovalenr cations, but do not distinguish beween similarly
[23].
Perhaps this is because its high conductance does nor allow
charged ions
it time to "discriminate"
between ions [23].
The RyR is named afte¡ the alkaloid ryanodine thar binds to it from the prant
þrmia
spcinsa F.yanod:ne binds near the c-terminus, which forms the central pore, consequentþ
interfering witlr RyR permeation characteristics by altering rhe diameter
of the pore[62-64]-
Generall¡ ryanodine maintains RyRs in a slow-gating subconductance state where rhe
current becomes 1/3 to % control [64]. The concentration-dependent response of RyRs to
ryanodine
is similar
across
all three isoforms [23]. Relativeþ lo¡v concentrations of
ryanodine, 10 nM, increase RyR open probability @) while 1 ¡iM ryanodine locks the
channel open and decreases its conductance
100 ¡¿M, lock the channel
[65]. Even higher concentrations of ryanodine,
in a closed state [66]. The varied response of RyR to the alkaloid
to cooperativìty at high (Ç-50 nL{) and low
(Ç-1
pÀd) affiniry binding sites
[6/-69]. Funhermore the binding of ryanodine to the high affinity
site may depend upon the
may be due
conformation of the channel [62].
Mechanisms of Activation and Inactivation
10
Aside from pharmacological agents like ryanodine, there are
a
number of
physiological affectors that regulate rhe ryanodine recepror. These include, bur are not
limited to molecular ligands such as Ca,., ATP, Mgr., and reactive oxygen species (ROS),
as
well as protein ligands like DFIPR, NC)q PKA and CaÀ4KII, Catr4, a¡rd FIGp. As
highlìghted earlier, RyRs are physiologicalþ acrivated during ECC. The process by which
RyRs are inactivated
will
be discussed.
Calciwm
Qnosolic Ca2* is known to regulate RyR P.. \(tithin 1 ms of DHpR activation Ca2*
concenrrarions can reach 10-15
¡iM in the junctional cleh
ca2* that enrers the cell is proponional to the amount of
l7a,7l.
ca'*
The amount of trigger
released from the
sR. vaСmg
1.. shows this dependence and indicates that a larger 1." may increase ca2*
release by
recruiting more RyRs 17,72,731. It has also been suggested that 1." may affect the amplirude
of Ca2* released [7,74]. Generalty, c¡tosolic
Ca2* activates RyRs between 1-10 AÀ4 Car* and
inhibirs RyRs by 1-10 nù4Ca'?.[23].
Lumenal Ca2* simultaneousþ modulates the RyR with q,tosolic Ca2*. Under
physiological conditions total ca2* within the sR is approximateþ
130
þM
ct.
is free [7 5-771. Above 40 ¡M
t mu
[23], of which 60-
fca'?*]ro o." there is an exponenriar rerationship
berween 1.. and the amount of Ca2n released from the SR [23, Z8-gO]. RyR
p.
and the
frequenry and amplitude of Ca'?* sparks vary directþ with SR Cah [11, g1-8g]. One
postulated mecha¡lism of how lumenal ca'* regulates these effects is that it may activate the
q.'tosolic side of the RyR [86, 87]. Another group of evidence suggests that SR ca2* acts on
the lumenal side of the RyR t88]. Activating and inactivating iumenal sires may exist on rhe
RyR that alter the sensitivþ of RyR to agonisrs [89]. Lumenal Ca2* also binds lumenal
proteins like calsequestrin and junoin [90, 91].
ATP and Mgz'
ATP and Mg'z. have opposing acrions on RyRs. Under physiological conditions free
qtosolic ATP (300 FM AlfP)
sensirises
the RyR to Ca2* 12, 23, 9Zl. rJnáer equivalent
conditions 0.5-1mM free Mg'z. desensitises Ryfu possibþ by competing with Car* at RyR
activation and inactivation sires 17,23,93]. The significance of the equilibrium thar musr
exist between these two affectors is seen during ischaemia when increased qnosolìc Mg.*,
due to decreased buffering by ATP, may delay the onser Ca2*-overload by decreasing RyR
Ca2*sensitivþ [94].
Reactiz; e Oxy gen Sp e ci e s
The oxidation state of the RyR is also thought to alter its activþ [95]. Each RyR
monomer has 80-100 rysteine residues some of which can potentialþ be oxidized [50, 53].
Oxidation may inhibit Mg'z* and cal¡nodulin effects [96, 9Z]. Nitric oxide (l'JO) likeþ alters
the redox status of RyRs
as
well [98].
DHPRs
DHPRs are located at the junctional clefts of myoclres and interact with RyRs in the
terminal cistemae [2, 99]. DFIPRs rra¡smits the acdon potentiaì signal to the RyRs through
an unknown direct or protein interaction
potentiâI ca¡rdidate
[2].
[2].
Triadin, a junctional SR protein may be a
Cardiac muscle is generalþ regulated by a Ca'?*-mediated DFIpR-
RyR signal while skeletal muscle has a more direct linkage through regions
II-III in the
DHPR alpha c¡oplasmic \oop [7,7,23]. This is puzzling since there is relative homolory
12
berween DIIPRs
in
skeletal and cardiac muscle. The physical disribution
of the two
channel rypes and their relative distribution may account for tåese different mechanisms of
activation [99, 100].
NCX
The NCX can potentially activate the RyR by two means [Z].
A
sharp rise
i¡ Na*
after
the action potentiâl may activate the NCX in the ourward diection and create a source of
trigger Ca2*
to activate SR Cah
release
[101-103]. Howeveq many argue that such
mechanism is not feasible [104-106] because
NC(s
a
are nor presenr in junctional clefts [107].
The other mecha¡rism of activating the RyR via the NCX may be by direct acrivarion of rhe
exchanger upon depolarisation
[i08, 109]. Still, the proximigz of NCXs to RyRs and
the
significantþ larger DFIPR 1." oppose the idea that the NCX is a physiological activaror of the
RyR The role of the NCX may
be ro porentiare SR Ca2* release or rrigger its release in the
absence of DFIPR activation [7].
PKA and CaMKII
DifferentiaÌ results have been obtained in response to kinases PI(A a¡rd CaMKII
acting on RyRs. PI(A has been shown to both activare and inactivate RyRl and RyR2 [7,
231. Phosphorylation
of RyR by PKA decreased the P. ar IOOnM Cah, but P. was increased
v'ith the rapid application of trigger Ca2* [1iO]. Studies by Marx et al prowde evidence that
P. RyR is increased due to FKBP 12.6 dissociation by phosphorylation at Ser28O9 I1111.
Il
comparison to the above lipid bilayer studies, inracr cells show PK-{ has no effect on resdng
Car* sparks [83]. Stili, PKA may mediate the RyR DTIPR interaction through sorcin, a 22
kDa SR accessory protein [112, 113]. Sorcin decreases Po and Ry binding which is reversed
by PKA phosphorylation [11a].
Experiments conducted on RyRs response ro CaMKII yielded similar responses as to
PKA. According to Marx, CaMKII phosphorylates rhe
same serine residue as
Likewise to PKA, CaMzuI has variable effects on RyRP.1115-1171.
PKA [115].
A possible explanation
may be that CaMKII potentiares Ca2* release from RyRs after their acrivation [7], since
phosphatases depress ECC gain and inhibition of CaMKII prevenrs Ca'?*-dependent SR Ca2*
release in a concentrâtion-dependent manner [118, 119].
Calmodwlin
Calmodulin (CaÀ4) binds Ryfu 10 nm from rhe entrance ro ihe TM pore [1201. Ir
activates RyRs at concentrations less that 2 pM, and
i¡hibits them in greater amounts. RyRl
binds 4-16 CaM inversely ro lca'z.] [121]. Contrary to RyRl, RyR2 binds 4 CaM at high
Cf
,
2Oo
pM,
*d
binds onþ 1 CaM at low Ca2*,
how marry CaÀ4 bind to
1OO
a tetrarner there is consensus
nM [Z]. -X/hile there is debate over
that Ca2* increases the
affinþ of CaM
for RyR2 [Z]. Effects of CaM on Ca2*-dependent activation of RyR are not v¡ell defìned
RyRl and RyR3 may be activated by CaM at low
whereas RyR2 is only
Ca2* and inhibited by
it
at high
inlibited by Cali{ [122, 123]. Alteratìon of RyR oxidation
CaM arrd interactions with DFIPRs could play a role
n
Ca'?.
stares by
its acrionlgT, 1241.
FK506 Binding Proteins
Immunophilin FK506 binding proteins (FKBPs) are 72 kDa proteins that associate
with RyRs in hean and skeletal muscle. FKBP12 and FKBP12.6 respecrively bind RyRl and
RyR2 preferentially due to their relative cJ'tosolic abturdanry. Each FKBP stoichiometrically
binds a RyR subunit, so four FKBPs bind per RyR ter ramer 11,25-727f. The binding site for
FKBP is about 10 nm from the cha¡nel opening [120]. Genera.l obserwations indicate that
FKBP acts to stabilize RyRs in an open or closed state 12, 125f. FKBP removal from RyRl
results in acdvârion of rhe channel
studies where FKBP
is
[tzs]. Its role in RyR2 function isn't as clear. In
cha¡rnel
removed, subconductance states are observed and normal
conductance is restored with its reintroducrìon [125,128-1,30]. FKBP gene knockout studies
rezult in embryonic lethality due to impaired hean, brain but not skeletal muscle fu¡ction
[131].
The function of FKBPs may be to coordinate the RyR rerrârner and neighbouring
channels 17, r32), or mediare DFIPR RyR coupling
[133]. FI(BPs could potentialþ inhibit
channel adaptation by increasing RyR Ca'?. sensitivity
suggested in hean failure.
[7]. A
role for FKBp has been
PI(Â phosphorylation of FKBP12.6 leads to its dissociation from
RyR which could result in defective RyR regulation
[i 11]. In support of this hlpothesis, the
FKBP antagonisr FK506 increases Caz* spark frequenry and decreases SR Car* 1134,
þR
1,351.
Inøctiøation
Three likeþ mecha¡isms have been proposed for terminating Ca2* release from
RyRs: RyR inactivation and adaptation, SR ca':* depletion, a¡rd stochastic attrition [1].
Fabiato proposed that a process of negative-feed back musr exisr for RyRs,
if nor CICR
would be a continuousþ amplìf4nC process [136]. Gating in RyR has been cornpared to
vGSCs where there are low affinþ acrivarion sites and high affiniry inactivation sites [21].
Inacdvation of a channel implies refractodness, there is a period in which the channel cannor
be reacdvated
11'37
-1'4a1. Reacdvation
of the
channel would require the removal
of
ca2*
from inactivation sires. A number of studies have produced evidence of refractorìness [g1,
L36, 137, 1411 while others have shoq'n that neighbouring sites can be activated [82].
Similarþ, the mechanism of adapration implìes that after a peak [Car.], is reached, an even
greater stimulus is needed to reâoivâre the channel [110,
1is].
These mechanisms could be
physiological significant
if they contribute to
decreased force-frequenry relationship
less spontaneous events and accôunr
that is obsewed with
for the
activation-dependent
inactivation [7].
Depletion of SR Ca'?* has been suggesred as a mecha¡ism for RyR closure simpþ
because there would be no more ca2*
the P"
Ry\
to release. Also, recall that lumenal sR ca2* regulates
so P. RyR may be effectiveþ decreased by Ca'?* depletion. Critics of rhis
hlpothesis point out that it doesnt account for repetitive ca'?* sparks that occur in the same
area
B3) or Ca2* sparks that last up to
2OO
ms [11], situations where Ca2* srores are nor
exhausted.
Stochastic atrririon, the "inherent random closing
perhaps the least probable mechanism
simultaneous closing
of
RyR closure
of an individual cha¡rnel,, is
[18]. It would requie
of RyRs and DFIPRs in the junaional cleft
the
thereby preventing
amplìfication of rhe Ca':* signal, the positive feedback mechanism of CICR
IIg, 137,1421.
This hlpothesis is not likeþ because a sufficient number of cha¡rels in a junction would
have to close at once. Ilnless the cha¡rnels are gated together this may not be a favourable
theory [143].
SARCOPIASMIC / ENDOPIASMIC RETICULUM CALCIUM
ADENOSINE TRIPHOSPATASE
Structure and Function
The characteristics of SERCA have been ertensiveþ studied over the past 50 years
(for review
see
ca¡afoli
l2l). in
1952 Marsh obsewed thar fractions
of
skeleral muscle
induced relaxation. Later in the 1960's Ebashi would show that relaxation was ATp-driven.
Hasselbach and Makinose
then deduced that ca2* was sequesrered into the SR during
relaxation (1961). By 1970 Maclennan was the firsr ro identif' SERC,A as a
in skeletal muscle that comprised Írom
SERCAs belong
7Oo/o
1OO
kDa protein
to 90% of SR membrane protein [144].
to the P-qpe ATPase famil¡ which are characterised
by
phosphorylation of an aspanate residue. Two subfamilies of Ca2*-AT?ases exist: plasma
membrane Ca2*-ATPases (PMC-{s) and SERCAs. These pumps couple Ca2* rransporr ro
ATP hydrolysis. The SERCA pump is constitutiveþ active in SR membranes [35]. It creares
a thousand-fold Ca2* concentrarion gradient across the SR membrane that is
non-
elecrogenic due to 2 H* release and anion exchangers [35].
At
least 3 SERCA genes have been identified that share some
homologr. SERCA1
is mostþ found in fast twitch muscle [145]. Its splice varia¡ts SERCAIa and SERCAIb
differ slightly at the C-terminus [146] and are found respecdvely in adult and neonaral
muscle. SERCA2 is also alternativeþ spliced into variants that differ at the carboxy terminus
11,47
-1491.
A
restricted pattern of SERCA2a expression occurs in the hean while SERCA2b
is elsewhere ubiquitousþ expressed
in smoorh and non-muscle tissue [147,1,50,151]. Less
is
known of SERCA 3 which is expressed in kidney [152], platelers [153], and endothelial cells
us4l.
The crystal strucrures of SERCA in its Cat* bound and free forms have recenrly been
derennined [155, 156]. The pump consists of 10 TM a-helices and three large q.toplasmic
domains[152]. The 3 cltoplasmic domains are referred to as the A-, P-, and N- domains.
The activation-domain or A-domain is a 125-residue loop connected to TM segments lvl2
and M3 [155]. The P-domain or phosphorylation domain is 410 residues, interacts
witl
the
M4 and M5 TM segments and contains Asp351 that is phosphorylated by ATP [158]. The
N-domain or nucleodde binding domain is encased by the P-domain
5.7
Ìr
apan are 2
Ci*
11581.
Approximateþ.
binding sites located between M4, M5, M6 and M8 [158]. Site
1
consisrs of oxygen ìigands from
TM
segmenrs M5,
M6 arìd M8 while site 2 is mostþ found
on M4 [158].
The ¡ate of Ca2* translocation by SERCA is much slower than an ion channel
because confornational changes
mu$ occur for cat* to reach high affimty binding
sites
from qtosol to TM, and then from the TM to the lumen t1581. A number of models exist
that try to predict the conformational changes that SERCA undergoes during ca2* transporr.
The E1-E2 model is adapted from the Post-Elbers model for (1.{a.-K.)-ATPase (described
in [158]). It predicts thar SERCA oscillares berween 2 conformarions, E1 and E2. During
EI 2 Ca'?t molecules f¡om the cttosol
can bind SERC"A,. Phosphorylation of Car*-bound
SERCA by ATP creates an inrermediate, E2PCa, that aligns the low affinity
C_ar.
binding site
with the lumen. Bourd Ca2* is then deposited into the SR during E2 and SERC,A
is
dephosphorylated. The E1 conformation is then rerycled.
The four-site model builds on rhe E1-E2 model by inco¡porating the low affinþ
Ca'. binding sìtes found on SERC,A's lumenal side [159]. It predias that Ca2* molecules are
transferred from the high affinity sites to the loq¡ affinity sites before release. Alternativeþ
the modified altemating four-site model agrees that ca2* is released from qtoplasmic sites,
but Ca2* can onþ bind to lumenal side in EI or E2 and not the ìntermediary srate [160].
Lipid Interactions
The lipid bilayer immediateþ surrounding SERCAs is called the annulus (for Review
see [158])
.
Amular lipid is immobile due rc its inreracrion wirh the SERCA protein aad
altered lipid packing in that region [161].
in the annulus [158]. Most SERCA
charged
It
has been predicted that 30 Iìpid molecules reside
residues are hydrophobic and reside
in the similarþ
lipid bilayer [158]. The few charged residues on sERCA maintain neutrality with its
surrounding either by forrning ionic bonds or presenring themselves at the lumenal or
ct'tosolìc faces of the SR membrane [158]. Relative tilting of a-helices during pump
rycles is
influenced by TM domain thickness, lipid chain length and composition of annular regions
L158, 1621. As constituents of the SR lipid bilayer, phospholipids, fatty acids, and cholesterol
could all be potential modulâtors of SERCA funcrion.
Pbospholipids
Phopholipids (PL$ are rapidly exchanged from annular to bulk lipid as ¡he Ca2*
pump changes conformarion [161].
Both
phosphatidylethanolamine (PE) impact SERCA
phosphatidylcholine
in
fluidig' due to
[163].
fast-rwitch versus slow-twitch muscle resultìng
decreased sphingomyelin conrenr
increased pump kinetics
and
function. Among PLs, PC bìnds
preferentially to SERCA over PE regardless of aryl chain lengú
times higher
(PC)
in
[164]. This may
in fast-twitch muscles. At the
PC content is 1.2
increased membra¡e
accounr
in paÍ for
same time that PC is necessary
for
SERCA activity, PE increases SR coupling [165]. Unsaturated PE is closely associated with
SERCA. The purpose of it may be ro maintain membrane fluidity in the an¡ulus and allow
for
to
easy transitions between
pump conformations 1166, 1671. r*4rile anionic PLs are rhoughr
increase the coupling ratio
of SERCA [168], methylation and gþosylation of PLs
have
been shown to decrease its activity [165].
Fatty Acids
Charged headgroups of fany acids also bind SERCA in the lipid annulus [169,
1ZO].
Binding in the annular layer is enhanced when SR lipid is in the liquid crysralline phase
opposed
to
gel phase lipid
[163]. The fatty acid profile of
as
SR membranes differs berween
longitudinal and terninal SR. The majority of unsanrated fatty acids are found in the
terminal cistemae [171]. Unsaturated famy acids are necessary for SERCA
^ctìvìry
17721.
Membrane thickness and fluidiry as determined by FA acyl chain length vary directþ with
SERCA activity U73, 1741. SR FAs may correlare ro muscle function, since fasr-rwitch
muscle contains much higher amounts
of
docosahexaenoic acid than slow-twitch muscle
L17s).
Cholesterol
Studies of cholesterol effects upon SERCA function have been controversial. Much
of the
debate has centred arou¡d wherher or
exchanging annular
lipid regon
117
not cholesterol is excluded from the slowþ
6-1791. Under norrnalþ simulated Ca2* transpon
conditions, cholesterol has no effecr on SERCA activtty [176, 177f. However, when SR
membranes are made permeable, an inhibitory effecr of cholesterol on SERCA activþ is
revealed [178, 179). Another poinr
ro consider is that cholesrerol could act ar orher non-
annular SERCA sites, as exemplified by the binding of thapsigargin [15S, 180]. Aìso, the
cholesterol content
of SR lipids
also varies between fasr- and slow-twitch muscle types,
which suggests that cholesterol does influence SERCA activiry [16a]. This data indicates
that cholesterol may interacr with the protein or affect its surrounding fluidity [1/8].
Phospholamban
First identified by Tada and colleagues
n
1975, phospholamban (PLB), an
endogenous inhibitor of SERCA function, is expressed in smooth muscle cells, slow-twitch
and cardiac muscle [2,
181]. It
is a 22-27 kDa hydrophobic prorein [44, 181]. PLB spans
the SR membrane with rwo alpha helical regions extended into the transmembrane region
and cltosolic space on either side of a ßtum (Lambenh and Carafoli 2OOO). PLB is
regulated by PI(A
md CaI4K phosphorylation at serine 16 and threonin e 17 12,
441.
Dephosphorylated PLB binds near the P-site on SERC"\
[iS2]. It
suppresses SERCA
activþ by interaction of a 30 residue q.toplasmic region with the pump's M6
transmembrane region
þg
immediateþ beneath its qnoplasmic N-domain L44, 183, 1g41.
This then inhibits SERCA by decreasing its Caz* atíinìtyl2l.
IM.OLVEM ENT OF TH E SARCOPLASMIC RETICULUM IN
ARRTIYTHMOGËNES1S
"When SR Cî. load ß ele,uated, it may be tbe rise in [Cd*]ro rohich triggers
tbe,so'called spontaneors sR c¿- release during cê* oaerload thal-can be dirictþ
arrlrythmogenic. " [7J
Inappropriate calcium mobilization is the cause of much ceÌlular pathologz including
necrosis, apoptosis, and cardiac ar:rhlthmias. Normalþ, an influ-r
of Ca:* from
the
interstitial fluid during cardiac excitation triggers the release of ca2* from the sarcoplasmic
reticulum. This free q,tosolic Ca2* activates the contraction of the myofilaments. One qrpe
of
cardiac arrhlthmia, termed delayed after depolarisations (DADs), srems
ca'?*
from improper
handling. This addirional excitation may âppear near the very end of repolarisation or
just after repolarìsation in the excitatory porential of the myocardium.
afterdepolarisations may themselves trigger propagated impulses.
Such
DADs are likely ro occur
when the basic rycle length of the initiating beats is shon and when the cardiac cells are
overloaded with Ca2*.
conditions that foster c,a2* overload present themselves during hean failure. Free
q,tosolic ca2o has been shown ro increase over several minutes during ischemia [1g5-1gz].
Ca'?.
is initialþ increased by release of Ca2* from the SR during ischaemia then the
extracellular space during reperfusion t1881. A number of factors contribute to this rise in
Ca2* including reduced for-ward acdviry
increased SR
car. leak.
Decreased
of
Ca2*-ATPases, ourward
NCX activiry,
and
ATP production during ischaemia prevents both
SERC,As and PMCAs
increases
from effectiveþ removing
in the outward direction due ro
a rise
Ca'z*
in
from the q.tosol [189]. NCX activity
p.Jal, thereby increasìng Car* influ-x
[190,
1911. C¡osolic Ca2* conrenr may also be increased by PKA-induced phosphorylation of
Ry\
which wou.ld dissociate FKBP and promote RyR-mediated Ca'?. leaks
[111].
Elevated
q,tosolic Ca'* will also be perpetuated by increasing RyR P, [192-195]. The net effect is a
depletion of SR Cat* store and a rise inrracellular Ca2*.
There
is porenrial for
overload by affectors of the
regulation
SR.
of
ischaemia-induced arrhlthmias
from
Ca2*
Cyclopiazonic acid and thapsigârgin, SERCA antagonists,
decrease the incidence of ventricular fibrillation arrhlthmias in rat heans after ischaemia and
reper{usion [196]. Ryanodine prevenrs ouabain-induced arrh]thmias
in rabbit and dog
heansf797, 1981 although it had no affect on ischaemia and electically ìnduced arrhthl'rnias in
dog hearts [198]. The RyR antagonisr da¡rtrolene may delay Ca2* overload development
during myocardial ischaemia [188]. Both SERCA and RyR are porenrial targets for antiarrhythmic agents.
EVID E NC E FOR CARDIOPROTECTION BY OM EGA-3
P O LYU N SATURAT E D FATW AC I D S
The omega-3 poþunsaturated fany acids (n-3 PUFA$ have received a grear deal of
attention due to their many porenrial health benefits. Among these benefits are improved
visual a¡d neural development, immune response, anti-inflammatory and anticancer actions,
protection against renal failure and improved lipoprotein levels.
are the cardiovascular effects
studies have lent
of
imponance ro rhis srudy
of the n-3 PUFAs. Epidemiological, dietary, and
cellular
to our understanding of how rhese lipids may function. To begin we will
examine their structure, metabolism, and potential dierary sources.
Essendal
humari
bofi
fary acids (EFA)
are termed so because they caraor be spthesised by the
and therefore mu$ be acqui¡ed in the diet. The two precursors ro rhe EFA
familes are linoleic acid (C18: 2 n-6) and linolenic acid (C18: 3 n-3). Both the n-j and n-6
fatq¡ acids belong to the family of pol¡,rnsaturated fary acids. PllFAs are charaoerìsed by
more than one double bond in the carbon chain that constitutes a fatty acid. Linolenic acid
(ArA)
is metabolised by a process of elongation and desaruration into the predominant n-3
PlJFAs, eicosapentaenoic acid (EPA, C20: 5) and docosahexaenoic acid (DFTA,, C22: 6).
n-6 PUFAs are distinguished from n-3 PUFAs by the location of their double bond
from the methyl
rerminus
.
n-6 fatty acids have a double bond six carbons in from the
methyl-terminus, while n-3 PUFAs have a double bond at the third carbon from the methyl-
terminus. A major n-6 fatty acid found in the body after conversion of linoleic acid is
arachidonic acid (AA, C2O: 4, n-6).
Metabolìsm is a collective term for the Ð.rrthesìs and degradation of a substance.
PIIFA metabolism is segregated into different locations of the cell. PI,FA sgrthesis occurs
in the c¡toplasm, while PUFÄ degradation occurs in the mitochondria and peroxisomes.
PIJFA s¡mthesis can arise from a varieg' of pathways in which aceryl CoA plays a central
role. The catabolism of fatry acids
is usually through the pathway
of ßoxidation.
The process by which PUFAs are metabolised is through a sequence of elongation
and desaruration. Both n-3 and n-6 PUFA metabolism is catalysed by the
desatu¡ases and elongase. Since
enzr.'rnes in
?
4-, ? 5-,
n-3 and n-6 poþunsaturated fary acids utilise the
? 6-
same
their metabolism, they compete with each other for these en4¡rnes. To maintain
homeostasis, an opdmal balance must be achieved berween the relative amounrs
of
these
two PIIFA groups in rhe diet. This equilibrium is often described by a ratio of n-6 to n-3
PLIFAs. The recommended rado is 4:1 U991.
23
A
general consensus
in regards to fat intake and improving one's health has been to
reduce total fat, saturated fat, and cholesterol intale [2OO]. Health and rX/elfare Ca¡ada
recommends that of total caloric intake, iO Yo consist of dietary
fat. Of that
30
o/o,
there
should be an equal distribution of saturated-, monounsaturated-, and poþtnsarurated- fatry
acids. Potential sources of n-3 PUFA intake include fany fìsh and flaxseed. The Adequate
Intake (AI) values for n-3 are L.6
g/d
and 1..7 g/d for men and women respectively [201].
On the upper end, the Nutrition Comminee of the Amerìcan Hean Association deems an n-
3 fatty
acid intake
acceptable
of 15 o/o of total
calorìes
ro be
excessive
while 10 % of calories to be
[200]. Epidemiological and controlled randomised trials indicate that
0.5-1.8 g/ d EPA and DFIA or 1.5
-
3 g/
d ALA
are preventive against
doses
of
CV monality [202].
EPIDEMIOLOGY AND CONTROLLED TRIALS
Several studies have shown that the consumption
of n-3 PUFAs in foods Iike fish
and flaxseed decrease CV monaliry. The premier studies in thh fìeld by Bang et al. [2A31
observed decreased cardiovascular monality in Greenland's Inuit which they attributed to
their n-3 PUFA enriched diet. This work set the stage for funher analyses to estabfish the
inverse relationshìp between populations consuming relativeþ high n-3 pl,IFA diets and
their monality and propensity for
cV
events l2o4-2a61. Proposed mechanisms for this
beneficial action initialþ focused on the prevention
of
coronary atherosclerosis [2oz];
however, direct actions on the myocardium are now proposed.
Some of the frequently cited studies on n-3 PllFAs and CV disease include the
Chicago rü/estem Electric Stud¡ which examined nearþ 2
years. Over a 3A year follow-up fish consumption
correlated q.ith death from CVD, especialþ from
OOO
men from the ages of 40-55
of more thart 35 g/d was inverseþ
MI t2OSl. A
srudy on women in the
Nurses Health Study also correlated n-3 PllFAs from fish v¡ith decreased incidence of
coronary hean disease and death [209]. The Dier and Reinfarction Trial (DART) advised
over 2 000 survivors of MI on diet over two yeaß and demonstrated that fish eaters, who
consumed 200-400 g famy fish,/week or
5OO-8OO
mg/ day n-3 PllFAs, had a 29% decrease in
monaliry [210]. Burr et aL reasoned this was due
fibrillation arìd not changes
in
serum lipids
to
decreased fatal
MIs, venricula¡
[211]. The GlSSl-Prevenrion Srudy
demonstrated a 2Q o/o decrease in all-cause moratalig' ar:'d 45o/o decrease
âlso
in sudden death
from n-3 PUFA supplemeiratìon f212f. Orher CV related effects establish that DFIA a¡rd
not EPA lowers blood pressure and heart rate significantly in humans [213]. Fish
reduce primary
c
atdtac anestl21,4l and decrease infarcr size 121.5,2161.
Specific data garhered on AI-A consumption associares it with lower risk for
MI
fatal ischaemic hean disease l212l. An inverseþ proponional relationship berween 1.4
g/ day
AIl'
meals
and risk of fatal ischaemic hean disease in women
established in addition
to
a similar relation
Lung, and Blood Institute Famiþ Hean
containing mustard
to coronary anery
Sildy [219].
l2l7l
disease
to
and
1.5
and men [218] was
in the National Hean,
AIso groups administered AI-A-
oil ând DHA- and EPA-containing fish oil both had
decreased
incidences of }dI12201. Using AI-A. supplements, the Lyon Sudy exhibited ZO% lower CV
monality at 5 years compared to conrrol withour changing senrm lìpids [221].
\X,/hile there is a great deal
of data that
cor.relares an effect
of n-3 PTIFA feeding with
positive CV outcomesJ some studies suggesr rhâr there is no effect [205, 222-224]. I{ns-
Ethenon et al. [2A2] propose rhât
a.n inverse
relation berween fish consumption and CV
monaliry is established onþ when a sufficientþ large non-fish earing popularion is used as a
control.
25
Evidence lends suppon that n-3 PUFAs may alter the myocardium. Both fish meals
and fish oil supplements increase hean rate variabilþ rvhich is associated with decreased
arrbthmia 1225, 2261. Also, purified DFIA a¡d EPA alter the hean rate and
diastolic fìlling
of the heart 12271.
Some postulated mecha¡risms
increase
for the positive CV
outcomes associated v¡ith n-3 PllFAs are decreased occrüïence of ventricular arrhlthmias
and atheroma progression, hypotrigþeridemic, anrirhrombogenic and
hypotensive
propefties and increased NO-induced relaxati onl228l.
DIETARYMECHANISMS
Dietary mrdies by Peter Mclennan on rar heans have shown rhar feeding a diet rich
in n-3 PllFAs significantly
ischaemia 1229,
nAl.
reduces the incidence and severiq'
of arrhlthmia occrirring in
PUFAs, including n-3 and n-6, but nor MUFAs or SATs prevent
ventricular fibrillation in a rat model of ischaemia and reperfusion [229]. More recent studies
by Mclennan indicate that an n-3 PUFA diet in rats
acidosis, K*, lactate,
CI!
decrease ischaemic markers like
and increase conrractile recovery during reperfusion due to reduced
myocardial oxygen consumption
(I4VO) [231]. Other physiological findings in the field
confirm that n-3 PUFA containing fish oils reduce ischaemic darnage 12321and arrh¡hmia
1233, 2341.
In
panicular, spontarìeous tachycardias, venrricular fibrillation, venrricular
tachycardn and the duration of arrhlthmias are all positiveþ reduced [233, 234]. However,
similar observations were nor described in models of acute recurrent ischaemia [235].
A handful of studies have looked at the effec* of dietary mariipulation of n-3 PLIFA
content in the SR. Pa¡ameters examined include Ca2* uptake, SERCA acriviry, PL and faay
acid composition, lipid order, membrârie fluidity, SR Cat* conrent, and Ca'?t sparks. The
mos comprehensive study to date by Taffet et al looked at CSR from rats fed diets
containing either menhaden oil or com oil, n-3 or n-6 PUFA sources [236]. Anaþsis of
cardiac SR (CSR) phospholipid membra¡es revealed an increased
DIIA,/AA rario ând
corresponding n-3ln-6 conrent. Both Ca'?* uprake and SERCA activþ were reduced in the
n-3 PIIFA group, which could nor be accounted for by changes in membrane permeability
or active pump
sites 1236). TafÍet showed that n-3 PUFAs are incorporated
into CSR PLs
widrout affecting permeability but interacting wirh membra¡re proteins, specificalþ SERCA
They proposed that dietary alteration of CSR lipids affects SERCA function by way of
changes
SR
in the structu¡e or composition of PL membra¡res. CSR may firnction like skeletal
which displaces membrane 1þid during conformational changes f237). Changes in n-3
PIIFA content in the Iipids immediateþ surounding SERCA and in the bulk lìpid
membrane could affecr this process by resisting displacement [236].
A¡other study in fish oil fed rats, examined Ca2* sparks in atrial myoq'tes [238].
Incorporation of n-3 PUFAs into these cells produced Ca2* sparks with shoner duration
than those formed in myocltes from rars fed a sarurated fat diet. The group anribured rheir
obseruations
to either decreased
àcti.vàtory
Cí-
(1,))
or faster RyR kinetics
(decreased p")
[238]. This study suggesrs a dìrect effect of n-3 PUFAs on SR protein fu¡ction.
Analysis
PUFAs,
DIIA
of CSR from mice fed menhaden oil also exhibit incorporation of
a¡rd EPA, and a decreased n-6/ n-3 ratio [239]. Both the initial rate a¡d
maximal raæ oÍ
Ci*
upÞke were depressed in the n-3 PLIFA group [239]. As in Taffett
smdy, this slowing of Ca'?* upmke
ráâs
to 1/6ú of control. They proposed that
change
l23el.
accounted for by SERC,A acriviry rhâr was decreased
since PC content and composition affects
SERCd
in PC with n-3 PIIFA conrenr may alter SERCA acriviry arìd render SR
suscepdble
n-3
to "large rapid" fÌxes in
Ca'?*
a
less
that car occur during reperfusion a¡ld ischaemia
A
second study on mice fed either
AI-4, EPA, or DFIA for 14 days showed that
CSR underwent marked membrane lìpid changes as compared
there was a compensarory increase
in
to control diets [240]. Here,
sarurated farry acids and
a decrease in oleic
acid.
Metabolism of precursor n-3 Pl,rFAs into DHA produced corresponding decreases in
and
lÁ.. In response, Ca2* uptake was decreased in all groups
Ca2* uptake was depressed
,{A
compared to an n-6 diet, but
to a lesser extenr in rhe AI-A-fed group. In conrra$ to
rhe
aforementioned studies by Swanson and Taffet, maximal SERC"\ activþ a¡rd SERCA Car"
affinity were not altered. Croset
e¿
a/ suggest that Ca'* uptake was decreased in n-3 pUFA
fed groups due to uncoupling of rhe membrane due to changes in membra¡e permeability
[240].
Diabetic cardiomyopathies may also be altered by n-3 PIIFA supplementation. ln
studies by Black et. al. coronary and aorric flow rates and diastolic function were improved
with the n-3 PIJFA preparation, Promega [241, 242]. Their work revealed improved SR Ca]
transpon activity which may explain the improved diastolic function in this group [241].
Fluorescence anisotropy was used ro assess membra¡e lipid order
in skeletal S\
which can serve as a model for less abundant and robust cSR, from fish oil fed rats 1243).
"Barely" significant changes in lipid order were established [2a3]. The aurhors rhoughr rhar
compensatory inco¡poration
of
cholesterol may allow
fo¡ continued ordering of
membra¡e [243] even though cholesterol normaJþ only accounrs for
Thq'
"suggest(ed)
5o/o
the
of SR Lpids [2aa].
that in natural membranes rhe complex mirture of phospholipìd
molecular species can, by itself, act as a passive buffer ro prevent the response from large
changes
in fany
acyl chain compositions and motional properries 12431."
rn contrasr to the
preceding CSR data, SERCA caraþ'tic aoiviry was not affected by n-3 PUFA feeding in this
skeletal SR preparation.
28
To date, n-3 PUFA feeding studies indicate that both CSR a¡rd skeletal SR fatry acids
to
are responsive
changes
n
diet 1236, 239, 241-2431. Confounding resuhs have been
reponed on Ca2* uanspon and SERCA catal)tic activity in these groups [236, 239, 241, 2431.
It is also debateable
that membrane permeabiliqy, fluidity ard order are not altered by
relativeþ modest changes in n-3 PIIFA content L236,2431. .W4-rether or not incorporarion of
n-3 PUFAs into SR phospholipìds contributes to their knoq/n antiarrhlthmic âcrion requùes
funher study.
I¡
addition, we have relativeþ litrle information on the effects that chronic
dietary interventions may have that would be expected to aher AI-A levels.
FREE FATTY ACID MECHANISM OF ACTION
The mechanism whereby unesterified n-3 PtlFAs exen rheir anti-arrhlthmic action
has been activeV researched over the pasr decade. The major findings have indicated that n-
3 PtIFAs act by enliancing the electrìcal stabiliry of hean cells. Alexa¡der Leaf and his
associates have conduoed many of the fundamental studies done in this area.
In
studies by
Billman and Leaf, infusion of n-3 PIIFA emulsions prevented the acute occurrence of
ischaemia-induced ventricular fibrillation
esterified
-AIA, -EPA
in
exercising dogs [245-247). Fish oil, non-
and -DFIA were each shown ro prevent fibrillation when given as an
infusion prior to a¡ exercise-ischaemia rest 1245-247).
Leaf and his group have also focused on the cellular mechanism of rhe acute addition
of unesterified
?
3 PITFAS (see Kang and Leaf
for review l2asl). Initial
srudìes
in neonaral
rat cardiomyoq.tes illustrated that EPA prevented aÐ'nchronous contracrilq/ induced by
high [Ca'?.] or ouabain [249]. Voltage-clamp experiments revealed thar n-3 PLIFAs reduce
the cônductarìce of VGSCs (ICs. 4.8 AM EPA, DIIA), L-t¡pe Ca'?* channels (ICso 0.8 pM
DHA) and initial outward K* currenr
(11J, and .Io delayed
rectifier (Q 1248,25A-253). Based
on their observations they hypothesise that n-3 PUFAs electricalþ stabilize cardiac myoq.tes
by increasing the electrical sdmulus required to elicit an acrion porential by hlperpolarizing
the membrane and raising the threshold of VGSC activation l24ïl. They arc also thought to
prolong the refractory period of cardiomyoq,tes [254].
Other studies have shown thar alternative pathways of arrhlthmogenesis may be
inhibited by n-3 PUFAs. Na.-H* exchanger inhibition by EPA and Dt{A could prevent the
unfavourable activation of the NCX during ischaemia [255]. Studies have also examined the
acute addidon of n-3 PUFAs to ventricular myoc)'tes to observe their effect on rhe sR [256-
2581. n-3 PUFAs appear to decrease the frequenry of both
ca2* ¡elease resulting
Ca'?* sparks
and sponraneous
in decreased amplirude of contraction. They concluded that puFAs
may exert their antiarrþthmic effects at the level of the SR in addition to rhe SL membra¡e.
Rodrigo's studies
of EPA on
permeabilised ventricular myoq.,res revealed a¡r
eventual decrease in contraction in both guinea pig and rat myoc)'res [256]. They proposed
that the decreased amplitude of contraction is due to eirher decreased ca2* uptake because
of L-type Ca2* channel inhibition or increased threshold for Ca2* release from rhe SR. They
correlated a reduction ìn the frequenry of spontaneous contractions v¡ith decreased P. RyR,
which is regulated by both SR and rytoso.lic
decrease P" RyR
Ct-
ï2561.
It
is suggested that EpA m4y
by âcting on SR K* channels which maintain electroneutrality, since K*
inhibition reduces ca2* uptake, ca2* content a¡d uldmateþ affects sponraneous RyR activitll25el.
Negretti
is,
it
et al.
propose that EPA, an n-3 fatg' acid, is a negative inotrope 12571. That
decreases the velociry and force
of contraction of the hean. These rwo pararnerers
are
functions of Ca2* concentration; as such rhe suspected mechanism by which EPA v¡orks is
through Ca'?*handling. The effects of rhe ts¡o n-3 PLlFAs, EPA, and DFIA, on sponraneous
and electrically stimulated contractions
in
single, isolated ventricular myoq.'tes from rat
heans were examined. The study concluded rhat EPA and
of propagating
DIIA ìnhibir sponraneous waves
Ca2* releæe, frequenry, amplìtude, and resting cell length
[252]. Another
paper by the same aurhors conc.luded that EPA and DFIA act through the inhibition of ca'?*
release and reduced availabilir¡
of ca,* to the SR [258]. These effects were thought ro be
simila¡ to tetracaine, a known antagonist
of the RyR
Tetracaine decreases the open
probability of the RyR and increases SR ca2* conrent [260]. overend and colleagues
describe this phenomenon as a compensatory mechanism where a decreased frequenry
of
spontaneous release from the SR in the presence of retracaine is made up for by an increase
in release of
Ca2n
s¡hen spontarìeous release does occur.
A review of the literature indicates thar n-3 PllFAs are and-arrhlthmic agenc. The
anti-arrhlthmic action may occur after dietary adminìstration, when we would expect n-3
PUFAs to be esterified to hyrdrophobic membra¡e constiruents, as indicated by the studies
of Black where SR
Ca':* uptake was enhanced
in
rats
with diabetic cardiomyo pathy
l2al.
Likewise, administration of n-3 PUFAs in their free or unesterified forms has been shown
alrcr ion channel conductance in studies by Leaf fz+sl.
our
studies anempted
t<.r
to examine
both of these aspects in relation to rhe SR. Isolated SR vesicles were assayed for function
after the exogenous addition of n-3 PllFAs and after chronic dietary interventions. These
studies on SR function formed pan of a larger study where n-3
PIIFA zupplementation in
rabbits v¡as assessed in tenns of arrhlthmia and atherosclerosis in cho]esterol-fed rabbits.
Since arrhythmias contdbute considerabþ ro morraliry from myocardial infarctions, n-3
PLIFAs may act as positive modulators of cardiovascular health.
31
II.
EXPERIMENTAL PROCEDIIRES
MATERIALS
Linoleic acid (I-4, C18:2 n-6), alphalinolenic acid
(AI-{,
C18:3 n-3),
eicosapenataenoic acid (EPA, C20;5 n-3), and docosahexaenoic acid @}JA\ C22ß n-3) were
purchased from Doosan Serdary
(Ioronto,
Ol$.
Standard rabbit chow wæ from CO-OP
Complete Rabbit Ration, Federated Co-operatives Limited (Sakatoon, SK) and Promega
Fla-x was
from Pola¡ Foods Inc. (Fisher Branch, MB). Roche Molecular Biochemicals (-aval,
PQ) supplìed; adenosine triphosphate (ATP), creatine kinase (CK) crearine phosphokinase
(CPK), dithiothreitol (DTl), leupeptin (Leu), nicotinamide adenine dinucleotide [.ÌADFÐ,
phenylrnethylsulfonyl fluoride (PMSF), and p1rrrvare kinase (PK). Calbiochem (San Diego,
CA provided lactate dehydrogenase (LDFÍ), ryanodine ßy),
phosphoenol (PEP) was supplied by Fluka (St. Louis,
*d
MO). Calcium
and Fwa-2 were made avaiìable by Molecular Probes (Eugene,
reagents and
DC prorein assay reagenß were
ionomycin (Io), while
G reen-2, }/rag Fura-2,
OR).
Electrophoresis
purchased from Bio-Rad Laboratories
(Ilercules, CA). Al1 of the gas chromatography standards were obtained from Nu-chek Prep
(Elysian, À4NÐ.
NEN Life
Sciences ftVoodbridge,
Olrt) supplied l'F!-ryanodine. Millipore
filters for the binding assay were obtained from Fisher Scienrific (Napean, Olrl). Atl other
reagents were of
anaþcal grade and from Sigma (St. Louis, MO).
METHODS
ACUTE STUDY
SKELETAL MUSCLE SARCOPIASMIC RETICULUM ISOIATION
Isolation
of
skeleral HSR from \X/trite New Zealand male rabbits followed the
method of Gilchrist et al. 126ll. Briefl¡ Frozen rabbit skeletal muscle (160 g) was ground
32
under liquid nitrogen wìth a monar and pestle. Ground muscle was then suspended in
mL of homogenization buffer conrâining
3OOmM sucrose, 2OmM
8OO
imid,zole, 1mM PMSF,
1mM DTT, 500 ¡r.M ATP-TRIS, 500 f¿M EGTA, 2uM Leupeptin, pH 7.4. The suspension
was then processed
in
a small lX/aring blender on
low for 40 seconds twice w.ith a 20 second
interval. The processed muscle was subsequentþ centrifuged at 10
15 minutes
OOO
¡pm (tS
3OO
x g) for
in a JA-14 Beckman Roror. The supemaranr was filtered through cheesecloth
and re-centrifuged in a Beckman JA-20 rotor âr 19
OOO
rym (+l
IOO
x g) for
t hour.
were then re-suspended in homogenization buffer containing 2 mM PMSF, 2 mM
Pellets
DTT, a¡d
4 ¡rM Leupeptin. Re-suspended pellets were gently homogenised under a glass monar and
pestle and volume was built up
to
18
mL. The
suspension was gently layered on previousþ
thawed 25-45"/" linear sucrose grad;ents. The gradients v/ere spun at 23
OAO
rpm on
a
Beckman S\Xu-28 swingìng bucker roror for rwenty-four hours. Following centrifugation, the
light SR (LSR) and hear,y SR (FISR) bands were harvested from rhe $adienrs. The two
bands q/ere then washed at 4 oC for one hour in 3OOmM sucrose and 2OOmM
salt mixtures were cenÍifuged
re-suspended
for t hour in
a 50.2
KCl. The wo
Ti rotor at 35 OOO rpm. The pellets were
in transpon buffer containing 3OOmM sucrose, 5OmM Dipotassium PIPES,
pH 7.0 and stored at -70
9C.
SKELETAL SR PROTEIN DETERMINATION
HSR protein concenrrations were determined from a bovine serum albumin standard
according to Harrington's modified Lowry method [262].
STOPPED-FLOW RAPID KINETIC FLUORIMETRIC ASSAY OF
EXTRALUMINAL CA2* TRANSPORT ANd SERCA1 CATALYTIC
ACTIVITYIN SKELETAL
SR
Freshly thawed HSR vesicles (0.25 mglml-) were pre-incubated with 0.S FM
Calcium Green-2 (CG-2) in transpon buffer (300 mM sucrose/SO mM dipotassium pipes,
pIì7.0, 25 t) containing 20 units/rnl PK,
FNI
Ci.,
14 units,/rnl
LDH,
2OO
FM NADH,
O
¡À4 or 20
a¡rd n-3 PIIFAs at varying concenrrarions. Ca2* transporr was initiared
combined addition
of 2.5 mM PEP and 1 mM Mg.ATP in a
Accessory from Hi-tech Scientific (Salisbury, U.K.)
with the
SFA-20 Stopped Flow
with pneumatic
dr-ive set
at 4
bars
pressure. Fluorimetric measuremenß of CaZ* transpon and SERCA catal¡ic activiry were
made in a Photon Technologr lntemational Quantimaster* steady state spectrofluorometer
with a Delta Ram upgrade. Data was acquired with
V/indov¡s @-based Felix-sofma¡e.
Extraluminal Ca2* transport was monitored with CG-2 (ExrrlEmr,) q¡hjle NADH
fluorescence (Ex,rrlEmrr) was used as an indicator of SERCA1
a coupled
hydroþis of ATP through
PEP/PYR enzr,rne pathway and ATP regenerating system.
\,X4rere skeletal SR
mg,/rrl) were
was assayed solely for SERCA activiry, HSR membranes (0.1
perrneabilized
with 10 ¡rM ionomycin, 20 units/ml PK,
200 pM NADH, 5 ùLCa2. free Q56
ylncl-/25A
14 units,/ml
LDH,
FM EGTA) prior to transpon and pre-
treated wirh varying concentrations of n-3 Pl,IFAs ìn transport buffer (:OO
mM
zucrose/SO
mM dipotassium pipes, pH 7.0,37'C). Car* trarspon was initiated with the combined
addition of 2.5 mM PEP and 1 mM Mg.ATP in a SFA-20 Stopped Flow Accessory.
fluorescence (Exrrr/Emnr) was used as an indicator of SERCAi
a coupled
NADH
hydroþis of ATP through
PEP/PYR enzl.rne pathway and ATP regeneraring qysrem.
FLUORIMETRIC ASSAY OF CA2* PULSE-LOADING IN
SKELETAL SR
Fluorimetric measuremenß
of Ca'* pulseJoading in
in
a
Photon Tech¡olog' Intemational Quantimaster'" steady stare specrrofluorometer with
a
skeletal SR were made
Delta Ram upgrade. Dara was acquired with \X/indows @-based Felix-software. The reaction
mirtr:re consisted of 0.8 ¡rM CG-2, 400 pM NADH, 14IJ/nL LDH, 20 U/mL PK, DF{¡!
and transpon buffer (300
mM
sucrose/SO
mM dipotassium pipes, pH 7.A, 25 'C) in a 3 mL
cuvene with magnedc fleâ. Ir was equilibrated for approximateþ 80 seconds, after which the
assay was
initiated by the simuhaneous addition of 1mM Mg.ATP md 2.5 mM pEp. \X/hen
a steady baseline was obtained, incremental boluses
of
10 ËM Ca'?* were administered
with
a
"repeator" pipette, retuming to baseline before the next pulse was given.
SKELETAL'1H¡ - nVaNODINE BINDING
Briefly, HSR (0.25 mglrnl-) were incubated ovemight in a reaction media consisting
of trarrspon buffer (:OO mM sucrose/5O mM dipotassium pipes, pH 7.0, ?5 'C), i.O3 mM
CaCl, and 250 pM EGTA (10 FM Cah free),
ryanodine, 10 mM creatine phosphate, 4
t
mM DTI, 4 FM leupeptin, 4
units/ml
creârine phosphokinase, and 1 mM
Mg.ATP. Aliquots (3 x 100 pl) were then vacuum filtered across
diameter
filter.
a 0.45 ¡rm
Each filter was v¡ashed rwìce with 5oo ¡rl transpon
dissolved overnight
"M irÉ!-
FIA\{?
13 mm
buffer. Filters were then
in 2 rnl CltoScint and 2OO ¡rl ethylene glycol. Radioactivþ of specific
[3F!-ryanodine was then deterrnined by liquid scintülation counring from total and nonspecific counts ìn the presence of tOO
¡M cold ryanodine.
FATTYACID PREPARATION
Stock solutions
(t
mNA)
of individual n-3 PllFAs were prepared in silanized
glass
tubes. Pipetted volumes of n-3 PUFA and HrO were combined in a glass test tube, sonicated
and vonexed at
4 C in a Branson
12OO
bath sonicaror @4arkham, ON) until the emulsion
resolved ìnto a uniform cloudy suspension [255]. \Vhere the n-3 PUFA,
I.{,
was suspended
in hexane, the solvenr was dried-off with nitrogen gas before re-suspension in F!0.
SDS-PAGE OF SR PROTEINS TREATED WITH n-3 PUFÄS
Sodium doderyl sulphate poþcrylamide gel electrophoresis (SDS-PAGE) of HSR
membra¡es was carried out according to Gilchrisr et at. 126ll. Briefl¡ HSR (1 mglml-) in
0.1
o/o
CFIAPS, 0.i% saponin,
temperaûre.
TIA
100
or 400 ¡M PI,IFA were vonexed for
Samples were then spun down
for 30 minutes ar Z5
OOO
t
hour ar room
rpm in a Beckman
rotor (150 000 x g). The supematanr wæ precipitated with trichloroacetic
deoxycholic acid on ice and spun ar 13
5OO
rpm in an IEC MicroMax
The resulting pellet was resuspended in ZS y"L of
1.
@
acid
/
desktop centifuge.
x sample buffer for anaþsis.
SR proteins
were stained with Coomassie brilliant blue R-250.
FEEDINGSTI-]DY
FEEDINGPROTOCOL
Male NZ!Ø rabbits were randomþ fed one of four diets for 8 weeks: regular chow,
or regular chow supplemented with 10% flaxseed (wtlwt), 0.5% cholesterol (w,/wt), or iO%
fla-xseed and 0.5olo
wih
cholesterol. Briefl¡ diets were weighed, ground, reconsrirured inro pellets
FIrO, drìed, and stored in the dark at 4
"C.
Rabbits that did not undergo an ischaemia-
reperfusion protocol were fed ad likam while rhose that were subjected ro ischaemiareperfision were [ed tzsg/ day.
PIASMA SAMPLING AND TISSUE FIARVESTING
Blood samples were taken from rabbit left marginal ear vein every 2 weeks beginning
at week zero and collected
for
in Vacutainer tubes. Samples were spun ar 5
10 minutes ât room remperarure
OOO
rpm (a
5OO
x
g)
in a¡ Eppendorf Centrifuge 5804R with A-4-44 rotor.
The upper plasma layer was pipened into eppendorf rubes and frozen at -80 .C for later
anaþsis. Cholesterol and trigþeride measuremenrs were made
witl
the VerTest
8OO8
blood
chemistry analyser (IDEXX Laboratories Inc., lü/esbrook, ME, USA). Samples were ready
for testing after they were brought to room temperârure and re-spun at 8
OOO
rpm (e
SOO
x
g) in an Eppendorf Centrifuge 5804R q¡irh F45-30-11 rotor.
At
8 weeks rabbits were anae$hetised
v/irh isoflurane. Flearts were rapidly excised,
rinsed in 0.09% NaCl, and frozen in liquid nitrogen. Alternativel¡ some heans underwenr
ischaemia-reperfision on a modified Langendorff
hearts were rapidþ excised and placed
NaCl 115.0,
NalICq
app
arus before quick freeze. These
into Tyrode's solution of pH 2.4 containìng
28.0, NaIlrPO4 0.5, glucose 20.0,
(nrN4)
KCI 4.0, CaClr 2.O, andMgCl,
O.Z.
The aorta was tied onto the cannula ofthe perfusion âpparatus. Retrograde perfusion of the
heans was initiated within 60 s
of excision. lleans were
perfused at a
flow
nl,/min with Tyrode's solution maintain ed at 37.A + 0.5'C a¡d bubbled wìrh
rate
95o/o
of
20
Or/ 5"k
COr. Heans were equilibrated for 50 min prior to a 30 min test ischaemia. Global
ischaemia was initiated
by bypassing the flow to rhe hean and retuming it to the buffer
reservoìr. The solution in the acrylic bath was bubbled with 95% Nr/solo CO, during
ischaemia. Hea¡ts were then reperfused
for 45 min by
re-establishment
of flow with
oxygenated Tpode's and the immersion bath v¡as again bubbled with 95% 02/ 5"/o
CARDIAC SARCOPLASMIC RETICULUM ISOIATION
CO
.
CSR was isolated according ro rhe merhod
of Feher [263] with some modifications.
Four rabbit heans from different feeding groups were prepared ar a rime. Flearts were
ground
to a fine powder under liquid
nitrogen and put
in 5x weight volume
ice-cold
homogenization buffer (1M KCl, 10 mM Imidazole containing 2 ¡;M leupeptin, 1 mM DTT,
and 1 mM PMSF). The suspension was rhen homogenized at 6
rpm using a PT31OO
OOO
Pobtron by Kinematica. The slurry was pulsed for 20 seconds rwo rimes and then spun
down
i¡
a Beckm¡r JA-20 roror at 9
OOO
rpm Íor 20 minures (10
P1, was rehomogenized 2 x 20 seconds at 6000 rpm
and centrifuged in a Beckman JA-20
x gmax). The peller,
OOO
with the poþon in the original volume
rolor at7 OOO rpm for
20 minutes (6
resulting supernarânr, 52, was then cenrrifuged for 25 minutes âr 14
OOO
OOO
gmax). Supematant, 53, was layered on a gradient of 3 mls of 35
homogenization buffer and 5 rnls 25 o/o sucrose in homogenization
gradient s/as cenrrifuged ar 18 5OO rprn for 2 hours (+1
OOO
x
g ma-r). The
rpm (2+
OOO
x
o/o sucrose in
buffer. The
layered
x gma-r). Pellet, p4, was built up
with an equal volume of homogenization buffer and centrifuged again (41 OOO x gmax) for
hour. The final pellet, P5, is resuspended in transpon buffer
(:OO
dipotassium pipes, pH Z.O) containing 2 ¡iM leupeptin and 1 mM
1
mM sucrose, 50 mM
DTT. Aliquots of
CSR
were quick frozen with liquid nitrogen and stored at -ZO 'C.
CARDIAC SARCOPIASMIC RETICULUM PROTEIN
DETERMINATION
CSR protein conrenr wâs detemined wirh the Detergenr-Corrpatible (DC) protein
Asay supplied by Bio-Rad using BSA
and samples (20 ¡.rL) were built
dipotassìum pipes,
pH 7.0)
to
as a
1OO
containing
standard. Both standards (0.2-1.0 mglnìL BSA)
¡rL in transpon buffer (3OOmM sucrose, 50mÀ4
2 ¡rM leupeptin, 1 mM DTT, and O.l%
SDS.
Dilutions were then mixed with reagenrs according ro the Bio-Rad DC Protein Assay
38
protocol and were read at 75a nm in a MRX' ReveLrdontt with remperature control
Microplate Spectrophotometer by D1.nex Techologies.
KINETIC MEASUREMENT OF EXTRALIIMINAL Ca,*
TRANSPORT BY CARDIAC SARCOPI-A.SMIC RETICULTJ.A4
Freshly thawed CSR vesicles (0.125 mg
transport buffer containing
/
20u
nn-
/
cK, a¡d 5
mL) were mired wirh 1 ¡M Fura-2 in
plv[.
cak
and pipened into separate wells
in a single column of a 96-well black flat bottom Microfluor@ 1 microplate. The samples
were then pre-incubated af 37 aC
for
10 minutes
in a SPECTRAmax @ Gemini XS Dual-
Scanning Microplate Fluorometer by Molecular Devices
Ca2*
coorporation. At time o seconds,
transpon was initiated by the combined addition of 10 mM Cp, 1 mM Mgr.ATp and 5
mM K*-oxalate from
a multichannel pipette
was then thorougtrly
mixed At time 30 seconds, data collection of extraluminal
transpon began. Fura-2 fluorescence (Exr*.
to the microplate column. The reaction volume
r*o
/
E..trr)
ca2*
data was acquìred in the microplate
fluorometer with SoFTrna-x*' PRo softv¡a¡e. A kinetic assay was run over 15 minures v¡ith
readings every 10 seconds. Each reading was an average of 6 reads/well. Dual-excitation
the dye occuned ar borh 340 nm a¡rd 380 nm wirh emission ar 510
nm.
of
The plate was
automixed before and berween readings for 1 second. The photo-multiplier tube was set to
medium. Fura-2 was calibrated with both 5 mM
media. Ratìos of the 34onm
/
cal
and 1oo mM EGTA in the reaction
38onm signals were computed in Microsoft@ Excel2ooo.
Free calcium conversions and rates were then executed in Graphpad prism'n, software.
KINETIC ASSAY OF SERCA2a CATALYTIC ACTTVITY
At
37 aC CSR membranes (O.O1O mg
containing 20 U
/
mL PK, 14U
/
rnl-
LDH,
/
mL) were incubated with transpon buffer
2OO
pM NADH and 10
¡M 4-Bromo-A2318/
in a 96-well black flat bomom Microfluor@ 1 microplate. The SERCA antagonis,
10
¡M
Thapsigargin, or arr equivalenr amount of vehicle was added to deterrnine specific SERCA
acriviry. ATP hydroþsis was iniriated with the combined addition of 2.5 mM pEp, 1mM
Mg'?.ATP, iO1
NADH
¡rM
Ca'?. and 1OO
fluoresc ence (Ex,r,
/
ATP through a coupled PEP
¡M EGTA from a multichannel pipette to the microplate.
Em*r) was used as an indicator of SERCA2a hydroþsis of
/
PYR enzyme parhwây and ATP regeneradng system. The
reaction was monitored 30 seconds after initiation with SPECTRAma-x
@
Gemini XS Dual
Scanning Microplate Fluoromerer by Molecular Devices Coorporarion using SOFTmax*
PRO softwa¡e. A kinetic assay was nrn over 2 minutes. Readings were mken every
seconds as an average
of 6 reads/well. The microplate was automixed for
reads. Data anaþsis was done in Graphpad
Prism''
10
1 second berween
sofrware.
SPECTROPHOTOMETRIC DETERMINATION OF
CHOLESTEROL COMPOSTITION OF CARDIAC SARCOPIASMIC
RETICULI]M
Previousþ frozen CSR was double-extracted in 2:1 CFICI: MeOH and resuspended
n
95o/o
EIOH.
CSR was buiÌt
CSR, 125 pg, s/as pipened into a borosilicated glass rube. The volume of
to 60 pL with dd HrO. Then 1.2 rnl- of 2:l CHCIr: MeOH was added to the
tube follwed by 0.25 mL of
A.73o/o
vortexed every 15 minutes for
NaCl. The tube was sealed,áirh
t hour.
Samples were spun
a
Teflon-lined cap and
for 5 minutes ar 5OOO rpm (4 5OO
x g) in an Eppendorf Centrifuge 5804R with Rotor A-4-44 at 22"C. The resulting bonom
layer
of CHCI, was transferred to a cuvene. The remaining
extracted with an additional 1.2 rrtr ol
2:1,
aqueous solution was re-
CHCI,: MeOH and1.Z5 rnl of
0.73olo
NaCl. The
tube was vo.nexed and spun as outlined above and the CHCI, layer was added
40
to
the
previous
efiract. The solvenr was removed with N, (g). The remaining lipids were then
dissolved
in
lQA
¡tL
95o/o
EIOH and stored ar -20'C unril
A reaction media consisting of
6.6
with 0.25 U
/
0.5olo
needed.
Triron-X, 3 mM sodium cholate, 0.1 M Tris, pH
mL cholesterol oxidase, 0.1 mg / rJ- o-Dianisidine and peroxidase
added to cSR exrracts ìn
EtoH.
was
The mirture was pipetted in triplicate into a 96-well 3595
microplate by costar@ and incubated at 37"C for 20 minutes in a SPECTRAmax
@
pLUS]s4
Microplate spectrophotometer by Molecular Devices coorporation. To determine free
cholesterol absorbance
of the
spectrophotometer using
samples was read
at 450 nm with the microplate
soFTmax- PRo software. To obtain rctal
meâsurements alìquots were incubated
with
1
u/
mL cholesterol
cholesterol
esterase at 37"C
îor 30
minutes and read again at 450 nm. Esterified cholesterol was rhen calculated from the total
and free cholesterol values.
FATTY ACID ANALYSIS BY GAS CHROMATOGRAPHY \TITH
FI-{ME IONISATION DETECTOR
CARDIAC SARCOPLASMIC RETICULUM AND PLASMA ANALYSß
Samples were prepared
for analysis according to the methods of Lepage et
al.
[264)
with adaptations by Ms. Andrea Edel. cSR and plasma samples v¡ere thawed and vonexed
Using a serological pipetre, 2 mL of methanol-benzen e 4:7 (v/v) (containing the intemal
standard) was added to a borosilicate glass tube. With a Drummond pipetter
or 50 pL of
SR was added
to the metha¡ol
1OO
benzene solution and vonexed
¡rL plasma
well. Aceryl
chloride, 200 ¡rL, was added to the mimrre over severai minutes during continuous agitation.
The tubes were sealed with Teflonlined caps and vorrexed again. Methanoþsis of the
sample occurred over 60 minures on a 9O-95'C heating
block.
Samples were vonexed every
15 minutes whjle being heated. Tubes we¡e then cooled ar room
41
temperan'e. To neurralize
the solution 5 mL of
6o/o
KrCO3 was added to the tubes using a serological pìpette. Each
tube was vonexed 5 times and centrifuged for 5 minures ar 45OO g at Z2'C
n
an Eppendorf
Centrifuge 5804R with Rotor A-4-44. An aliquot of the upper benzene layer was then
transferred with a Pasteur pipene to a lov¡ volume insen of an autosampler vial.
Samples were analysed
in duplicate on
a Varian CP-3800 GC equipped
wìth
1777
injecors, CP-8400 Autosampler, Flame lonization Detector (FID), 10 ¡iL injection s¡ringe,
and CP-Sil 88 Column (50m x 0.25mm
ID). Helium was used as rhe carrier
as the make-up gas. Column flow was 1.5
mllmin.
gas and nitrogen
The splìt rario used for injection is
shoqm in the table below
Time (minutes)
Split State
Split Ratio
Initial
JN
0.01
ON
ON
5
50
1.00
5
The injection port temperature was 250'C and the FID remperatlre was ser âr 270"C. The
column oven temperature followed the parameters outlined below.
I emp ("C)
Hold (min.)
l(ate {"u/mm}
80
740
240
5.0
225
5.0
1.00
0.00
0.00
10.00
30
I otal Min.
1.00
3.00
15.00
30.00
The instnrment was calibrated using a standard mixture of FAMES and an intemal standard
was used to check recoveries.
BßAIN, HEART, LIVER,AND KTDNEY ANALYSIS
Previousþ extracred samples of brain, hean, liver and kidney stored in CFIC! at -80
oC
were removed frorn the freezer ald vonexed. -ùühen samples reached O "C they were
pipetted into pre-weighed rubes at varying volumes depending on rhe rissue extrâct
42
(see
Table insen) using a Dmmmond Micropipette. Each sample .was rhen purged ï¡irh
N, ro
remove the solvent. Once the tube was reequilibrated (approximateþ 5 minures later), the
tube was reweighed rc obtain lipid weight. Next, a Calibra Macropipene was used to add
nI- oÍ
3:2
1
MeOH: Benzene solution containing an intemal standa¡d to each tube. Samples
were vortexed and stored at -20 'C until the remaining samples were processed. Then 1 mL
of 5:100 (v,/v)
acetyl chlorideMeOH was added ro rhe tubes, rubes were vonexed a¡rd
weighed. Tubes were heated between 95-1OO "C
cataþse methanolysis
of fatty acids.
reweighed to determine
wih 5 mL of
6%
if
Samples were retumed
any sample was
ÇCOr. Finall¡
for t hour a¡d vonexed every
15 minures ro
to room temperature
lost. The reaction in
and
each rube was neu¡ralized
tubes were vorrexed rwice and centrifuged for 5 minutes
at 4500 g, 22"C. An aliquot of the resolved benzene layer was removed with a pasteur
pipene and transferred into a low volume insert in an autosampler
vial.
Samples were
injected into the gas chromatograph as outlined above.
I rssue
Stored
in X
mL
Volume
ol
I issue
Grams
ot
CHCF
Extract Used for
Used
Brarn
3mL
Derivatization
100 pL
1g
Hean
2mL
100 ¡rL
0.25-0.50 g
Llver
3mL
5A ¡L.L
lg
Krdney
3mL
100
1g
43
¡rL
I rssue
III.
RESULTS
Recent studies suggest omega-3 polymsarurated fatty acids (n-3 PLIFA) reduce the
arrhl'thmogenic potenrial
of the myocardium.
Since disturbances
in
intracellula¡ Ca,*
regulation are arhlthmyogenic, we examined effects of exogenousþ added unesterified n-3
PLIFAs on skeleral SR vesicles ftISR) Ca2* rranspon. HSR membranes isolated in
abundance
from fast-rwitch skeletal muscle were chosen because they allov¡ for sudy of
both SERCA pump function and RyR mediated
Ca'?* leak
wirhin the same membra¡re
preparation. HSR vesicles are enriched in both SERC,A pumps and RyR channels, and thoT
respond dlmamicalþ in Ca':. transpon assays [261].
The effects of esterified n-3 PUFAs on Ca2* handling by the SR were also studied in
a
dietary model
of n-3 PUFA enriched feed.
Since our interest was
to
elucidate the anti-
arrhlthmogenic action of n-3 PllFAs in the hean, Ca'* transport by the SR was assessed in
isolated cardiac SR vesicles (CSR).
In
vesicles is less robust and its
is less abundant. Despite rhese shortcomings
leld
conrrasr ro HSR vesicles, Ca'?* transpon by CSR
transpon assâys were developed ro caprure
Ca'?.
Ca2*
handling. In addition, our studies on CSR
were part of a larger study that assessed the electrophysiological and atherosclerotic effects
of this dietary model.
EFFECTS OF EXOGENOUSLYAPPLIED PUFAs ON ISOLATED
SKELETAL HSRVESICLES
To quantif, the response of HSR vesicles to PfJFAs we empþed recentþ described
fluorimetric methods [261] adapted for stopped-flow rapid kinetic anaþis (See Merhod$.
Extraluminal Ca2* transport by isolated SR vesicles was monitored with the fluorophore
Ca.lcium Green-2 (CG-2, Exroo
/
Emr.,). NÁDH oxidatio n (Exrr,
indicator of SERCA hydrolysis of ATP through a coupled PEP
ATP regenerating sysrem. Flere,
functional coupling were studied (Figure 1).
a¡rd
LA.
Em*r,) was used as a¡r
PYR enzr..rne pathway and
Ca2* rrânsporr and SERCA activiry v/ere srnchronously
monitored and concentration-dependent effects
DIIA,
/
/
1We
of
exogenousþ added PUFAs upon
tested the n-3 artd n-6 PLTFAs:
AIA, EpA,
HSR vesicles were pre-treated with varying concenrrarions of PUFAs and
rapidþ mixed with ATP to activare Ca2* uptake.
The rate of Ca2* accumulation by HSR was measured by the decrease of CG-2
fluorescence. Thus relative to control rraces, a leftward shift in the time-dependent Car*
uptake curve indicated an improvement in the rate of Caz* uptake, and a righffiârd shift
indicated a delay in the rare of ca2* uprake. The rate of ca2* sequesrrarion by HSR vesicles
was not unifonn in response to the
PUFAs. PllFAs increased the rate of ca2* accumulation
by HSR vesicles below a cenain concentrârion threshold of PUFAs, but decreased the rate
Ca2* uptake above that
Fig're
limit (Figures 2-4, representative traces are shown). For example,
4 demonstrates that relative
byHS\
to control, 10 pM DFIA increased the rate of Car* uptake
whìle 75 ¡M DFIA decreased Ca2* uprake. PUFA concenrrarions rhat delayed FISR
Ca2* sequestration exceeded 50
¡M with n-3 PLTFAs and
PUFA, f-A, (Figure 5). This data may indicate thar
Ci*
approached 50 ¡rM with the n-6
transporr is enhanced by pUFAs
(<50 Él\4.
The SERCA catalltic activiq/ rhar corresponded with rhe Ca2* sequesrrarion data was
measured
from the rate of NADH oxidation. A rapid
decrease
in the concentration of
NADH was indicative of high SERCA activity, similarþ a slow decline in NADH
concentration represented
low SERCA activity. Rates of ATP hydroþsis ar PUFA
concenffadons that improved the rate of ca2*sequestrarion were unaffec¡ed when compared
@**
\
v
7"531
Figure 1. Syncfuonous Fluorimeory of SR Ca'?* transport activity and ATP hydrolysis
Ertraluminal Ca2* transpon by isolated SR vesicles was monitored with the fluorophore CG(Exro,
Emrr,). NADH oxidation (Ex.r, F,m*,.,) was used as an indicator of SERCA
hydrolysis of ATP through a coupled PEP / PYR enr¡,rne pathway a¡rd ATP regenerating
2
qFstem.
/
/
A,
1
(\¡
gË
E9l
ËH,
Fä
tr=
olL
z
0.5
0.0
10
Time (seconds)
B.
=1
-
o1
z
Figure 2. The Effects of Alpha-Linolenic Acid on A. Extraluminal
Ca2* Transport and B. SERCA ATP Hydrolysis by HSR Membranes
HSR membranes (0.25 mg,/m'l-) were pre-treated wirh varying concentrations of
alpha-linolenic acid (¡À4, Ct8;3 n-3). Ca:* úânspor¡ (Calcium Green-2 traces) and
corre sponding SERCA1 cataì¡ic actility (NADH trace$ were synchronousþ
monitored as described in "Metho&". Ca2* transpon was rrutiated by the
combined addition of Mg.ATP a¡d PEP.
4"1
(\
.io
õË
E9¡
Ëã,
Fb
¡-olt
z
l_
t
o
10(
z
Figure 3. The Effects of Eicosapentaenoic Acid onA. Extraluminal
Ca2* Transport and B. SERCA ATP Hydrolysis by HSR Membranes
HSR membranes (0.25 mg/ mI-) were pre-rreared with varying concentra¡ions of
eicospentaenoic acid (¡À4, C20:5 n-3). Ca2+ transporr (Calcium Green-2 traces)
and corresponding SERCA1 cataly'tic activity (IIADH traces) were sl.nchronousþ
monitored as descril¡ed in "Merhods". Ca2* trarspon was initiated by the
combined addition of Ms ATP and PEP.
A.
(\¡
gË
ç8
ÈE
Fb
tr5
zolr
0.5
10
Time (seconds)
¿
I
o
100
z
10
Time (seconds)
Figure 4. The Effects of Docosahexaenoic Acid on A. Extraluminal
Ca2* Transport and B. SERCA ATP Hydrolysis by HSR Membranes
HSR membra¡res (0.25 mglmI-) were pre-treated with varying concentrations of
docosahexaenoic acid, ÇM, C22:6 n-3). Ca2* transport (Calcìum Green-2 traces)
and corresponding SERCA1 cata.lltic activþ SJADH traces) we.e qmchronouiþ
monitored as described in "Methods". Ca2* rr¿nsporr w¿s iruriated by the
combi¡ed addìtion of Mg.ATP a¡d PEP.
49
I
ô¡
óq
r¡Ë
*O
ËH
Fã
È=
zolr
0
J-
o
100
z
10
Time (seconds)
Figure 5. The Effects of Linoleic Acid on A. Extraluminal Ca2*
Transport and B. SERCAATP Hydrolysis by HSR Membranes
HSR membra¡es (0.25 mg/rr'L) were pre-treated with varying concentrations of
linoleic acid (pM, C18:2 n-6). Ca2* traaspon (Calcium Green-2 traces) and
corresponding SERCA1 catalltic activiry SJADH traces) were s¡mchronousþ
monitored as described in "Methods". Ca2* trarspon was initiated by the combined
addition of Mg.ATP and PEP.
50
to control NADH consumprion (Figures 2-5). For example, Figre 4 shows that the rate of
NADH oxidation was unchanged from control despite its effect on
Ca2* transpon.
llowever, the related cataþic acivity seen wirh PI,IFA concentrations that delayed Ca,*
uptâke was variable. rWhen Caz* uprake was delayed, comparativeþ long chain n-3 PllFAs
(EPA and DFIA) hydroþed ATP slov¡er than control (Figures 3-4). Comparativeþ shoner
chain PUFAs (AI-A and I*A) consumed ATP faster than control under the same conditions
(Figures 2,
5).
Despite this variabiliry, a susrained acrivarion of the terminal phase of the
NADH trace (ast
10 seconds) is a common feature
of all of the
rates
of ATP hydroþsis
when Ca2* uptake is prolonged by PUFAs. This may indicate that SERCA is sequestering
Ca'* into the SR in the presence of an opposing Ca2* leak created by PlIFAs. These initial
characterisations
concenüations
of both
Ca'?* handling a¡rd SERC.A âcriviry suggesr
of PllFAs improve coupling of
that relatively low
Ca2* transport whereas relativeþ higher
PUFA concentrations impair coupling of Ca2* transpon.
To account for the observed enha¡rced sequesrrâtion of
concentrations
of PUFAs, uncoupled
Ca']* by HSR
Ca2*-dependent SERCAIa acriviry
with activating
in
ionomycin-
permeabilised HSR vesicles was assessed using a stopped-flow rapìd kinetic assay of
NADH
fluorescence. This method a]lowed us to examine SERCAIa activig' independentþ of RyRl
leak status. Maximum acdvation rates of SERCA 1a catalttic activiq/ were derived from
linear regression analysis of the
NADH oxidadon
rares using Graphpad
þee Method$. Generaþ a concenrrarion-dependent increase
Prism-,
sofrware
in rhe rare of NADH
oxidation by permeabilised HSR vesicles was observed in response to PUFAs (Fìgures 6-9).
These small, albeit statisticaþ significant, increases
overall ATP
onþ accounted for a
5-lAo/o increase
hydroþis. This suggests that PIIFA effects on SERCA activity
consequentþ they may act at a different site in the SR membrzrie.
in
are limited
o
.!C
los
o
o
o
s loo
. P < 0.001
Figure 6. Effect of Alpha-Linolenic Acid on SERCA1 ATP Hydrolysis
HSR mernbranes (0.10 mglmÌ) were pre-treated with varying concenrrations of alpha-linolenic
acid (AI-4;
pM).
SERCA1 activiq, as measured by
perrneabilized HSR membranes is shown. * P
(
NADH consumption in ionomycintM ALA
0.001 vs. 0
115
õ
c
o
fo
110
tos
àe
100
0102550
[EPA] pM
* P < 0.001
Figwe 7. Effect of Eicosapentaenoic Acid on SERCAT ATP Hydrolysis
HSR membranes (0.10 mglrnl) were pre-rreared with varying concentrarions oI
eicosapentaenoic acid (EPA, pl.4). SERCA1 aaivity as measu¡ed by NADH consumption in
ionomycin-perrneabilized FISR membranes is shown. * P < 0.001 vs. O gM EPA
53
115
õ
110
q
o
fo ros
s
100
.
P < 0.001
Figure 8. Effect of Docosahexaenoic Acid on SERCA1 ATP Hydrolysis
HSR membrane s (0.10 mg,/rnl) were pre-rreated with varying concentrations of
docosahexaenoic acid (DIl,\ pL!. SERCA1 activity as measured by NÂDH consumption in
ionomycin-permeabilized F{SR membra¡es is shown. * P ( 0.00 1 vs. O ¡lM D} IA
54
õ
110
c
o
fo
ros
s
0102550
* P < 0.001
[LA] ¡rM
Figure 9. Effect of Linoleic Acid on SERCA1 ATp Hydrolysis
HSR membranes (0.10 mglrnl) were pre-treared Í¡irh varying concentrarions of Lnoreic acid
(tA, AIVÐ .
activþ as measured by NADH consumption in ionomycin-S-ERCAI .
permeabiÌized HSR
membranes is shown. "- P ( 0.001 vs. O ¡rM l,4.
Since PIIFA-induced enhanced sequesrrârion
decreased leak starus
of
vesicles, rather
of
Ca'?*
in HSR could be due
rhar an increased rate of SERC-{ acrivity,
ro
we
examined this altemate hlpothesis. Ca'?* leak pathways in SR membra¡les are often mediated
by RyR subcondudance srâres, so a lrF!-ryanodine binding assay was used to determine the
effect of the selected PllFAs on RyR inregrity. Specific effects of [3F!-ryanodine binding
were derived from total and non-specifìc binding data conducred under maximal binding
Ca:* buffered conditions þee Methods). In all cases, n-3 and n-6 PLTFAs decreased []F!ryanodine binding
in a concentrarion-dependent manner
(Figures 10-13). lnterestingþ
concentrations of PUFAs that delayed Ca2* transport significantþ decreased []Fll-ryanodine
binding suggesting that relatively high PLrFA concenffarions disrupt RyR functional status
and in so doing potentiate Ca'?* leal<s. Still, the improved coupling of Ca2* transpon and
ATP hydrolysis seen with lower PIJFA concenrrârions was not explained by this data.
To funher examine the effect of PUFAs on
Ca2* release processes
from FIS\ we
incrementally loaded vesicles with Ca'z* ro acrivare CICR using a previousþ published pulseloading protocol
[261]. Usualy
3-4 pulses
of rc pMCt., represented by rapid
increases
in
CG-2 fluorescence, were needed to srimulare Ca2* release depending on rhe prepararion of
HSR vesicles and residual Ca'?* found in the buffers (approximateþ 4OO nÀ.4). CICR was
characteizedby an increase in CG-2 fluorescence and a slow retum to baseline
as
compared
to initial Ca2* pulses. Figure 14, Panel A shows that u¡der control conditions, with vehicle, a
CICRlike phenomenon was obserued after four pulses
loading level
of
40.4
ñI
Ca
wirh
O.25
of
10 ¡¿M Ca'?* (equivalent
mglml- HSR). A progressive i¡hibìdon of CICR
observed with relativeþ low concentrations
(¡M
a
was
of DFIA; addition of 2.5 ¡M DFIA to the
reaction media delayed the CICR response by one pulse, but 5
(Figure 14 Panel
to
¡M DFIA inhibited CICR
A). In a similar preparation of HSR, where a comparable control of CICR
o
o
o
o
s
. P < 0.001
Figure 10. Effect of Alpha-Linolenic Acid []Hl-ryanodine Binding
HSR membranes (0.2 5 mglml) were pre-treated wirh varying concentrations of alpha-Linolenic
acid (AI-4, C18:3 n-3). Specific [tF{]-ry4odine binding was determì¡ed from non-specific
binding, as deter:rnined with 100 pM ryanodine, subtracted from total binding (n : 3). * P <
0.001 vs. 0 ¡À4
AIA
õ
c
o
o
o
s
0
+P<0.05
-
P < 0.00'1
Figure 11. Effect of Eicosapentaenoic Acid on []Hl-ryanodine Binding
HSR membranes (0.25 mglml) were pre-rreated with varying concen¡r¿ions of
eicosapentaenoic acid (EP,A, C20:5 n-3). Specific [;Il-ryanodine binding was determined
from non-specific binding, as deterrm¡ed with 100 pM ryalrodne, subtiacted from total
binding (n : l). + P < 0.05 vs. 0 pM EPA + P < O.OO1 vs. 0 sM EPA
õ
o
o
o
s
0
10
25
[DHA] pM
50
+P<0.05
. P < 0.001
Figure 12. Effect of Docosahexaenoic Acid on [3H]-ryanodine Binding
HSR membra¡les (0.25 nig,/ml) were pre-treated with varying concentrations of
docosahexaenoic acid (DFI{ C22:6 n-3). Specific []F!-ryanodine binding was determined
from non-specific binding, as derermr¡ed with 100 ¡M ryanodine, subtiacted from total
binding (n:3). + P < 0.05 vs. 0 ¡rM DHA '¡ P < 0.001 vs.0sI4DFIA
õ
tr
o
o
o
àe
Figure 13. Effect of Linoleic Acid on []Hl-ryanodine Binding
HSR membranes (0.25 mglÍü were pre-treared with varying concenrrations of linoleic
(LA, C18:2 n-6). Specìfic
acid
ftil-Vmodi". binding was detår.rritred from non-specific binding,
as derermined with 100 pM ryanodine, subt¡aded from totaì binding (n : f). ? p < 0.001 vi.
0ÉMl-A
A,
1.3x1006
n,
CL
o
o
o
o)
o
an
o)
t-
6.3x 10 05
o
E
(\I
o
o
0.0x 10'oo
125.0
212.5
300.0
Time (seconds)
B.
1
.0x 10 o{
(t,
o.
o
o
o
o
o
u,
o
o
5.0x 10 0l
J
tr
(\
o
o
0.0x'10-oo
120
600
360
Time (seconds)
Figure 14. Effects of Docosahexaenoic Acid on
Membranes
Ca'?*
PulseJoading in HSR
memb¡a¡es (0.25 mg/¡nl-) were pre-treated *'ith varying concentræions of docosahexaenoic acid
*3). Ca2* transpon (CG-2 traces) was monirored as described in "Methods". Transpon was
initiated by rhe combined addition of Mg ATP and PEP. Caz* additions were made with a repeater pipette
as 10 pM pulses aod are seen as rapid rises in CG-2 fluo¡escence. CICR is cha¡acterised by a rapid rise in
CG-2 fluo¡escence and a time-dependent delay in a ¡erurn to baseline fluorescence. CICR uas
suppressed by lotu-end (A.) ønd potentiated by high-end (8.) concentrations of docosahexzenoic
øcid.
FTSR
(C22:6
61
occuffed after three pulses of 10
tM Câ'. (equìvalent to a loading
A.25 mg/rrJ, HSR) relatively high concentrations
of DFIA porenriared
concentratiôn-dependent manner (Figure 14 Panel
increase in the Ca2n threshold needed
level of 30.4 ¡rM Ca'?. s¡irh
B).
for CICR (Figure
Ca2* release
in
a
Results that exhibited a gradual
14 Panel
A) were remarkabþ similar
to the known effects of ruthenium red, a RyR antagonist (Figure 15). Perhaps PLIFAs
specificalþ inhibit a RyR response at relativeþ low concenrrations, bur prevenr normal
kinetics of RyR activarion and inactivation at higher concentrarions.
\Ve then speculated that PllFA-activated Ca'?*leak conditions were mosrly likely
caused by a loss
of
SR membrane integrity in the
bulk lipid rather than direct effects on RyR
To determine if the obserwed PIIFA effects on FISR vesicles v¡ere due to a disruption of
HSR membrane protein, HSR vesicles were incubated for an hour with various PllFAs,
centrifuged
to
separate out protein, the supematants were then precipitated and analysed
PUFAs appeared to have no effect on SR protein levels as compared to untreated HSR
vesicles. Detergents known to solubilise HSR membra¡es, like CFIAPS and saponin, were
used as outside controls to show that solubilisation of SR membranes increased the recovery
of SERCA proteins from
Qualitative analysis
of
supematarìts
of reated HSR on SDS-PAGE gels (Figure
16).
in
rhe
SERCA protein (110 kDa) and calsequestrin (44 kDa)
preparations indicated that PUFAs do not ìncrease the recovery of these proteins (Figure
16). Therefore PtIFAs do nor disrupr HSR membranes to such an exrenr that inregral SR
membrane proteins are removed.
EFFECTS OFCHOLESTEROLAND FLAXSEED FEEDING ON
CARDIAC SR FUNCTION
The second half of this study anempted to modulare the lipid compostion of
cardiac SR membra¡res wirh fatry acids and cholesterol feeding. Fany acid content of the
1.5x100(
0
U'
o.
o
o
o
c
o
o
at,
o
o
7.5x l0
,
05
J
tr
(\
t-rM
\',vîq
,li, :\.rlt
\
*;çl\*l"S{'
o
o
0,0x 10-oo
120
225
Time (seconds)
Figure 15. Effect of Ruthenium Red Caz* pulse-loading
HSR membranes (0.25 mg/ .:;-L) were pre-treated with varyrng concentrations of mthenium red
úrM). Cl- transport (CG-2 rraces) was monitored as described in "Methods". Transpon was
initiaed by the combined addition of Mg.ATP and PEP. Car* additions were made with a
repeater pipette as 10 ¡¡M pulses and are seen as rapid rises in CG-2 fluorescence. CICR is
characterised by a rapid rise in CG-2 fluorescence and a time-dependent delay in a retum to
baseline fluorescence.
7
I] U J-
+
11'), 1
U
f
J.O
a1 ¡
26û
lq rî
lLA
Figure
16.
SDS-PAGE of SR response to PUFAs
Læres 1-Z respectiveþ contain untreated HSR, 0.1% CF{APS + HSR, 0.1 %
Saponin + HSR, 400 i/M AfA / mg HSR, 400 aM EPA / mg HSR, 400 ¡,M DFL{
/ mg HSR, and 400 aÀ{ IA / mg HSR Marker 1 indicæes SERCA (110) kDa
wh.ile ma¡ker 2 indicæes CSQ (a0 kDa).
SERCA
CSQ
diets was varied
with flaxseed incorpontion. In addition cholesterol chow was fed to alter
both membra¡re rigidiry ard fany acid content. Finaþ a cholesterol,/flaxseed diet was given
to obsewe ¡heir combined effects.
Plasma and tissue analyses were used as indicators
of
incorporation of dietary subsdruents. Isolated CSR vesicles were then characterise d for fatq,
acid, cholesterol content, and corresponding Ca2* transpon and SERCA activities.
DIETARYANALYSIS
NZ!í
fla-xseed,
rabbits were fed a regular chow, or a regular chow supplemented with 10%
or 0.5% cholesterol, or 10% flaxseed and 5% cholesterol fo¡
8 v¡eeks. Promega
flaxseed was chosen as a rich-source of n-3 PIJFAs because it was fonified in
Al-A
(70o/o
of
total faty acids compared to 55% in regularþ grown seed). Based on the studies of Prasad
12651 and
our preliminary studies, which indicated that the texture and palate of flaxseed-
augmented regular chow could be maintained, we chose to supplement rabbit diets
with 10%
flarseed (equival ent to 72.5 g for a qpical 125gdiet). This was less thâri r.he 7.5 gflaxseed/
1rg
body weight (equivalent to 78.75 gfor a 2.5 kg rabbit) fed by Prasad[265]. However, we
still expected significant levels of n-3 PUFAs to be reached in the rabbits since the fla*tseed
was ground
to aid in its digestion and the rabbits did not
experience
the gastrointestinal
disress he reportedly observed. In the interest of obsewing changes that were clinically
relevalt, dìets were also reconstituted with o.s % cholesterol, which we knew prornoted
atherosclerotic plaque formation in NZSø rabbits over a time-course of 8 weeks [266]. In
addition, the cholesterol may be incorporated into membra¡res like CSR [262] and alter its
lìncLion and physical
ch aracr erisr ic s.
Analysis of the fatty acid composition of the diets confirmed AIA-enrichment in the
flaxseed diets.
Al,A conrent
increased approximately ten-fold
in diets containing
fla-xseed.
The fla-xseed and cholesterol/fla-xseed diets respectiveþ conained 20.077 + 0.841 mg AJA,/
gand22.535+ 0.679 mg ALL/
g diet
in comparison to the regular and cholesterol diets that
contained 7.9%+ A.720 mg Al-A,/ g and 2.179x 0.036 mg 1J-4'/ g respectiveþ (Iable 1).
Significant increases in palrnitic acid (C16:0), and oleic acid (C18: 1) were also observed in
the fla-xseed diets (Iable 1).
The amount of dier received by the animals was not controlled in the study where
heans did not undergo ischaemia-reperfusion, since the resulting weight of the a¡imals at
weeks did
8
not affect our abiliq¡ to normalise the leld of isolated CSR vesicles. Floweve¡
during the ischaemia-reperfusion study rabbits v¡ere rationed 125 g feed /day to allow for
the comparison of electrophysiological data between like groups (fhis data will not be
discussed here).
limited
to
725 g
After 8 weeks of feeding, rabbit weights were normal between rabbits
ol
dret/ day (Figure
17). Caloric intake [or I25 g of each of the prepared
diem did not differ statistically. Regular chow, 10olo flaxseed, 0.5% cholesterol, a¡rd to%
flaxseed
/
5% cholesterol
c
ontatned 423, 445, 42I,450 calories respectively.
CFIARACTERISATION OF RABBITS
Over the course of the
stufi
rabbits were monitored for plasma fatty acid and
cholesterol composition as an indication of the effectiveness of the feeding prorocol.
steady time-dependent increase
feeding
in n-3 PUFAs from fl¿x-fed rabbits was noted during
period. Gas chromatography
significant increases in plasma
AIA
revealed
as compared
A
the
that cholesterol/flax-fed rabbits exhibited
to all other fed rabbits (Iable 2). Plasma
from cholesterol-fed groups was easiþ distinguished from others by its cloudy colour.
Indicators of plasma cholesterol, as measured by the VetTest 8008, confirmed that the study
design promoted cholesterol absorption ìn fed animals. Total plasma
66
fary
acids, cholesterol
C14:0
4.293 + 0.01.4
14:1
0.111 + 0.001
+ A.247
0.403 + 0.024
2.240 + 0.147
10.693 + 4325
6.502
C16;0
16:1
C18:0
C18:1 (n9) Oleic
+ 0.148
+ 4.01.3
+ 0.0000
v.Jh/ + u-J+v
0.540 + 0.031
3.942 + 0.1.57
17 .455 + 0.862
+
0.267
0.0000
0.033 + 0.033
0.0000 + 0.0000
6.118 + 0.044
9364 + A.n2
+
2.048 +
0.001
u.+41 + u.uu4
0.006
3.643 + 0.186
0.354
1.644
11.191 + Q.343
11.781 + 4.513
12.328
C20:0
0.113 + 0.011
0.165
C18:3 (n6) Gr¿.
C20:1 (n-
0.000 + 0.000
0.115
1..779
A.AA2
ru-ltz l0.a
2.775 + 4.402
C] 18:1
4337 + 0.016
0.371.
tu
+ 0.064
+ 0.840
16.361
2.611
+
4.067
Vaccenic
ulð:l
(n-
+ 0.170
73.713
+ 4.490
6) I-c'
+ 0.009
+ 0.002
U.UUU
+
C18:3 (n-
1.993
+ 0.12a
]) AIA
LIU:Z (n
0.092 + 0.012
0.125 + 0.009
u.lbu +
0.112 +
0.000 +
0.000 +
u.ulz
0.032
0.000
0.000
0.000
0.214 + 0.004
U.UUU
0.117
0.000
0.000 + 0.000
+
+
0.001
4.1.59
+ 0.025
0.000
u.Lz5
t
u.uu4
0.000 + 0.000
0.000 + 0.000
+
22.535 + 0.679
9)
20.477
+ 0.847
119
0.036
0.082 + 0.002
0.106
A.D6 + 0.042
0.166 + 0.006
0.292 + 0.0A9
U.UY5
0.199 + 0.036
0.094 + 0.042
+
0.002
Áì
C22:A
C22:l
,ZUIJ
C22:6 (n3) DHA
+
0.000 +
0.105 +
+ 0.000
U.UUY
0.000
0.193
0.105
0.115
+
+
0.003
0.115
Table 1. Faay Acid Composition of Rabbit Diets
Fatty acid composition of rabbit diets was measured as mg of fatty acid methyl esrers (FAME) per
gram of diet + SEM. Abbreviæions: LA- linoleic acicl GL-A- gamma linoleic acid AIA- alpha
linolenic acid, DF{A- docosahexaenoic acid.
6'7
II'
T Regular Chow
M'10
CD
.Y
% Flaxseed
0.5% Cholesterol
w
I0.5%
.o¡
Cholesterol
10% Flaxseed
o
È
=3
-o
-o
G'
ú.
Figure
17.
Mean Rabbit Veights of Feeding Groups at 8 lü/eeks
Data shown represents weight in kg of rabbits fed 125 g/ day for 8 weeks. There were
no significant changes in rabbit weights after 8 weeks of feeding.
/
C8:0
+ 0.000
0.000 + 0.000
0.049 + 0.002
0.007 + 0-002
0.000 + 0.000
0.150 + 0.0i 5
0.019 + 0.003
0.002 + 0.001
0.000 + 0.000
0.149 + 0.010
0.130 + 0.030
0.000
C10:0
C72:0
Cl4:0
CL4:L
C16:0
C 6:7
C :0
C 1
C 8:0
C18:1 (n-9)
0.000
+ 0.000
+ 0.000
+ 0.002
+ 0.001
+ 0.000
+ 0.011
+ 0.001
+ 0.000
U.UUU
I
0.000
0.000
0.034
0.002
0.000
0.096
0.008
U.UUU
+ 0.008
0.089 + 0.009
0.109
u.uuo
I
u.uuu
U.UUU
t
U.UUU
+ 0.000
0.000 + 0.000
0.036
+
0.029 + 0.015
0.009
0.726 + 4.922
0.088 + 0.012
+
0.001
I
0.002
U.UUO
0.019 + 0.002
u.ul,/
I
o.uul
0.000
+ 0.000
+ 0.072\b
o'194 + 0'019 4ì'
0.042+0.0054b
0.003 + 0.002
0.284 + 0.0244b
U.UUU
+
U.UUU
A.798
4.795 + 4.062
U,UUU
4
0.737 + A.A1.4 4
0.040 + 0.003 c¿
0.000 + 0.000
A3$ + 0.029 4
u.uu+
t
u.uuu
a
0.516
+
0.152 4
0.48+
!
0.821.
+ 0.073 4
Oleic
C18:1
Vaccenic
C18:2 (n-6)
0.000
0.774 + 0.087
+t
¿
0'229 n"'o
75l +
4.228 4d
LA
+ 0.000
u.uuu
t
u.uuu
0.009
+
0.001
0.010 + 0.001
0.000 + 0.000
0.000
+ 0.000
U.UUU
t
U.UUU
0.000 + 0.000
C2O:1
0.000 + 0.000
U.UUU
I
U.UUU
0.002
+
0.001
0.000 + 0.000
C18:3 (n-
0.005
+
0.003
0.061
+ 0.010
u.1bl
I
u.uly
0.994 + 0.!95
()A:2
0.000
+ 0.000
u.uuu
I
u.uuu
0.013
+
0.002
C22:0
0.000 + 0.000
0.000 + 0.000
U.UUU
I
U,UUU
C20:3
0.000 + 0.000
0.000 + 0.000
0.+22
I
0.0+5 4b
+ 0.003
0.000 + 0.000
0.300 + 0.047 4d
C20:0
C18:3 (n-
0.000
6)
3)
AIA
r,'o
0.011
tt^mña
C.22:1
c2a3
|
+ 0.000
U,UUU
t
0.000 + 0.000
0.000
+ 0.000
+ 0.000
0.000 + 0.000
0.011 + 0.000
o'063 + o'oo5
0.000
0.000 + 0.000
0.000
U.UUU
0.008
+
0.003
74-17
I
u.uuj
C20:4
u.uzu
C.24:O
0.000 + 0.000
u.uuu
t
u.uuu
C20:5 (n3l
0.000
+ 0.000
U.UUU
t
U.UUU
C24:1
U.UUU
I
0.000 + 0.000
A2:6 (n-3)
0.000 + 0.000
U.UUU
U.UUU
I
0.053 + 0.003 cd
c1'
+ 0.000
0.000 + 0.000
0.000 + 0-000
0.060 + 0.002 a¡
0.068+0.005c¿
0.000 + 0.000
0.000
0.000
U.UUU
0.003 + 0.002
+ 0.000
T)HA
Table 2. Effects of Feeding on Plasma Fatty Acid Profiles at S Veeks
Fatry acid composition of rabbit plasma at 8 weeks 'was measu¡ed as mg of fatry acid methyl esters
(FAME) per mL of plasma + SEM. Abbreviations: LA- linoleic acid. AIA- alpha linolenic acid,
DFIA- docosahexaenoic acid Sratistica-l Significance: RG vs. FX, OL, CF
vs.CF:c;FXvs.CF:d
69
:
4 FX
vs-
OL
:
b;
OL
and
trigþerides in cholesterol-fed animals were significantþ higher than non-cholesterol fed
animals (Figure 18,
19). Substantiaþ higher
levels
of
famy acids \¡/ere present
in
bôrh
cholesterol-fed and cholesterol /flax-fed animals because cholesterol diets contained
additional fatry acids esterified
to cholesterol molecules. These results provided
evidence
that components of both fl¿xseed and cholesterol were digested and relativeþ enriched in
these subjects.
The fatty acid profile of the haruested dssues from both flax-fed rabbit groups
showed an incorporation of
Al-A and downstream metabolites. Brains from flax-fed rabbits
demonstrated significant levels of
3). Kidneys from the
AIA,
EPA, and docosapentaenoic acid (C22:5, n-3) (fable
same flax-fed anìmals also demonstrated an increase in the n-3 PUFAs
AI-A., EPA, and DFIA, and a concomitant decrease in
ffable +). This increase in
n-3 /n-6
6 PLIFAs are in competition
AA
(arachidonic acid C20:4, n-6)
ratio is consistent with our understanding that n-3 and n-
for the same en4¡rnes. Livers also had significant levels of
AIA
and EPA in both flax groups (I'able S). Both livers and kidneys had a striking increase in the
amount of oleic acid in cholesterol-fed animals from these tissues (Iables 4, 5). This could
be a compensatory reaction to cholesterol feeding since the cholesterol diet does not include
significant amounts of oleic acid as compared to the other diets (fable
1). Both non-
ischaemic/perfused hearu and ischaemic-perfused heans successfully included
AI-A into
their lipid fractìons. Non-ischaemic heans were significantly enhanced with AI-A and had a
small decrease in
AA (Iable 6).
Ischaemic-reperfused heans also showed an increase in
AI-A in the fla-x-fed group, but a similar increase was not observed in the cholesterol/flax
group (fable
'were seen
7).
Overall, the most significant incorporations of ALA (mgl per g tissue)
in liver; which was not suryrising since it is the major site for lipid metabolism in
animals. These data established that cholesterol a¡d flaxseed feeding in rabbìts changed the
r----j Regular Chow
r..:-¡ 10 % Flaxseed
G=
t!t
G\
Fd)
tt\
I
(s
-1
dø
rE
w
w
3
0.5 % Cholesterol
0.5 % Cholesterol /
10 % Flaxseed
'-
ó<
F
Figure 18. Total Plasma Fatty Acid Concentrations After
8
Veeks of Feeding
Total fany acid concentration of rabbit plasma at 8 weeks was measured as mg of latty acid
methyl esters (FAME) per mL of plasma + SEM. Statistical Significance: RG vs. FX, OL, CF
:4 FXvs. OL: b; OLvs.CF:c;FXvs.CF:d
1000
J
G\
tro)
tlt c
fttv
EE
GO
OU'
t- -9
o
uoo
o
0
Diet
Regular Chow
,:---i.10
% Flaxseed
ffi 0.5 7 Cholesterol
w 0.5 % Cholesterol /
10 % Flaxseed
'150
J
9ol
t/D 5
!ø
O- o)
IUU
tE':*=
È$
so
lp
F
0
Figure 19. Total Plasma Cholesterol and Triglyceride Concentrations after
8
Veeks Feeding
Total plasma choleserol (,\.) and trigþeride (8.) concentrations of rabbit plasma at 8 weeks
r.ere measured in mg / dL per mL + SEM. Statisricaì Sigruficance: RG vs. FX, OL, CF : a FX
vs. OL: b; OLvs. CF: c; FX vs. CF: d
72
+
CI4:0
0.0576
A.AA1.9
4.0672 + 0.0045
0.0638:ì 0.0028
0.0596
C14:7
0.0000 + 0.0000
0.0000 + 0.0000
0.0000
0.0000
C16;0
4.9853
+ 0.0983
+ 0.0046
5.6523 + 0-1319
4.9+30 + 0.1.01.6
C16:1
0.0961
Q.1,261,
+ Q.012
5.34U + 4.147
5.YJðU + U.Jb4?
+ 0.0000
4.8583 + 0.1328
0.L267 + 4.0057
).5JvJ t U.Z lU)
6.393r + 0.201
+ 0.0361
0.5502 + 0.0238 "
C18:0
C18:1
(".9)
tJ.U-tUð
ol
I
U.+ZUI
4.5909
0.1101
5.0987
5.7161
+ 0.0038
+ 0.0000
+ 0.1486
+ 0.0062
+ 0.1Á66
+ 0.2245
C18:1 Vac
L\ai¿ (n-b)
+ 0.0492
0.+22/ + 0.011,2
+ 0.4493
0.602i + 0.0536
(20:O
0.1104
+ 0.0149
0.1050 + 0.0130
0.1265
0.0000 + 0.0000
0.0000
0.4464 + 0.0568
0.5523
+ 0.0410
0.0068 + 0.0046 r',.
0.4
0.7217 + 0.0715
0.0992 + 0.0084
0.2+15 + 0.0748
1.L697
l-{
C18:3 ("
GIA
6)
C20:1 (n-9)
C1
8:3
+ 0.0690
0.0000 + 0.0000
0.1682 + 0.0489
0.0890 + 0.0102
0.1025
0.4630
(n-3
1.4937
1..2392
"
",b
+ 0.0113
+ 0.0000
0.9930 + 0.02+4
"
0.5873 + 0.0135
0.1009
0.0083
+
+
0.0095
0.0083
i63 + 0.0395
0.1091 + 0.0162 a.
AI-A
C20:2 ln-o)
C22:0
+ 4.4127
0.3607
t
0.0243
0.27+l + 0.Aß8
6)
0.1448
+ 0.0079
0.1637 + 0.0112
0.3230 + 0.02+4 "
0.1608 + 0.0106
C20:3 ("-3)
0.0000
+ 0.0000
0.0132 + 0.0056
A.AA67
(203
("
^
*
0.1834 + 0.0093
8-11-14
1t-14-17
C2A:4 ln-6\
+ 0.4467
0.0025
2.6983 + 0.1467
2.JJO0
2.90ß + 4.7a59
0.2586 + 0.0311
0.0000 + 0.0000
4.2346 + 4.4366
C24:7 (n-9\
0.4328 + 0.0712
0.3734 + A.A$1
0.4902 + 0.0488
C22:6 ("J)
2.2416 + 0.0967
2.3974
+ A.UA3
2.3382 + 0.0867
C24:A
C2A:5
(n-3)
2
.4139 + 0 .1.066
0.0280 + 0.0052
^
0.2987 + 0.0363
,"b
0.0A77
+ 0.0077 h
+
0.0025
+ 0.1220 ¿
0.2A48 + 0.024
0.0239 + 0.0056,
EPA
u.J)/ ð + u.u44b
2.3769 + 4.1369
D}14
Table 3. Effect of Feeding on Brain Fatty Acid Profile at 8lùíeeks
Fany acid composition of rabbit brains were measr¡¡ed as mg of fany acid methyl esters (FAME) per
gram of tissue + SEM. Abbreviations: LA- Iinoleic acid, GI-A- gamma linoleic acid, AìtA- alpha
linolenic acid, EPA- eicosapentaenoic acid DFIA- docosahexaenoic acid Statistìcal Significance: RG
vs. FX, OL, CF a FX vs. OL
b; OL vs. CF c; trX vs. CF = d
:
:
:
13
C14:0
0.0617 + 0.0096
C1.4:1
0.0000 + 0.0000
Ci6:0
C18:0
C18:l (n-9) Ol
0.1736 + 0.0228
3.2784 + 0.0847
3.1656
3.5885 + 0.1656
3.23V + A.ß60
5.1458
+
+ 0.0410
0.4540 + 0.0161
0.2819
+
5.7473 + 0.2484
J,Jr/'J + 0./U+¿
0.0824 + 0.0042
6.1.094
0.0813 + 0.0032
u.uyl+
0.0000 + 0.0000
0.0000
+ 0.0000
0.0154
+
0.0042
U.UUUU
0.0711 + 0.0058
0.4577 + 0.0026
0. 1344
+
0.0143
4.1279
+
0.01.14
0.2128 + 0.0148
0.8693
7.6J29
+
A.IA37
0.1010 + 0.0034
0.0817
0.1293
+
0.4087
0.1538 + 0.0023
0.1396 + 0.0083
0.1510
0.1393 + 0.0095 "
0.3299
0.5312
C18:1Vac
LA
C20:0
C18:3
o.2r73 + 4.432'3
U.U' Z+
+ 0.0062
+ 4.4440
0.0092
4.5937
(n-6)
+ 0.0852
C20:1 (n-9)
(n-l
+
0.0354
+
0.4949
0.4954
4.2853 + 0.0299
J .7375
+
4.2493
0.4045
4.652t3
+
0.3120
0.0558
4.5722
+
+ 0.2477
6.7557
+ 0.4430
t
0.1050
3.0982 + 0.0910
GI-\
C 18:3
+ U.U ILU
0.0000 + 0.0000
i.9677 + 0.251
0.7423 + 0.0377
b
C16:1
C18:2 ln-6)
3.5842 + 0.29L5
+ 0.0098
+ 0.0000
3.2856 + 4.2554
0.0596
0.0000
u.uu4u
0.3264 + 0.0483
0.0372
+
+
0.0082
U.UUUU
AI-A
C2A:2 (n-6)
'J2:0
-zU:J (n-b)
tt-74
C20:3 (n-3)
ð-
11
0.2227
+ A.A06l
+
0.0063
0.1
ß7 +
+
4.0174
0.1504 + 0.0104
0.0048
+
0.A17
|
0.2794
+
4.0ú7
0.0173 + 0.0447
0.1004
+
0.0095
0.0149 + 0.0061
0.1646
+
0.0106
3.6501 + 0.1163
2.8567
+
0.7216
3.5198
+
3.0396
+
0.'1117
+ 0.00/1
0.1188 + 0.0071
0.0884 + 0.0024
0.1207 + 0.0140
0.1024
0.1518 + 0.0084
0.2143
14-77
C2A:4 (n-6)
(24:0
LIU:5
0.0889
(n-J,
0.3071
+
0.0098
0.0585
0.07+7 + 0.0044
0.A957
0.1055 + 0.0034
0.5273
+
+
A.AA69
0.11l8 + 0.009!
0.0103
4.1.486
+ 0.0067
+
0.4155
EPA
(24:l (n-9\
L¿t:6
(n-J
+
0.0088
+
0.4740
4.1220
0.1360
+ 0.0a77
DFIA
Table 4. Effect of Feeding on Kidney Fafty Acid Profile ât S lVeeks
Fatry acid composidon of rabbit kidneys were measu¡ed as mg of fatty acid methyl esters (FAME)
per gram of tissue 1 SEM. Abbreviations: LA- linoleic acid GIA- gamma linoleic acid AI-4- alpha
linolenic acid EPA- eicosapentaenoic acid, DFIA- docosahexaenoic acid. Statistrcal Significance: RG
vs. FX, OL, CF : 4 FX vs. OL = b; OL vs. CF : c; FX vs. CF = d
74
C14:0
0.5726 + A.A473
CI4:7
C16:0
4.07c3 + 4.0469
8.129+
+
+ 0-7548
0.0840 + 0.0560
11.+887 + 3.9729
t.0584 + 0.3164
0.7067
1.12A2
0.9066 + 0.1838
C16:1
1766 + 4.4443
C18:0
C18:1
(n-9)
7.1323
+ 0.8955
7.7674
+
1.0342
9.0362 + 2.6771
1.2253
+
0.161.4
7.307 5
0.61i0 + 0.0210
0.5684 + 0.0330
+ 4.4576
10.8560 + 0./503
2.74L8 + 0.1769
U.Ut
0.2776
4.7677
+ 4.2547
ðl I
U.U454
I
ru.,/uvb
1.9986
+
u.)+u+
0.1489
5.5854 + 0.2602
17.2156 + 2.L619
19.8279 + 0.9423
2.3949 + 0.7765
2.1130
10.2335 + 0.5608
10.5330
ol
C18:1 Vac
C18:2
(n-6)
+ 0.2685
+
0.0851
10.3348 + 0.853:
11.1191.
0.0000 + 0.0000
0.0430
+ 0.0286
0.2L+3 + 4.0245
4.2337 + 0.A025
0.0000 + 0.0000
U,UUUU
1
0.0000 + 0.0000
0.0000
4.235+ + 0.0642
0.0000
+ 0.0000
0.4002
+
0.0089
0.0000 + 0.0000
0.7849 + 0.1.142
4.8023
+
1.2094
+ 0.0861
8.2242 + 4.4645
+
+ 0.0154
+ 0.0052
4.4328
+
0.0172
0.+139
43342 +
4.Q379
0.3438 + 0.0041
+ 2.0993
+ 0.3000
l-A.
C20:0
Ci8:3
(n-6)
U.UUUU
+ 0.0000
GI-A.
C20;1 (n-9)
C18;3
(n-3)
1.5053
AIA
C2A:2 in-6)
0.4353
4.432+
0.1844
C22:O
0.3885 + 0.0068
0.3546
(n-6)
N/m
N,im
\/m
N/m
(n-3)
N/m
\/m
t\/
.N,/m
( 2O:4 /n-6\
2.6550 + 4.1147
1.9899
C24:O
I\./ m
l\/m
C20:3
+
0.0137
8-1.1-1.4
C20:3
m
71.-14-1.7
C20:5
(n-3
+ 0.2397
^
1..7740
+
4.051.5
^
1.5160
N,/m
1\/m
+ 0.0699 .
v.¿¿Jb
!
0.uJ/'J
o.+uzb
t
u.uJð,/
0.0820 + 0.0418
0.4/65 + 0.0105
0.1529
+
4.4779
0.0504
+
0.0504
0.1531+ 0.0280
0.4678 + 0.0522
4.5675 + 0.0722
0.6582
+ 0.0545
EPA
C24:1 (n-9)
C22:6 (n-3)
DHA
r,
0.4415
+
0.0096
0.7555 + 0.0960.
Table 5. Effect of Feeding on Liver Fâtty Acid Profile at 8 lØeeks
Fatry acid composition of rabbit livers were measu¡ed as mg of fary acid methyl esrers fAME) per
grarn of tissue t SEM. Abbreviations: l-A- linoleic acid GLA- gamma Linoleic acicl Al-A- alpha
linolenic acìd, EPA- eicosapentaenoic acid, DHA- docosahexaenoic acid. Statistical Significarce: RG
vs. trX, OL, CF
:
4 FX vs. OL
:
b; OL vs. CF
:
c; FX vs. CF
15
:
d
C1.4:L
+ 0.0495
0,0585 + 0.0209
C16:0
5.J,/U+
I
U.5ðU
C16:l
C18:0
C18:1 ln-9) OI
C18:1Vac
(n e)
C
u./04,
I
u-v/64
C20:0
0.4658
C14:0
l8:2
IA
C18:3
(n-0)
C18:3
(n-Ð
+ 4.T44
0.2205 + 0.0713
9.6224 + 2.0754
1..8674 + 0.4046
3.4253 + 0.2994
ó.') / ¿! l.ðuð/
1.6044 + 0.i981
l-1.052
1.0768 + 0.2757
4.0707 + 0.0+77
+ 2.5926
t.5590 + 0.4546
3.7663 + 0.3697
9.8353 + 2.1973
1,.+345 + 0.V04
9.61,67 + 0.9072
10.4529
1.1909 + 0.116
J-br/5lo.Jo//
5.6605
+ 0.6167
+ 0.0206
7 .6573 + A3685
9.9385 + ?.2124
1.0551
L5121 + O.2\7 4
9.2899
+
1.1.469
4.0779
+ 4.4778
u-tzu,
!
a.vJ Iy
U.UUUU
I
U.UUUU
+
U.UUUU
u.uuou
I
o.uuuu
+ 0.07A4
0.44i2 + 4.0278
0.0000 + 0.0000
0.1469
+
0.0448
0.0000 + 0.0000
+
GIA
C20:1 (n-9)
+ 0.2428
0.1537 + 0.4552
1O.O+59 + 2.4056
1.4567 + 43301
1.1073
U.UUUU
0.0000 + 0.0000
Y.UU/J
a
t
l.Uyðer
0.1455 + 0.0440
4.1.742
o.t3243
+ 0.\270
4.8547
+
7.1819
1.1365
4.2648
5.0081 + 0.6255
0.1888
+ 0.0255
0.3550
+
0.017
0.3846 + 0.0188
4.2547 + 0.0096
AI-A
C20:2 (n-6)
0.ß24 +
C22:A
C20:3 (n-6) 87r-14
C20:3 ("-3)
t
0.3929 + 0.0150
U.+-tU/
N/m
N/m
N/m
N/m
1\/m
N,/m
N/m
1\/ m
A.A345
U.Uleìb
0.2691,
+ 4.0069
71-1.4-17
C2A:4 (n-6)
C24:A
(24:5
("-3)
+ 0.1080
J.Uy+,/
1\,/m
N/m
N/m
0.2430 + 0.0363
0.6789 + 0.0177
0.2981
+ 0.0000
03867 + 0.0473
0.0000 + 0.0000
0.0000 + 0.0000
+
0.6258 + 0.0148
3.5188
+
0.041
2.5688
1
U.UbðU
+
0.0651
2.8636 + 0.0806
l\,/m
0.6?64 + 0.4304
EP,\
C24:1 ln-9]l
C2
("-3)
2:6
0.0000
4.5932
0.0263
DHA
t
u.z+u)) t
u.uuuu
Table 6. Effects of Feeding on Fatty Acid Profile of Non-ischaemic Hearts at
Veeks
u.uuuu
u.uu),/
8
Fatry acid composition of non-ischaemic rabbit hea-rts were measu¡ed as mg of farty acrd methyl
esters (FAME) per gram of dssue 1 SEM. Abbreviations: l,\- Iinoleic acid, GIA- gamma lino.leic
acid, ALA.- alpha linolenic acid EPA- eicosapentaenoic acid DFIA- docosahexaenoic acid Statistica-l
Signficance: RG vs. FX, OL, CF : 4 FX vs. OL = b; OL vs. CF : c; FX vs. CF : d
76
+
A.2525
4.8945 + 0.7521
t.5OZl + 0.2564,
0.5988 + 0.0213
0.1839 + 0.0446
0.106i + 0-0319
0.2927 + 4.Q405
t).rJJy
+ 2.7792
1..6192 + 0.3017
4.0223 + 0.4841.
13.8393 + 3.0498
9.1968
L.+639
+ 0.1782
C78:2 (" 6)
+ 0.2759
12.0125 + 2.4462
9.6363
+
C20:0
0.1.69+
4.441.6
C18:3 ("-6)
0.0418 + 0.0284
0.1859 + 0.0484
0.0000 + 0.0000
U-JU/
2.0883 + 0.4853
5.8914
C20:2 (n-6)
0.3668 + 0.0228
(12:A
C14:0
1.2261.
C74:1
C16:0
12.5761.
C16:1
C18:0
C18:1 ("-9)
+
J
I
U.U¿66
i.5056
14.6030 + 2.5380.
5.813/ + 0.6835
+ 0.1591
3.5663 + 0.1053
2.2835 + 0.+6476'.
0.8232 + 0.081
4.1504 + 0.+247
2.8378 + 4.V71
1.7777
+
+ 2.9+0+.
6.1201
2.056+
+ A.2829 .
73.8677 + 2.0713,
0.9545
4.7349 + 0.0297
0.2424 + 0.0342
0.1497 + 0.0294
0.0000 + 0.0000
4.0441 + 0.0299
U.UUUU
1
V.A+í.J
0.0000
+ 0.0000
2.6473 + 0.5199
¿.6óJV
!
4.2387 + 0.042A 4b
0.J933 + 0.01I
0.2741 + Q.0239
4.2709 + A.A459
0.2607 + 0.0278
0.3500
("-6)
N/m
1\/m
N/m
N/m
("-3)
N/m
.L\/m
N/m
N/m
2.1031 + 0.0480
l.)+öz
1\/m
I
0.3146
+ 0.0400
u.55ðb
U.UUUU
I
U,UUUU
0.0000 + 0.0000
A.+918 + 0.Q171
0.4577 + 0.0381
14.8639
1.8124
16.5863
+ 4.6705
01
C18:1 Vac
1.8490
IA
+
1.t693
+ 0.0723
6.9836 + 0.5712
U.UUUU
GI-A
C20:1 (n-9)
C18:3 ("-3)
+
1.4354
/!
U,+/ t5')
AI,A
(20:3
+ 0.0149
v.¿iJó + u-ul+Y
8-11- 14
C2a3
11-14-17
C20:4 (n-6\
2.5435
A4:A
C2O:5 (n-J
N/m
N/m
0.3192 + 0.0533
0.5571
!
0.0217
C24:7 (n-9\
0.0000 + 0.0000
u.uuuu
t
u.uuuu
C22:6
0.5n4
+ A.U99
u.luJ)
2.1559 + 4.1830
N,/m
I
U.Urty4
EPA
(n-3
!
0.0079
0.5001 + 0.0268
DFIA
TableT. Effects of Feeding on Fâtty Acid Profile of Ischaemic-Repedused Hearts ât
8
Veeks
Fatty acid composition of ischaemic-reperfused ¡abbit heans were measu¡ed as mg of fatty acid
methyl esters (FAME) per gram of tissue + SEM. Abbreviations: LA- linoleic acid, GIA- gamma
linoleic acid AJA- a.lpha linolenic acid, EPA- eicosapentaenoic acid" DHA- docosahexaenoic acid
Statisticaì Significance: RG vs. FX, OL, CF : 4 FX vs. OL : b; OL vs. CF : c; FX vs. CF : d
fatry acid compostion of major organs; especialþ heans from which CSR were isolated.
CFIARACTERISATION OF CSR
stufi
Before analysis of CSR ìsolated from the above
characterised
for
control CSR vesicles were
specific protein content, Ca2u transport propefties, and fatry acid
composition. The SR proteins
Ry\
CSQ, and SERCA were sparse in CSR as compared to
skeletal SR vesicles. CSR vesicles were compared
to HSR
stained with Coomassie brilliant blue R-250 (Figure
20). Qualitative
vesicles
in a SDS-PAGE
gel
observarions revealed
that the CSR preparation was relativeþ devoid of RyR monomer (550 kDa) and CSQ (aa
kDa) opposed to
HSR
SERCA protein bands (110 kDa) were visible in the CSR lane, but
were less abund¿nt than HSR
CSR were also characterised
for
responses
to known
SR Ca2* transpon affectors.
Pharmacological agents acdng on SERC"A,, but not RyR nor IPrR affected Ca2* transport.
Ca2* transpon assay was refined
simultaneous monitoring of up
to
in a microplate
8 assays and
spectrofluorometer
a
the
to accommodate the snall feld of CSR (see
Method$. Potassium oxalate was used to facilitate conductance of
in
to allow for
A
Ca2* into the SR lumen
timely fashion, since CSR vesicles had very limle CSQ. Figure 21 shows that CSR was
unresponsive to the RyR inhibitor ruthenium red (Panel
A).
Moreover, RyR activating
concenûations of ryanodine produced only a slight delay in
Ca'?*
uptake (Figure 21, Panel E).
Ca2* transpon
in isolated CSR vesicles was not responsive to changes in RyR conductance
induced by RyR ligands, RR and
ryanodine.
IP,,R antagoniss, xesrospongin C and heparin
had no effect on Ca2* tr¿nsport, which indicated rhar these CSR preparations were not
enriched
in IPrRs. (Fìgure 21, Panels C and D). As
antagonist inhibited
Ca'?*
expected, thapsigargin, a SERC,A
uptake by these vesicles (Figure 21, Panel
18
B). The
Ca2*
transpon
1234567
I
g 10
Ë'#-@
"rn
l\
r=*"o
(110 kDA)
+
(44kDa)
'
Figure 20. Comparison of CSR and HSR by SDS-PAGE Gel
Lane 1-10 respectiveþ represent HSR, HSR, CSR, CSR, Pellet 1, Pellet 2, PelÌet 3, Supernatant
Supematant 2, and Super:narant 3 as sampled from CSR isol¿ion.
1,
"t
,.1 Ì..,
ìl
"
;i'l \\ \..-ì.
-
"l
x
cônt'or
,çì:i;:ìi:;"..-j:"r-ì.,-:,,r:!.:,
{,
i,l
10!MtìaÞsqafqin
g
,
5 PM
,
Rurìen'umFPd
r':¡"_
l
i----- -.ü------;,
rift
"i,
{seco.ds)
i\,
t,,
.ì,,00."/.1,,".,,"
!:
ì'E.l
: rouM
\
\
\
l''
I
'\
,\-.-_
".",.
,1"-...-"-.,.*.-
E.
..1
'\
,.]
I
rim{*conds)
Figure
21.
Characterisation
of
Extraluminal Ca'?* Transport by Isolated CSR
Vesicles
HSR membra¡es (0.25 mg/mf) were pre-treated with varying concentrarions of A. ruthenium
red B. thapsigargin, C. xestospongin, D. heparin, E. ryanodine. Ca2* transport (CG-2 trace$
q'¿s monitored as described in "Methods". tanspon çr'as initiated by the combined addition of
Mg.ATP and PEP.
data suggests that CSR vesicles isolated in this manner and under these conditions (see
Method$ are responsive to alterations in SERCA acdvity, bur not channel conduoance.
CSR from both non-ischaemic and ischaemic-reperfused rabbit heaÍs fed one
four diets were characterised to
see
if
ofthe
dietary aherarions in lipid profile could modifr their
function. CSR were isolated from 8-week rabbit heans and
assayed
for protein content
recovery as an indication of the comparability of the prepararions. Diet did not affect the
leld of CSR
(mg CSR
/
g hean), but perfusion status
did.
Isolated CSR vesicles from non-
ischaemic reperfused heans had similar yields between all feeding groups (Figure 22, Panel
B). Likewise,
CSR yields were similar amongsr ischaemic-reperfused heans (Figure 22, Panel
B). However, protein
recovery of CSR from ischaemic-reperfused heafis was significantþ
reduced compared to CSR from non-ischaemic reperfused heans (P<0.05) (Figure 22, Panel
A)
To measure if dietary
vesicles, gas chromatography
fla-rseed changed
the fatty acid composition of isolated CSR
anaþis was performed on erlracred lipids. Fatry acid analysis
of CSR from non-ischaemic heans exhibited a significant increase in the contenr of
and its metabolite EPA in CSR from both groups
analysis
AIA
of flax-fed rabbits (Table 8). Fatty acid
of isolated CSR from ischaemic-reperfused heans also indicated that AIA, EPA,
and even DFIA (in the 10olo flax/ O.5o/" cholesterol group) were incorporated into flax-fed
animals (Iable 9). Hence, dietary fl¿xseed supplementation successfully increased the n-3
PIIFA content of isolated CSR vesicles from rabbit and v¡as not altered bv
ischaemia-
reperfusion.
'lüe
were also interesed in the exrent to which cholesterol was ìncorporated inro SR
membra¡es during feeding. For the most pan there is a trend of increasing amounts of free,
esterìfìed, and total cholesterol in CSR vesicles from rabbits fed cholesterol, although tliese
050
Ë
IE
ae m i c
lT,----*1
No n- i sc
M
lschaemic
h
ro)
ú,
É.
Ø
õ)
E
0.00
r---r Control
wza 10ok Flax
w0.5 % Cholesterol
w0.5 % Cholesterol /
10 ok Flax
Non-ischaem¡c
Figr:re 22. Yield of CSR from Feeding Groups
A. Comparison of CSR yield between CSR isolated from non-ischaemic hea¡ts and ischaemiareperfused hearts (mg SR,/ g heart). " P < 0.05 B. Relative comparison of CSR yield from nonìschaemic hearts and ischaemia-re perfused hearts from rabbits fed similar diets, measured as o/o
control (regular chow diet). Not sigruficantþ different.
0.0000 + 0.0000
0.0000 + 0.0000
0.0000
+ 0.0000
+ 0.0018
0.0129 + A.A026
0.0694 + 0.0013
0.0456 + 0.0020
0.0000 + 0.0000
0.0610
0.0498
+ 0.0000
+ 0.0000
+ 0.0020
+ 0.4422
+ 0.04ß
+ 0.0016
+ 0.0038
+ 0.0041
0.0280 + 0-0036
0.0460 + 0.003i
0.0121
+ 0.0005
0.0190
+ 0.0007
0.0183 + 0.0011
0.i404 + 0.0046
0.1388
+ 0.0030
0.1535 + 0.0052
+ 0-0000
0.0000 + 0.0000
0.0000 + 0.0000
0.0000 + 0.0000
0.0000 + 0.0000
0.0000 + 0.0000
C14:0
0.0000 + 0.0000
0.0000
CL4:7
0.0000 + 0.0000
0.0629 + 0.4424
0.0000
C16:O
C16:1
0.0098
+ 0.0013
0.0731 + 0.0020
0.0511 + 0.0020
o.AIn
0.0192 + 0.0006
C18:0
C18:1 (" 9)
0.0602
0.0709
0.0645
0.0042
ol
C18:1 Vac
C18:2
IA
(n-6)
0.1486
+
0.0038
C20;0
0.0000 + 0.0000
C18:3 (" 6)
0.0000 + 0.0000
0.0000
GI.,\
C20:1 (n-9)
0.0000
c 18:3 ("-3)
0.0056
+ 0.0000
+ 0.0001
0.0000
+ 0.0000
o'0161 + o'ooo4
0.0000 + 0.0000
4 r'
0.0018
+
0.0008
U,UUUU
1
U.UUUU
q.
U.UUUU
I
U.UUUU
0.0162
+
0.0006 "
U,UUUU
t
U.UUUU
AI-A
C2a:2 ln-61
0.0000 + 0.0000
(22:A
0.0000 + 0.0000
C20:4 (n-6)
C20:5 ("J)
0.1040
+
0.0029
+ 0-0000
0.0000 + 0.0000
0.0280 + 0.0022
U.UUUU
t
U.UUUU
0.0102
0.0000
I
0.0003
0.0000 + 0.0000
0.0001 + 0.0001
0.0903 + 0.0019
0.0892 + A.AA47
0.0000
+ 0.0000
0.0108
+
0.0005
EPA
C24:1 (n-9)
0.0000 + 0.0000
Llli6
DHA
u.uu5z
(n-J,
I
u_uuu+
+ 0.0000
0.0041 + 0.0001
0.0000
0.0000 + 0.0000
0.0000 + 0.0000
0.0038 + 0.0006
0.0048 + 0.0006
8. Effects of Feeding on Fatty Acid Profiles of CSR from Non-ischaemic
Hearts at 8 \/eeks
Table
Fatq' acid composition of CSR was measu¡ed as mg of fatty acid methyl esters (FAME) per gram of
1 SEM. n-3 PUFA antøx of CSR lipds rtun Jkx-fd ral*ß itlcrusad sigaifaffitlj, ìnclding ALA
(C18:3) anÅ iß mdakl;re EPA (C20:5), possib[, at the exparc of AA (C20:4 2,6/. Abbreviarions: LAlinoleic acid GIA- gamma linoleic acid, AI-A- alpha linolenic acid, EPA- eicosapentaenoic acid,
DHA- docosahexaenoic acìd. Statistical Signìficance: RG vs. FX, OL, CF : 4 FX vs. OL = b; OL
vs. CF = c; FXvs. CF : d
tissue
83
C14:0
0.0000
C1,4:1
0.0000
C16:0
Cl6:1,
C18:0
0.0526
0.0000
C18:1 ("-9)
0.0389
+ 0.0000
+ 0.0000
+ 0.0020
+ 0.0024
+ 0.0024
+ 0.0012
0.0123
+
0.0000 + 0.0000
0.0000
u_uuuu
0.0000 + 0.0000
0.0000
0.0641 + 0.0063
0.0552 + 0.0037
0.0534
+ 0.0018
0.4776 + A.AA65 ^
0.0486 + 0.0038
u.uuuu
1 u.uu5j
u.ubjz t u.uuj+
0.0092
0.0J9 + 0.0024
0.0388
0.0008
a.o2r5 + o.ao27
0.0182 + 0.0013
0.0152 + 0.0001
0.1121 + 0.0042
0.1384 + 0.0121
0.1191
+ 0.0056
0.1393 + 0.0019
t
0.0000
0.4594
ol
C18:1 Vac
C l8:2
IA
("-6)
t
U,UUUU
U.UUUU
u.uuuu
I
0.0036
+ 0.0000
+ 0.0000
U.UUUU
+ U,UUUU
0.0002 + 0.0002
U.UUUU
1
U,UUUU
u.ulðu
t
u.uulu
C20:0
0.0000
C18:3 ("-6)
0.0000
U,UUUU
0.0000 + 0.0000
0.0/02
+ 0.0000
+ 0.0000
+ 0.0027
+ 0.0035 ^
+ 0.0030
+ 0.0021
+ 0.0000
0.0000
0.0000 + 0.0000
0.0000
+ 0.0000
0.0000 + 0.0000
0.0000 + 0.0000
0.0000 + 0.0000
0.0000
+ 0.0000
0.4779 + 0.00+A
0.0000
+ 0.0000
+ 0.0000
GI-A.
c2jt
/n-9\
U.UUUU
C18;3 ("-3)
AIA
C20:2 (n-6)
U.UUUU
C22:O
0.0000
C2A:4 (n-6)
C20:5 ("-3)
:!
+
U.UUUU
0.0000 + 0.0000
0.0000
u.uuuu
I
u.uuuu
4.4740 + 0.0024
0.0/85 + 0.0069
0.0000 + 0.0000
0.0082
+
0.0000
0.0000
0.0148 + 0.0007
+ 0.0000
+ 0.0000
4.0748 + 0.0026
I
0.0011
0.0000
+ 0.0000
0.0000 + 0.0000
U.UUUU
I
U.UUUU
0.0000 + 0.0000
0-0009 + 0.0004
0.0000
+ 0.0000
0.0027 + 0.0004
u.uuuyl
u.uuuy
EPA
C24:1 ln-9\
(22:6 (n-Ð
0.0000
0.0003
+ 0.0000
+ 0.0002
DFIA
Table
9.
Effects
of
Feeding on Fatty Acid Profiles
of CSR from
Ischaemia-
Reperfr.rsed Hearts at 8 'JØeeks
Fatry acid composition of CSR q'as measu¡ed as mg of fatry acid methyl esters (FAME) per gram of
tissue t SEM. n-3 PUFA cøttmt of CSR lpuls fon fkx-fe! r¿rWzts Ìnoeßøì signifrÁntb, atclutlìng ALA
(C18:3) anà i¡s nulnli¡es EPA p20:5) and DHA (C22:6). A66rev'Lations: l-4- linoleic acìd GIAgam,rna linoleic acid, AI-A- alpha linolenic acid, EPA- eicosapentaenoic acid, DF{A- docosahexaenoic
acid Staristical Significance : RG vs. FX, OL, CF : 4 FX vs. OL : b; OL vs. CF : c; FX vs. CF =
¿
84
results are not significârìt.
A colorimetric
assay was adapted
to meâsure both free and totai
cholesterol, and consequentþ esterified cholesterol in a microplate reader (see Method$.
Cholesterol content of CSR isolated from rabbits fed a flax, cholesterol, or cholesterol/ flax
supplemented diets are shown
in Figure 23. There were not any significant changes in
cholesterol composition of CSR from non-ischaemic heans (Figure 23, Panel A). Within the
reper{used-ischaemic group, CSR from cholesterol
/
flax-fed rabbits had significantþ higher
amolùrts of cholesterol over control and fla-x fed rabbits (Figure 23, Panel
B).
reperfused-ischaemic heans had significantþ lower levels of total cholesterol
tha¡ CSR from
non-ischaemic heans
increase
(P:
CSR from
0.0156), so the groups were not comparable. There was a general
ìn cholesterol content in all cholesrerol-fed animals; however rhe trend was onþ
significant in one case.
After deternining that the CSR lipid composition q/as altered by changes in diet, we
assessed
their response to
Ca'?*
handling. Extraluminal Ca2* uptake of CSR isolated from
rabbits fed a flax, cholesterol,
or
cholesterol/flax supplemented diet from both non-
ìschaemic and ischaemic-reperfused hea¡ts was monitored using a novel microplate assay
þee
Method$. The
A.
Ca'Z* uptake
averaged values of Ca'* upake are shown as traces
in Fìgure 24, Panel
by non-ischaemic CSR was not affected by diet, but Ca':* uprake by
reperfused-ischaemic CSR was changed.
Figte
24, Panel B compares rhe râte constants
of
extraluminal Ca2* uptake as determined by exponential decay in Graphpad Prism software.
A""lytit of these
rates revealed that neither flaxseed nor cholesterol supplementationto the
diet altered Ca2* transpon in CSR isolated from non-ischaemic heans. However, a srriking
improvement in Ca2* transpon was shown in CSR from hearts of fla-rseed-fed rabbits that
were subjected
to
ischaemia
reperd:sion. This may suggesr rhar flaxseed feeding may
improve Ca2* handling in CSR after ischaemìa-reperfusion
É.
Ø
o,
õt-
o)
IJ'
-9
o
,c
o
E)
É.
(t)
ct
0.04
õL
Iu, nnz
- --
Reperfused-ischaem ic
t:- Regular Chow
t::10% Flax
@r 0.5 % Cholesterol
@ 0.5 % Choleste¡ol/
10 a/"
Fla\
_9
!
o
o
ct
0.00
23.
Cholesterol content of CSR isolated from rabbits fed a fla-x,
cholesterol, or cholesterol / flax supplemented diet frorn both non-ischaemic
and ischaemic reperfused hearts
Figure
CSR ìipids were ertracted in CHCII : MeOH a¡rd assayed for frce and total cholesterol
content as in'Methods'. No significant cbanges in cbolesterol composition of CSR
from non-ischøemic hearts zoere obsetøed. CSR from reperfused-iscbaemic bearts
bad significantly lozøer leuels of total cholesterol thøn CSR from non-íschaemic
bearts. V/itbin tbe reperfused-iscbaemic group, CSR from cholesterol / flax-fed
rabbits had significdntl! bigher amounts of cholesterol oøer control and flax fed
rabbits.'t P < 0.A5
86
Reperfused-ischaem ic
Non-isc haem ic
=
+
ô{õ
o
=
+
-__
ð/5
(g
o
500
0
0
1000
500
Time (seconds)
Time (seconds)
2.5x1O'æ
r---¡ Regular Chow
r,.::¡ .10% Flax
Ma
(ú^
w
9':o
ö8
s3
(ú-
1000
1
.3x10'02
0.5 % Cholesterol
0.5 % Cholesterol /
10 o/o Flax
É
0.0x'10
Non-ischaemic
lschaemic-reperfused
Figure 24. Panel A. E)'-traluminâl Ca'?* uptake by CSR isolated from rabbits fed a flax,
cholesterol, or cholesterol / flax supplemented diet from both non-ischaemic and
ischaemic-reperfused hearts, Mean values of oxalate-dependent uptake of 5 ¡rM Ca'?* by 0.125 mg
mL CSR æ 37 € are shown. As described in 'Methods' Ca2* tra¡spon was initiated by the combined
addition of MgATP, CP and K*-oxala¡e. Y-axis represents free extraluminal Ca2* as convened from
Fu¡a-2 ratio. Panel B. Comparison of rates of extraluminal Ca2*uptake by CSR isolated
from rabbits fed a flax, cholesterol or cholesterol / flax supplemented diet from both
non-ischaemic and ischaemic-reperfused hearts. lVhile there were no significant differences in
rhe rates of Ca2*uptake within the non-ischaemic group, flax-fed ischaemrc-reper{used CSR sequestered
Ca2* at a significantþ higher rate than all other groups (p < 0.00Ð.
/
87
This may suggest that flaxseed feeding may improve Ca2t handling in CSR after ischaemia-
reperfision.
SERCA activity in these preparations of CSR vesicles was also srudied. The rate of
SERCA activity as measured in nmol
NADH/
ischaemic reperfused preparadons (Figure
minute.mg SR was similar
25). This was in
agreemenr
in the
with the CSR
nondata
that showed no change in Ca2* transport râtes. Overall, the ischaemic reperfused-group had
higher rates
of ATP hydrolysis compared to the non-ischaemic reperfused
groups.
However, the râtes of NADH oxidation were significantþ slower in the rwo flax-fed groups.
A discussion of these results follows.
88
r---l Control
É,
U'
F:-;r 10% Flax
0.5% Cholesterol
w0.5% Cholesterol
loo/o Flax
ffil
õ,
E
-c
Oc
z
Stêtistical Signif icance:
Non-ischaer¡c CF vs.
l'lon-ischaemic OL = a
lscahenic-reperf used RG, OL
vs. lschaerúc-reperf used FX,
o
E
C,
CF= b
Non-ischaemic
lschaemic-Reperfused
Figure 25. Comparison of SERCA catal¡,tic activity between CSR isolated from
rabbits fed a flax, cholesterol, or cholesterol / llax supplemented diet from both
non-ischaemic and ischaemic-reperfused hearts. NADH fluorescence decay was
used as a measure of SERCA câtabnic activiry by 0.010 mg / rJ- CSR ar 3/ oC, see
Methods'. Specific SERCA2a activity was determined in the presence of the Ca2*
ionophore, 4-bromo-A23787 (10 pM) and SERCA inhibitor, Thapsigargin (10 rrÀ4). CSR
from perfused-ischaemic hearts showed significantly faster cataþic activity þ < O.OO1)
than CSR from non-ischaemic heans regardless of diet.
89
IV. DISCUSSION
\X/e sought
to elucidate the effects of dietary flaxseed, enriched in n-3 PUFAs, on
function. Our experimental
design examined both free fatty acid
a¡d esterfied Íany
SR
acrd
effects; by exogenous addition of n-3 PUFAs to isolated SR preparations and isolation of SR
from animals fed diets high in n-3 PlIFAs, respectively. Both srudies suggest that n-3
PLIFAs improve SR function. Unesterified n-3 PLIFAs enhanced Ca2t sequestration of the
SR at least in pan by impairing Ca2* efflux via RyRs. Esterified n-3
Ca2n uptake
PllFAs also improved
in isolated SR vesicles, but onþ afier ischaemia-reperfusion. The implìcation of
how these fatty acids act pertains to their potential clinical relevance as anti-arrhythmic
agents and their recommended route
of
administration as nutraceuticals
or
dietary
supplements.
One theory reasons that n-3 PfIFAs prevent arrhJthmia in their free
form. A
number of studies suggest that FAs may simpþ panition into the lipid membrane to exert
antiarrhlthmic effects because these actions are rapidly reversed with the FA chelator BSA
[245]. This could mea¡ that
esterified
these acting n-3 PUFAs are not covalentþ bound nor are
to any membrane compontenß [245]. A long-chain FFA
thry
mechanism may be
physiologicalþ releva¡rt since they are liberated from phospholipids during ischaemia by
phospholipase
A,
1245, 268-2711. AJso, mitochondrìal fatcy acid oxidation leads to
accumulation of C16 ând Ci8 chains a¡d camitine derivatives in q,tosol, which may explain
pump failure in ischaemic hean
127 7-27
31. In fact q,tosolic FFA concentrations may reach
up to 20 pM at which point we would expect to see direct effects on SR function l2Z4l.
Unesterified n-3 PUFA effects on SR funcdon generally point towards an inhibiton
of RyR
Ca'z. release
[258]. These SR effects are manifested in permeabilsed ventricular
90
myoc)'tes by decreased frequenry, velocìty, and amplitude
of contraction [?56,257]. Our
initial studies also predicted that n-3 PllFAs reduce the open probabiliry (PJ of RyRs in
isolated HSR vesicles. lX/hen HSR vesicles were pre-treated
with relativeþ
low
concentrations of n-3 PUFAs v¡e observed an increase in the rate of Ca2* uptake without an
increase in the corresponding
catal¡ic activity. This is consistent with improved coupling of
Cat* transpon attd/ or a decrease in the RyR P,,. More efficient rycling of Ca2* into HSR
vesicles per mole of
increasing the rate
AT? would increase the rate of Ca'* sequestration without
of ATP hydrolysis.
Reagents known
necessarily
to improve coupling can act by
inhibiting a tonic leak of Ca2* out of the SR. For example, the knov¡n RyR antagonis
ruthenìum red SR) produces a sharp increase in the rate at which the SR accumulates Ca2*
without increasing SERC,A âctivity. It can be reasoned that RyR antagonists increase
Ca2*
uptake by blocking the inherent leak of Cat* out of the SR. Similarl¡ improved coupling
Catn transpon may arise
of
from a decrease in the mean open dme of RyRs. llence, under
these conditions, n-3 PllFAs appear
to inhibit RyR leaks in a similar
manner to
pharmacological RyR antagonists.
At relativeþ higher n-3 PIIFA concentrations, we observed
rates,
a decrease in Ca2* uptake
while SERCA1 activþ increased. This may be due to uncouplin g of Cal* transpoft or
an increase in RyR P. may explain this effect.
A
leak of Ca2* out of the SR would result in
futile rycling as can be seen with Ca2* ionophores Iike ionomycin. Likewise,
leak would also decrease coupling
a RyR-mediated
of Ca'* sequestration to ATP hydroþsis. The
general
effect of these n-3 PIJFAs appears to be improved coupling below a threshold, a¡rd above
which the ratio of Ca2* Íanspon to ATP hydroþsis decreases. This is consistent with the
idea thar ampiphìles stabilize membra¡e lipids at
lipids at high concentrâtions Í275-2771.
9l
low concentrations and disrupt membrare
Oxidized forms of both I-A and AA have been shown to decrease the rate of Ca2*
accumulation
by SR vesicles independentþ of SERCA or detergentlike
e[fectsl278f.
Flowever, our n-3 PIIFA prepârations were purged with nitrogen gas to attenuate their
oxidation. Altemativeþ palmitic acid (C16:0), and not oleic acid (CtS:t), enha¡ced
Ca2*
uptake in isolated light skeletal SR fractions [27L-273]. \T,4rile these preparations wouid not
be enriched in RyRs, they still provide insight into a possible mecha¡ism of action
for fany
acids. They too hlpothesised that enhanced Cat* uptake could be due ro an alteradon in
SERCA activity, leak status of the vesicles, or both factors [2/1].
To account for the increased rate of
relatively low amo¡.rnts
Ca'?*
uptake in HSR vesicles in the presence of
of n-3 PLrFAs, we funher examined the possibility that intrinsic
SERCA1 àctiviry fray have increased thereby ìncreasing the rate of Ca2* uptake. Under
completeþ uncoupled conditions
increase in the rates of
of
Ca2* transport, n-3 PUFAs induced
NADH oxidation
as a marker
a slight
5-1Oo/o
of SERCA1 activity þ<0.001). This
indicated that n-3 Pl,IFAs might ìnfluence SERCAi kinetics by ligand-mediated actions on
the pump dìrectþ. However we reasoned that a faster rate of ATP hydroþsis did not
account for increased Ca'?* uptake, since others have shown rhat even though SERC,A
acdvity was stìmulated by more than 75o/o with palmitic acid, less than lo/o of palmitic acid
was associated with the annular layer, so it was not reasonable
þ
suggest
that improved
SERCA activity accounted for increased Ca2n trarsport 1279, 2801. Funhermore, palmitic
acid onþ increased the rate of E2P decay and did not affect phosphoen4nne intermediate
conformations of the pump [280].
An ahemative hlpothesis to our observations in Figures 2-5
s¡as that n-3 PllFAs
inhibited RyR-mediated leaks. To examine this h1'pothesis we used a []Fll-ryanodine binding
assay. The data indicated that n-3 PllFAs decreased []F!-ryanodine binding in
a
concentradon-dependent marìner. The results of this experìmenr did not necessari.ly display
competitive binding berween []Fll-ryanodine and n-3 PUFAs, bur perhaps a cha:rge in the
functional characteristics of RyR such that
it
decrease ìn [3F!-ryanodine binding which occurs
could no longer bind []Fl]ryanodine. A
in the presence of RyR channel inhibirors
may indicate that n-3 PUFAs decrease RyR P. [67].
Observed effects of n-3 PUFAs on RyR P" are supponed in the literature. Rodrigo
posfulated that an observed decrease in contrâcdon in permeabilised ventricular myoc¡tes in
the presence of EPA s¿as due to an increased threshold for Ca2* release from the SR or
blockade
of SR membrane K* channels that maintain electrical homeostasis 12561. The
second hlporhesis may be discounted on rhe basis that valinomycin, a known K*-selective
ionophore, has no effects on the HSR Ca2* transport [281]. Funhermore, both EPA a¡d
DHA have been shown to inhibit
ventriculâ-r myoq'tes
[257].
spontaneous Ca2* release causing contraction of
Subsequent studies by
ONeill indicate that these n-3 PUFAs
inhibit the SR Ca2* release mechanism [258].
To funher examine the h1'pothesis that n-3 PUFAs inhibit RyR we investigated their
effects upon CICR We chose to examine DHA, since
potent of the n-3 PtIFAs
[282].
'ùØe
it
has been shown
to be the most
observed a concentration-dependent inhibirion of
Ca'?*
release. Like the Ca2* transporr traces, the response of HSR vesicles to Ca2* pulseJoading
with DHA was similar to the ruthenium red-response, which also increased the threshold for
Ca2* release (Figure
15). A contrary effect was observed when pulseJoading HSR with
higher concentrations of
rate
of
re-accumulation
DHA (Figure
of
1a Panel
B).
Ca2* release v/âs potentiated, bur rhe
Ca2* was unchanged, as indicated
by the slope of the
Ca2*
tra¡sient. These results suggest that RyR kinetics, but not SERCA kinetics, were altered by
n-3 PtIFAs. This is consistent ¡vith results of a study by Leifen in which aÐ'nchronous
93
contractility of ventricular myoq.tes was decreased by feeding n-3 PLIFAs. This study also
showed that changes in the decay rate of the Cat* transient were independent of SERCA a¡rd
SR Ca2* content
RyRs.
AA
[283]. AdditionaÌ data concurs that FAs may alter subconducürce states of
(20 50 FLQ, but
not 5 tM A,{, has been shown to induce panially RR-sensitive
Ca2* release
from CSR [284]. Also, oleic acid and not palmitic acid accelerated spontaneous
Ca2* release
from
SR vesicles [280].
The mechanism by which unesterified n-3 PUFAs exert their action on the SR is not
well defined. Unesterified n-3 PUFAs most likeþ act by panitioning into the hydrophobic
domains of SR lipid" panicularþ the discrete TM domain regions of ion channels, since its
effects on SR function are reversed ìn the presence of BSA [257, 285]. Furthermore, n-3
PLIFAs may act by increæing membra¡e fluidity [282, 286]. Electron microscopy ìndicates
that FFAs that delay Cat* uptake may increase membra¡e permeability by solubilising SR
membra¡re vesicles [279]. However, FFAs that enhanced Ca2* trarspoft had no affect on SR
membrane morpholory, which suggests that these lower concentrations do
membrane packin
gl279l. An ncrease in fluidity
to Ca'*. Both pumps and
of the SR
channels may malfunction under such high
concentradons. At lower concentrations, n-3 PUFAs may facilitate
as
alter
due to higher concentrations of n-3 PUFAs
may disrupt normal membra¡re protein function or increase the permeability
membrane
not
Ca2*
trânspon by acting
Ca':*-precipitating anions or buffers in the SR [229 ,280,2871. FFAs that improve SR Ca'z*
tÌìansport form aggregates
exchangeable Ca':* pool
in
response
to high
Ca2* concentrations and create
V8A,2871. It is reasonable then to
a poorþ
suggest that n-3 PLIFAs may
uncouple SR Ca2* transporc by increasing membrane fluidity and permeabiJity to the point of
dysfunction in the range of 25-100
¡M.
Howeve¡
94
a more specific effect may occur at
low-
end n-3 PLIFA concentrations that decrease RyR P" possibly by decreasing the free pool ol
SR Cah.
The physiological ex-tension of these findings could find its place in the attenuation
of arrhl.thmogenesis. rü4ren incorporated into phospholipids n-3 PLlFAs, acñated by PLA,
may mediate enla¡rced sequestration of Cat* durìng ischaemia [288,
289]. In
the
cardiomyoq'te this would lower resting q.'tosolic Ca'* and thereby decrease the probabilty
of spontaneous Ca'*
release
from the SR. Additionall¡ decreased RyR P. would increase the
threshold for Ca2* release from the SR and possibþ prevent unwanrcd Ca2n release, since
ischaemic SR is less effective at sequestering Ca'* due to Cat* efflux occurring at RyRs [290].
Our interests in the potential for creating a store of n-3 PfIFAs available to the SR
lead us
to pursue a flaxseed-supplemented dietary model from which we could
assess the
function of isolated SR vesicles. Flarseed enriched with the n-3 PLIFA AI-A was chosen
over sources of EPA a¡d DFIA because it has a bemer taste, odor and is more resistant to
auto-oxidation [245]. However, AI,A may not be stored as well as the longer-chain n-3
PlIFAs, since it is metabolised more readtly 1297,2921. n-6 PlÆAs have also been shown to
be less potent antiarrhlthmic agents because they can forrn pro-arq.thmic metabolites [282,
293). A""lyrir of rabbit tissues confirmed that changes in n-3 PI,IFA content in the
diet
were reflected in major organs and especially CSR vesicles. Lfüewise, other studies that have
attempted
to alter SR lipid composion with dietary feeding have had simila¡
decreasing the n-6/n-3 ratio
success
in
in SR membrarìes 1236,240-2421. Fary acids can change the
physical structure of PL membranes and affect transmembra¡e ion cha¡rnels 1285,2941.
Cholesterol feeding was included in this study to create both fatry streaks in animals
that could potendally create ischaemic conditions in vùn
nd
to alter membrane lluidity and
function. We were not successful in achieving significant changes in cholesterol compostion
95
of SR membra¡les with
a 0.5% cholesterol diet
swine have shov¡n that the effects of a
1olo
over 8 weeks. Previous studies in Yorkshire
cholesterol diet on SR function are controversìal.
AIter 7-77 months of feeding a cholesterol-enriched dieq SR Caa upmte was increased
[295]. Another study used a cholesterol-supplemented diet for up ro
decrease in Ca2* uptake and a corresponding decrease
L yeâr and
reponed a
in SERCA activir¡ 12671. Ow lack of
significant changes in cholesterol content and SR function in cholesrerol-fed groups may
indicate that the given dose
of cholesterol was too low, the myocardium was capable of
resisting changes to cholesterol côntent, or most likely the duration of feeding was too shon
to induce
increases
in membrane cholesterol content so SR activþ could not be altered.
llowever, a combined cholesterol/fla-xseed diet masked the flaxseed effects on
Ca2'
trârìspoft in CSR from ischaemia-reperfused heans.
To
assess SR
funaion we used the rate of net Ca2* upake in isolated SR vesicles,
which represents the equilbrium between Ca'?* influ-x a¡d efflu-r. These studies on isolated
SR vesicles reveal a limited amount
of information about
SR lunction since
thq'
are
removed from a physiological setting. llowever, they do allow for d1'namic analysis of the
interacting pumps and chan¡rels
of the SR. The use of
conditions is advantageous because
ìt
augmenrs
a
oxalate
in our
Ca2* transporr
relatively slow process.
A¡
obvious
limitation to our study is the lack of negative control, SR from reperfused heans, for the
ischaemia-reperfusion group. Nevenheless, we were able to observe some significant trends
relative to the positive control, SR from ischaemia-reperfused hearts. Feeding rabbits
witl
AlA-enriched flaxseed protected SR function from ischaemia-reperfusion-induced injury
measured by Ca2* transpon
studies. This
that showed that n-3 Pl,ìFAs, including
agrees
AIA,
with our FFA study on isolated
enhance Ca2* uptake. However,
of dietary intervention, Ca'* uptake was improved relative to control by
96
as
SR vesicles
in our model
flaxseed feeding
onþ in the post-ischâemic period. It could be that CSR is altered by ischaemia-reperfusion
in
zuch a way that newþ incorporated n-3 PUFAs
cÍr
p¡otect against impaired
Ca'?*
handling. More likeþ, the changes in FA content in the SR membrane did not alær normal
basal SR
function. lloweve¡ when
stressed, as
in I/R challenge, the FA in the membrane
induced significant protective effects on SR function.
The response of SR Cat* trânsport to ischaemia hæ been extensively studied but is
not well defined.
It is generally agreed upon that oxalate-dependent Ca2* transport is
depressed after ischaemia due
to a combination of reduced SERCA activity and concurrent
efflux of Ca'z* via RyRs 1290, 2964At. llowever, some
s
is unchanged after bouts of ischaemia lasting 60 minutes
dies indicate that Cat* transpon
or
less [300,
3A2304]. Ischaemia
may not affect SERCA activity after 60 minutes either 1296, 3001. These data are in
agreement with the present rezults.
of our data should be examined.
Since each assay was
same buffers before Ca2* uptake was initiated,
we can rule out the
Some potential limitations
equilibrated
in the
possibilìty that confounding concentradons of ions like Ca'*, Ht, Mg2* were present in olrr
ischaemic-reperfused SR preparations
[298]. We can also
exclude the possibiJìty that our
isolating process may select for "good" SR, since this procedure has been shown to re't'eal
damaged SR v¡hen
it is assayed 1296). There is evidence that variability exists beween
SR
isolated from different areas of the myocardium [305]. Our SR vesicles were not isolâted
from a single area of the hean,but rather
a whole hean was used
for the isolation of
SR.
Thus our results could not detect any variabiliry in SR function within specific regions of the
hean. Another limitation to consider in the present study is that
pathv/ay cannot be observed in this preparation, because
RvR
97
changes
in the efflux
it is unresponsive to effectors of
There are a number of mechanisms that could be occurring in our isolated CSR
vesicles that would account for the beneficial effects
of dietary
fla-xseed
on Cat* transport
a{ter ischaemia-reperfusion. For example, alterations in phosphorylation status couid be a
cause
for altered
Ca2* handling.
CaMKII has been implicated in the maìntenance of
SR
function after ischaemic preconditioning by phosphorylation of both phospholamban and
SERCA [306]. Since the SERCA pump s/as not maximally activated in the Ca']* transport
experìments, we would expect
to
see differences
ìn
rates
of
uptake as affected by
phosphorylation status [298]. Mechanisic data also points towards a reactive orygen species
(R.OS)-induced damage of SR that is mediated by CaMK [307].
It
has been suggested that
"reoxygenation upon reperfusion of the myocardium may then result in promodng ìnÕrganic
as
well as organic-free radical chain reacdons ultimately affecting unsaturated fatty acids and
membrane biìayer functions" [300].
h follows then that newþ incorporated PIIFAs and./or
cholesterol may be oxidized during reperfusion and thereby alter Ca'Z* uptake propenies.
As discussed earlìer, membrane fluidity as determined by cholesterol and fatty acrd
compostition may or may not affect SERCA activþ [174, 283,286, 308]. According to
Almeida, cholesterol decreased Ca'?* uptake and SERCA activiry is only slightþ decreased
most likely due to a loss of efficienry in the ionophoretic channel of the pump [308]. n-3
PUFA-induced changes
in membrare fluidity did not
account
for the
ter:rnination of
aÐmchronous contractility in cardiomyoc¡tes. Rather, mechanisms independent
of SERCA
affected Ca2* handling [283]. SERCA activþ is also modulated by membrane thickness as
determined by esterified fatty acids and alkanes
[1/3]. Ultimateþ, homoviscous
adaptation,
the maintenance of membrane integrity, may ailow for continued funcdon of
despite dietary modihcation
177
4, 2æ, 3A91.
98
SERC,As
Direct effects of flaxseed feeding on SERCA function were determined by a unique
assay. To exclude contaiminating ATPase activþ from our analysis, we used the SERC.A
antagonist thapsigargin to find specìfic SERCA activþ as a fùnction of NADH oxidation
over dme. Other sudies in rhe literarure have anempted to assay SERC,A activity as a
measure of inorganic phosphate liberation in isolated SR preparations, but the source of this
aaivity is not clear 1296, 31A1. Our data indicates that SERCA activity after ischaemiareperfusion
in control and cholesterol-fed rabbits is significantþ higher than in SR from
non-ischaemic hearts. As discussed above, this contradicts studies by Singl that establish
that both Ca2* transpon and SERCA activity are unchanged after ischaemia reperfusion
within our given time-frame [300]. This data did not give arry indication
as
to why CSR from
ischaemia-reperfused heans from flaxseed-supplemented rabbits sequestered Ca2* at a higher
rate. It is likeþ that the
Ca2ntranspon-related SERCA effects may have been masked
because the SERCA pump was ma-rimalþ stimulated ¡.rnder our assay conditions
t298]. ln
fact, SERCA activity was significantþ slower in CSR from ìschaemia-reperfused flax-fed
animals than control ischaemia-reperfused CSR. 'Ve can hlpothesise based on our study
of
the literature that oxidation of n-3 PLEAs after reperfusion may affect SERCA function.
Future smdies may account for differences in Ca2* transpon by assaying SERCA activþ
under similar conditions.
Improved SR
Cal
handling after ischaemia-reperftsion may have clinical
applications, since impaired SR Ca'?. ha¡rdling may result in increased diastolic Cat*, which
may cause spontaneous Ca2* release, arrhlthmias, and
a
weaker amplitude of contraction.
In
fact, the corresponding electrophysiological data from ischaemia-reperfused heans indicates
that fl¿t-fed rabbits showed delayed contracture during the onset of ischaemia and were not
susceptible
to ventrìcular fibrillation during the ischaemia
phase as were controls [311].
Cholesterol-flax fed rabbits were also resista¡t to ventricular fibrillation during reperfusion
[311].
A
number
of
SR-mediated mechanisms may contribute
to these beneficial effects
including alterations in coupling, RyR efflu-x, lumenal free Ca'?*, phosphorylation of SERC,A,
ROS a¡rd membra¡e fluidity. Overall ,these data suggest that n-3 PllFAs in borh unesterified
and esterified forms may attenuate changes in SR Ca'?. handling associated with ischaemia.
V.
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