Am J Physiol Heart Circ Physiol 302: H1584–H1590, 2012. First published February 3, 2012; doi:10.1152/ajpheart.00887.2011. Adrenomedullin-epinephrine cotreatment enhances cardiac output and left ventricular function by energetically neutral mechanisms Thor Allan Stenberg,1,2 Anders Benjamin Kildal,1 Ole-Jakob How,1 and Truls Myrmel1,2 1 2 Surgical Research Laboratory, Department of Clinical Medicine, Faculty of Health Sciences, University of Tromsø, and Department of Cardiothoracic and Vascular Surgery, University Hospital of North Norway, Tromsø, Norway Submitted 6 September 2011; accepted in final form 2 February 2012 Stenberg TA, Kildal AB, How OJ, Myrmel T. Adrenomedullinepinephrine cotreatment enhances cardiac output and left ventricular function by energetically neutral mechanisms. Am J Physiol Heart Circ Physiol 302: H1584 –H1590, 2012. First published February 3, 2012; doi:10.1152/ajpheart.00887.2011.—Adrenomedullin (AM) used therapeutically reduces mortality in the acute phase of experimental myocardial infarction. However, AM is potentially deleterious in acute heart failure as it is vasodilative and inotropically neutral. AM and epinephrine (EPI) are cosecreted from chromaffin cells, indicating a physiological interaction. We assessed the hemodynamic and energetic profile of AM-EPI cotreatment, exploring whether drug interaction improves cardiac function. Left ventricular (LV) mechanoenergetics were evaluated in 14 open-chest pigs using pressure-volume analysis and the pressure-volume area-myocardial O2 consumption (PVA-MV̇O2) framework. AM (15 ng·kg⫺1·min⫺1, n ⫽ 8) or saline (controls, n ⫽ 6) was infused for 120 min. Subsequently, a concurrent infusion of EPI (50 ng·kg⫺1·min⫺1) was added in both groups (AM-EPI vs. EPI). AM increased cardiac output (CO) and coronary blood flow by 20 ⫾ 10% and 39 ⫾ 14% (means ⫾ SD, P ⬍ 0.05 vs. baseline), whereas controls were unaffected. AM-EPI increased CO and coronary blood flow by 55 ⫾ 17% and 75 ⫾ 16% (P ⬍ 0.05, AM-EPI interaction) compared with 13 ⫾ 12% (P ⬍ 0.05 vs. baseline) and 18 ⫾ 31% (P ⫽ not significant) with EPI. LV systolic capacitance decreased by ⫺37 ⫾ 22% and peak positive derivative of LV pressure (dP/dtmax) increased by 32 ⫾ 7% with AM-EPI (P ⬍ 0.05, AM-EPI interaction), whereas no significant effects were observed with EPI. Mean arterial pressure was maintained by AM-EPI and tended to decrease with EPI (⫹2 ⫾ 13% vs. ⫺11 ⫾ 10%, P ⫽ not significant). PVA-MV̇O2 relationships were unaffected by all treatments. In conclusion, AM-EPI cotreatment has an inodilator profile with CO and LV function augmented beyond individual drug effects and is not associated with relative increases in energetic cost. This can possibly take the inodilator treatment strategy beyond hemodynamic goals and exploit the cardioprotective effects of AM in acute heart failure. left ventricular energetics; conductance catheter ADRENOMEDULLIN (AM) is a 52-amino acid peptide originally isolated by its ability to elevate cAMP in rat platelet assays (20). This peptide participates in cardiovascular homeostasis as an autocrine or paracrine factor with vasodilative and chronotropic properties (15). AM administration consistently augments cardiac output (CO) and facilitates forward flow in vivo (5, 29, 34), but experimental findings with respect to the direct cardiac effects of AM are diverging (14, 41). Evidence has suggested, however, that AM is inotropically neutral within what is potentially the therapeutic dose range (24). AM reduces ischemia-induced arrhythmias, limits infarct size, attenuates ischemia-reperfusion injury, and lowers mortality in rodent models (25, 30, 31, 33). The Address for reprint requests and other correspondence: T. A. Stenberg, Dept. of Clinical Medicine, Univ. of Tromsø, NO-9037 Tromsø, Norway (e-mail: [email protected]). H1584 cardioprotective effects are therapeutically attractive, and the hemodynamic profile of AM is advantageous in experimental heart failure models (35). The hypotensive effect, however, is possibly detrimental in ischemic heart failure, and profound hypotension after AM administration to patients with acute myocardial infarction has been reported (16). Interestingly, AM and catecholamines are both secreted from chromaffin cells through Ca2⫹-dependent regulated exocytosis after cholinergic stimulation (17). Adrenal medullary basal catecholamine release and catecholamine levels in plasma are both increased by AM (1, 43), whereas AM release from chromaffin cells is augmented by dibutyryl cAMP, a membrane-permeable analog of cAMP (21). AM release from cultured rat vascular smooth muscle cells is also augmented by epinephrine (EPI) (40). Thus, there is evidence indicating a physiological interaction between AM and catecholamines such as EPI. Furthermore, cAMP is a well-recognized second messenger in AM signal transduction, and both negative and positive modulation of -adrenergic signaling by AM has been reported in vitro (10, 13). It is presently unknown whether AM can modulate hemodynamic and inotropic responses to -adrenergic stimulation in vivo. In addition to hemodynamic and potentially cytoprotective effects, AM appears to be involved in insulin regulation, fatty acid mobilization, and glucose metabolism (13, 26). These metabolic effects of AM, either alone or in combination with EPI, can potentially influence left ventricular (LV) energetics due to alterations in the concentration of free fatty acids in plasma (23). There is evidence indicating that AM has favorable energetic properties (29), but the relation between total mechanical work and myocardial O2 consumption (MV̇O2) has not been quantified. In the present study, we assessed the hemodynamic and contractile effects of potentially therapeutic levels of AM in a porcine model. In addition, we analyzed whether low-dose AM infusion modulates the hemodynamic and contractile effects of -adrenergic stimulation by a low-dose EPI infusion in physiologically intact animals. The effects of both individual and combined treatments on LV energetics were quantified through the assessment of total mechanical work and MV̇O2 within the pressure-volume area (PVA)-MV̇O2 framework. METHODS The experimental protocol was approved by the local steering committee of the Norwegian Animal Research Authority and was conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996). Fourteen castrated male pigs (Norwegian Landrace, Sus scrofa domesticus) weighing 27 ⫾ 2 kg were habituated to the animal facilities for 4 –7 days and fasted overnight before the experiment with free access to water. The premedication, anesthetic protocol, surgical preparation of the 0363-6135/12 Copyright © 2012 the American Physiological Society http://www.ajpheart.org HEMODYNAMIC INTERACTIONS WITH AM-EPI COTREATMENT open-chest model, conductance volumetric technique, and LV energetic assessment method have been previously detailed (28). Instrumentation. Animals were anesthetized, tracheostomized, and ventilated via a volume-controlled ventilator (Servo 900 D, ElemaSchönander). A 7-Fr balloon catheter for preload reductions and transient caval occlusions was placed in the inferior caval vein. The hemiazygos vein was ligated to avoid mixture of systemic blood into the coronary sinus. Transit-time flow probes (Medi-Stim PB series, Medi-Stim) were placed on the pulmonary trunk, coronary, left femoral, and left carotid arteries for measurements of CO, coronary blood flow (CBF), muscular blood flow, and cerebral blood flow. The great cardiac vein was catheterized through the superior caval vein and coronary sinus for blood sampling. A 7-Fr dual-field combined pressure-conductance catheter (CD Leycom) was inserted into the LV via the right carotid artery to measure LV volume and pressure. Intravascular volume was maintained with a glucose-enriched (1.25 g/l) saline infusion (20 ml·kg⫺1·h⫺1). Animals received 150 mg amiodaron to prevent arrhythmias, and 2,500 IE heparin was administered to prevent catheter clotting. Experimental protocol. In preliminary experiments, the porcine dose-response relationship to intravenous infusions of human AM1–52 (10 –150 ng·kg⫺1·min⫺1, Bachem) was established (Fig. 1). Low-dose AM (15 ng·kg⫺1·min⫺1) was chosen to achieve a moderate increase in CO while minimizing heart rate (HR) and mean arterial pressure (MAP) alterations, thus aiming at a potentially tolerable level in settings with acute ischemia and hemodynamic instability. The isolated effect of AM and the interaction between AM and a concurrent infusion of EPI (AM-EPI) were compared with the individual effect of EPI in a time-matched control group receiving saline as vehicle. After baseline measurements, the AM group (n ⫽ 8) received a continuous infusion of AM, whereas the control group (n ⫽ 6) received a continuous infusion of saline. A second set of measurements was obtained 120 min after baseline recordings. Subsequently, both groups received a concurrent infusion of EPI (50 ng·kg⫺1·min⫺1) with the third set of measurements obtained at steady state (⬃15 min). LV energetics. LV MV̇O2 was plotted as a function of PVA as described by Suga (39). The PVA-MV̇O2 relation at each measurement set was assessed by preload alteration and differing levels of steady-state LV mechanical work. MAP and CBF were allowed to reach uninfluenced levels between the successive preload reductions to ensure unimpaired LV function. At each preload, steady-state H1585 pressure-volume data were recorded for 10 s with simultaneous blood sampling from the great cardiac vein to calculate cardiac O2 extraction. PVA and MV̇O2 were calculated as previously described (28). LV function and ventriculoarterial matching. Indexes of global LV function [preload recruitable stroke work (PRSW)], inotropy [slope (Ees) and volume axis intercept (V0) of the end-systolic pressurevolume relationship (ESPVR)], and lusitropy [curve fitting constant (␣) and diastolic stiffness constant () of the end-diastolic pressurevolume relationship (EDPVR)] derived from the pressure-volume loops were evaluated by transient caval occlusions, whereas indexes derived at steady state [peak positive and negative derivatives of LV pressure (dP/dtmax and dP/dtmin, respectively), and isovolumic time constant ()] were assessed using the initial steady-state recording at each measurement set. PRSW, which incorporates both systolic and diastolic properties, is a relatively load-insensitive index of global LV function (9). The ESPVR was defined as follows: ESP ⫽ Ees(ESV ⫺ V0), where ESP and ESV are end-systolic pressure and volume, respectively. The EDPVR was defined by the following exponential relation: EDP ⫽ ␣e(EDV) where EDP and EDV are end-diastolic pressure and volume, respectively. Due to covariance between Ees and V0, as well as nonlinearities in the ESPVR, positive inotropic effects may manifest as increased slope (Ees) or a leftward ESPVR shift (V0). Importantly, with the EDPVR being nonlinear in nature, there is no agreed upon method to compare EDPVRs without logarithmic transformation (3). Calculating capacitance yields variables that reflect altered LV function by integrating altered slope (Ees and ) and/or shifts in the intercept (V0 and ␣) (37). LV systolic capacitance (ESV120) and LV diastolic capacitance (EDV20) were defined as follows: ESV120 ⫽ (120/Ees) ⫹ V0 and EDV20 ⫽ [ln(20/␣)]/. Arterial elastance (Ea) was calculated as follows: ESP/stroke volume. Systemic vascular resistance (SVR) was calculated as mean systemic perfusion pressure/CO and expressed as dyn·s·cm⫺5. Ventriculoarterial (VA) matching was assessed by the ratios of Ea/Ees and PRSW/ SVR (12). Statistics. Groups and drug interactions were assessed by two-way repeated-measurements ANOVA (least-squares linear regression with dummy variables, subject identifier as random effect). Pooled PVAMV̇O2 data were analyzed by two-way repeated-measurements analysis of covariance (ANCOVA; dummy variables and random effect as above). Tukey’s highly significant difference test was used to adjust for multiple comparisons, and P values of ⬍0.05 were considered statistically significant. Statistical analysis was performed using JMP 7 (SAS Institute) with absolute or relative data (i.e., percent change from baseline) consistent with the data presented. Values are reported as means ⫾ SD. RESULTS Fig. 1. Data from preliminary experiments showing the dose-response relationship to incremental adrenomedulin (AM) infusions from 10 ng·kg⫺1·min⫺1 (n ⫽ 4) to 150 ng·kg⫺1·min⫺1 (n ⫽ 2) as percent changes from baseline (BL) values. MAP, mean arterial pressure; CO, cardiac output; HR, heart rate; CBF, coronary blood flow. The hemodynamic variables were matched between groups at baseline with the exception of a higher HR in the control group (62 ⫾ 7 vs. 88 ⫾ 6 beats/min, P ⬍ 0.05). LV systolic and diastolic function, LV energetics, and VA matching were similar in both groups at baseline. Hemodynamic and LV functional data are shown as percent changes from baseline values in Fig. 2. Corresponding absolute data and PVA-MV̇O2 parameters are shown in Tables 1–3. LV function and VA matching. The effects of AM, EPI, and AM-EPI on LV function are shown in Figs. 2 and 3. LV function, as assessed by dP/dtmax, showed that AM alone had no observable effect on contractility. AM-EPI increased dP/ dtmax by 32 ⫾ 14% compared with baseline (P ⬍ 0.05), whereas the control group had an initial ⫺14 ⫾ 12% decrease with saline (P ⬍ 0.05) and with EPI dP/dtmax was still ⫺2 ⫾ 14% [P ⫽ not significant (NS)] compared with baseline. Interaction analysis showed a significant interaction with AMEPI (P ⬍ 0.05). Global LV function, as measured by PRSW, AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00887.2011 • www.ajpheart.org H1586 HEMODYNAMIC INTERACTIONS WITH AM-EPI COTREATMENT Fig. 2. Hemodynamics and left ventricular (LV) function shown as percent changes from BL values. A: CO and HR were augmented to a greater extent with AM-epinephrine (EPI) cotreatment (AM-EPI) than with the individual drugs. MAP was also preserved with AM-EPI. B: CBF was greatly enhanced by AM-EPI cotreatment, whereas carotid (Car) and femoral (Fem) blood flows were less affected. C: LV systolic capacitance (ESV120), peak positive derivative of LV pressure (dP/dtmax), and preload recruitable stroke work (PRSW) results showing that AM-EPI cotreatment improved LV function to a greater degree than the individual drugs. Open circles, control group; filled circles, AM group. Within-group differences: *P ⬍ 0.05 vs. BL values and †P ⬍ 0.05 vs. AM or control; between-group differences: ‡P ⬍ 0.05; interaction: §P ⬍ 0.05 (by two-way repeated-measures ANOVA). AM-EPI / EPI showed that AM had no effect on LV function when administered alone. With AM-EPI, PRSW increased by 40 ⫾ 14% compared with baseline values (P ⬍ 0.05), whereas EPI alone produced a nonsignificant 23 ⫾ 27% increase. Through interaction analysis using absolute data, an interaction between AM-EPI was observed (P ⬍ 0.05; Table 2). ESV120 decreased by ⫺37 ⫾ 22% with AM-EPI compared with baseline (P ⬍ 0.05), whereas it was unaltered with the individual drugs (19 ⫾ 18% vs. 16 ⫾ 42%, AM vs. EPI, P ⫽ NS). The effect of AM-EPI cotreatment was a greater reduction in ESV120 compared with EPI infusion in the control group, and a significant interaction was detected (P ⬍ 0.05; Fig. 2 and Table 2). AM-EPI / EPI AM-EPI / EPI VA matching was assessed using the ratios of Ea/Ees and PRSW/SVR. Afterload, as assessed by Ea, predominantly reflecting arterial compliance, showed no systematic changes. The ratio of Ea/Ees was unaltered across all conditions. SVR primarily reflects peripheral vasoconstriction, and, in contrast to Ea, SVR was reduced with AM, EPI, and AM-EPI (⫺14 ⫾ 13%, ⫺23 ⫾ 10%, and ⫺34 ⫾ 14%, P ⬍ 0.05 vs. baseline by one-way ANOVA). The ratio of PRSW/SVR increased with AM, EPI, and AM-EPI (22 ⫾ 18%, 58 ⫾ 44%, and 120 ⫾ 41%, P ⬍ 0.05). A significant interaction was detected with PRSW/SVR, and thus forward flow facilitation was augmented to a greater extent by AM-EPI than by the individual drugs (P ⬍ 0.05). Table 1. Hemodynamics AM Group Mean arterial pressure, mmHg Mean pulmonary artery pressure, mmHg Central venous pressure, mmHg Cardiac output, ml/min Heart rate, beats/min End-systolic volume, ml End-diastolic volume, ml End-systolic pressure, mmHg End-diastolic pressure, mmHg Coronary blood flow, ml/min Carotid blood flow, ml/min Femoral blood flow, ml/min Ea, mmHg/ml SVR, dyn 䡠 s 䡠 cm⫺5 Control Group Baseline AM AM-EPI Baseline Control EPI 93 ⫾ 8 17 ⫾ 2 6⫾1 2,028 ⫾ 443 62 ⫾ 7‡ 25 ⫾ 6 57 ⫾ 7 106 ⫾ 8 15 ⫾ 2 87 ⫾ 13 214 ⫾ 62 94 ⫾ 27 3.3 ⫾ 0.4 3,516 ⫾ 484 95 ⫾ 13 19 ⫾ 2 6⫾1 2,441 ⫾ 625* 80 ⫾ 15* 29 ⫾ 9 59 ⫾ 10 107 ⫾ 12 13 ⫾ 2 122 ⫾ 24* 227 ⫾ 55 111 ⫾ 39 3.6 ⫾ 0.6 3,047 ⫾ 705 94 ⫾ 14 20 ⫾ 4 8⫾3 3,106 ⫾ 648*†§ 101 ⫾ 18*† 20 ⫾ 10 49 ⫾ 10 100 ⫾ 15 11 ⫾ 4 151 ⫾ 19*† 277 ⫾ 77 129 ⫾ 45* 3.3 ⫾ 0.6 2,315 ⫾ 615 89 ⫾ 10 19 ⫾ 2 6⫾1 2,318 ⫾ 331 88 ⫾ 6‡ 20 ⫾ 5 45 ⫾ 7 96 ⫾ 10 12 ⫾ 2 108 ⫾ 23 176 ⫾ 18 95 ⫾ 43 3.7 ⫾ 0.3 2,888 ⫾ 307 83 ⫾ 8 22 ⫾ 1 6⫾1 2,222 ⫾ 371 93 ⫾ 5 22 ⫾ 3 45 ⫾ 6 90 ⫾ 9 11 ⫾ 2 120 ⫾ 32 188 ⫾ 22 85 ⫾ 21 3.9 ⫾ 0.8 2,818 ⫾ 566 78 ⫾ 6 21 ⫾ 4 6⫾1 2,612 ⫾ 366† 104 ⫾ 10* 18 ⫾ 3 42 ⫾ 6 86 ⫾ 8 11 ⫾ 2 127 ⫾ 40 218 ⫾ 23 101 ⫾ 22 3.5 ⫾ 0.7 2,231 ⫾ 357 Values are means ⫾ SD; n ⫽ 8 animals in the adrenomedulin (AM) group and 6 animals in the control group. EPI, epinephrine; AM-EPI, AM and EPI cotreatment; Ea, arterial elastance; SVR, systemic vascular resistance. Within-group differences: *P ⬍ 0.05 vs. baseline and †P ⬍ 0.05 vs. AM or control; between-group differences: ‡P ⬍ 0.05; interaction: §P ⬍ 0.05 (by two-way repeated-measures ANOVA). AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00887.2011 • www.ajpheart.org H1587 HEMODYNAMIC INTERACTIONS WITH AM-EPI COTREATMENT Table 2. LV function and ventriculoarterial matching AM Group Ees, mmHg/ml V0, ml ESV120, ml PRSW, mmHg dP/dtmax, mmHg/s dP/dtmin, mmHg/s , mmHg/ml ␣ EVD20, ml , ms Ea/Ees PRSW/SVR, l/min Control Group Baseline AM AM-EPI Baseline Control EPI 2.5 ⫾ 1.3 ⫺26 ⫾ 21 29 ⫾ 9 57 ⫾ 7 1,410 ⫾ 208 ⫺1,762 ⫾ 217 0.068 ⫾ 0.026 0.66 ⫾ 0.79 62 ⫾ 9 49 ⫾ 6 1.5 ⫾ 0.4 1.32 ⫾ 0.17 2.5 ⫾ 1.4 ⫺24 ⫾ 27 35 ⫾ 10 60 ⫾ 12 1,459 ⫾ 265 ⫺1,891 ⫾ 249 0.072 ⫾ 0.036 0.86 ⫾ 1.29 67 ⫾ 10 44 ⫾ 4 1.7 ⫾ 0.6 1.61 ⫾ 0.38 3.3 ⫾ 1.2 ⫺19 ⫾ 22 23 ⫾ 12*† 80 ⫾ 13*†‡§ 1,868 ⫾ 317*†§ ⫺2,042 ⫾ 322* 0.092 ⫾ 0.048 0.45 ⫾ 0.51 55 ⫾ 10† 38 ⫾ 3 1.1 ⫾ 0.4 2.93 ⫾ 0.81*†§ 2.0 ⫾ 0.6 ⫺33 ⫾ 8 29 ⫾ 10 54 ⫾ 15 1,546 ⫾ 224 ⫺1,908 ⫾ 254 0.083 ⫾ 0.028 0.47 ⫾ 0.34 50 ⫾ 8 40 ⫾ 5 1.9 ⫾ 0.4 1.50 ⫾ 0.37 1.9 ⫾ 0.5 ⫺33 ⫾ 10 33 ⫾ 8 54 ⫾ 8 1,328 ⫾ 279* ⫺1,778 ⫾ 179 0.071 ⫾ 0.038 0.75 ⫾ 0.47 57 ⫾ 14 39 ⫾ 3 2.1 ⫾ 0.4 1.56 ⫾ 0.31 2.1 ⫾ 0.7 ⫺34 ⫾ 14 30 ⫾ 8 62 ⫾ 5‡ 1,511 ⫾ 312 ⫺1,825 ⫾ 182 0.067 ⫾ 0.039 1.05 ⫾ 0.67 54 ⫾ 11 35 ⫾ 3 1.8 ⫾ 0.3 2.27 ⫾ 0.39*† Values are means ⫾ SD; n ⫽ 8 animals in the AM group and 6 animals in the control group. LV, left ventricular; Ees, end-systolic elastance [slope of the end-systolic pressure-volume relationship (ESPVR)]; V0, initial volume (volume axis intercept of the ESPVR); PRSW, preload recruitable stroke work; Ea/Ees and PRSW/SVR, measures of ventriculoarterial matching (see text for details). Within-group differences: *P ⬍ 0.05 vs. baseline and †P ⬍ 0.05 vs. AM or control; between-group differences: ‡P ⬍ 0.05; interaction: §P ⬍ 0.05 (by two-way repeated-measures ANOVA). LV energetics. PVA-MV̇O2 relationships within each group are shown as pooled scatterplots in Fig. 4. Mean PVA-MV̇O2 parameters are shown in Table 3. There was no observable effect on the slope (i.e., cardiac energetic efficiency) or y-intercept (i.e., unloaded MV̇O2) of the PVA-MV̇O2 relationship with AM, EPI, or AM-EPI. Individual slope and y-intercept values showed no significant between- or within-group differences, and repeated-measurements ANCOVA of pooled PVAMV̇O2 data showed no significant main or interaction effects. Thus, LV energetics were unaffected by AM, EPI, or AM-EPI. DISCUSSION Our study supports evidence suggesting physiologically relevant interactions between AM, -adrenergic signaling, and the sympathetic nervous system. As stated, an interrelationship between the signaling and release of AM and catecholamines has been observed in several models (1, 40, 43), and modulation of -adrenergic signaling has been reported (10, 13). In addition, increased sympathetic activity has been seen with intravenous administration of AM (36). However, whether cotreatment could produce hemodynamically relevant interaction effects has not been previously described. Our data show that the increase in CO is paralleled by a chronotropic response, and altered HR could potentially explain the increased contractility observed with AM-EPI. However, a higher absolute HR was seen in the control group at baseline, and with AM-EPI and EPI, the HR was similar between groups, thereby indicating that other mechanisms are responsible for the increase in LV systolic function during AM-EPI cotreatment. AM can enhance the baroreceptor reflex response through cAMP- and PKA-dependent signaling in the nucleus tractus solitarius, the terminal site for primary baroreceptor afferents (11). Increased HR and cardiac sympathetic nerve activity have also been reported, with the effect being more pronounced with AM than with pressure-matched nitroprusside administration (4). Interestingly, the inotropic effect of CGRP has been attributed to indirect myocardial sympathetic activation through an increased interstitial concentration of norepinephrine (18). AM belongs to the CGRP peptide superfamily and partly shares receptor complexes with calcitonin receptor-like receptor (CL) and receptor activity-modifying protein (RAMP)2 and RAMP3, which constitute AM receptors, whereas CL and RAMP1 form the CGRP receptor (15). To which extent AM may stimulate myocardial sympathetic signaling remains to be clarified. Adrenal chromaffin cells used as models of catecholaminereleasing neurons have been shown to release AM through cAMP-mediated mechanisms (22), and catecholamine release after AM administration has also been reported (1). Thus, a hypothetical feedback system regulating myocardial sympathetic activity may be envisioned. AM reduces Ea and SVR and affects potent vasodilation in resistance vessels (6, 24, 34), whereas preload is maintained (24). Thus, in addition to the chronotropic response, reduced afterload with concomitantly less effect on capacitance vessels and sustained venous return may also explain why AM potently augments CO. It is uncertain whether AM has inotropic properties in vivo as there are reports of both positive and unaltered inotropy (24, 29). Positive inotropic effects in vivo Table 3. LV energetics AM Group Pressure-volume area, J 䡠 beat MV̇O2, J 䡠 beat⫺1 䡠 100 g⫺1 y-Intercept Slope R2 ⫺1 䡠 100 g ⫺1 Control Group Baseline AM AM-EPI Baseline Control EPI 0.64 ⫾ 0.13 1.76 ⫾ 0.26 0.51 ⫾ 0.12 1.93 ⫾ 0.14 0.96 ⫾ 0.02 0.68 ⫾ 0.21 1.87 ⫾ 0.29 0.53 ⫾ 0.10 2.01 ⫾ 0.29 0.98 ⫾ 0.01 0.61 ⫾ 0.17 1.75 ⫾ 0.31 0.44 ⫾ 0.12 2.19 ⫾ 0.42 0.98 ⫾ 0.02 0.59 ⫾ 0.09 1.58 ⫾ 0.28 0.35 ⫾ 0.12 2.17 ⫾ 0.68 0.97 ⫾ 0.04 0.55 ⫾ 0.09 1.55 ⫾ 0.37 0.39 ⫾ 0.18 2.00 ⫾ 0.72 0.95 ⫾ 0.05 0.51 ⫾ 0.09 1.44 ⫾ 0.39 0.38 ⫾ 0.10 2.07 ⫾ 0.48 0.96 ⫾ 0.03 Values are means ⫾ SD; n ⫽ 8 animals in the AM group and 6 animals in the control group. MV̇O2, myocardial O2 consumption; y-intercept, unloaded MV̇O2; slope, contractile efficiency; R2, coefficient of determination. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00887.2011 • www.ajpheart.org H1588 HEMODYNAMIC INTERACTIONS WITH AM-EPI COTREATMENT Fig. 3. Pressure-volume loops from a representative experiment in each group. A: AM group; B: control group. AM-EPI improved LV function, as shown by the leftward shift of the end-systolic pressure-volume relationship together with an increased slope (see text for details). AM-EPI MVO2 (J•beat-1•100 g-1) Fig. 4. Pooled scatterplots of the pressurevolume area myocardial O2 consumption (PVA-MV̇O2) relationships within each group from all experiments. A: AM group; B: control group. No significant differences were detected by repeated-measures analysis of covariance (see text for details). bining lipolytic drugs that can increase free fatty acids in plasma could potentially induce a mechanoenergetic inefficiency that would be unwanted therapeutically in the ischemically challenged heart (23). This is the first study to characterize LV energetics using total mechanical work related to MV̇O2 with systemic administration of AM. Favorable energetic properties of AM have been reported with an increase in contractility paralleling a reduction in MV̇O2, thus implying an increased mechanoenergetic efficiency (29). However, afterload reduction and relatively less energetic cost with volume versus pressure work may explain the finding, and the relationship between total mechanical work and MV̇O2 must be assessed to describe any influence on LV energetics (39). We found no evidence of altered mechanoenergetic efficiency or unloaded MV̇O2 with AM, EPI, or AM-EPI. The vascular response after AM administration is heterogeneous with increased blood flow in the heart, lungs, kidneys, adrenal glands, and spleen, whereas the splanchnic bed is relatively unaffected (19). In agreement with earlier studies (7, 44) showing that AM infusion augments CBF, we found that low-dose AM produced a relatively large increase in CBF. This effect was further enhanced by AM-EPI cotreatment, and the extent of CBF elevation suggests that the drugs interact. Importantly, PVA-MV̇O2 data indicate that the observed increase is not metabolically induced, as unloaded MV̇O2, contractile efficiency, and total mechanical work were similar between groups. In vitro studies (2, 42) have shown that AM MVO2 (J•beat-1•100 g-1) have predominantly been observed with relatively load-sensitive measures of LV function. AM in doses ranging from 20 to 200 ng·kg⫺1·min⫺1 confer no inotropic effect, as assessed by the ESPVR (24); thus, AM is probably inotropically neutral within the potentially therapeutic dose range. We found no evidence of any inotropic effect with low-dose AM, and preliminary dose-response experiments revealed no inotropic effect with doses ranging from 10 to 150 ng·kg⫺1·min⫺1 (data not shown). AM-EPI cotreatment caused moderate elevations of dP/dtmax and PRSW, with both being larger than with EPI as the only active drug, and a significant AM-EPI interaction was thus observed. The ESPVR was unaltered, as assessed by Ees and V0, and there were no differences in ␣ or  describing the EDPVR. The calculation of systolic capacitance accounts for the potential covariance between Ees and V0 (3, 37), and the reduction in ESV120 with AM-EPI exceeded the sum of the individual drug effects, thus implying that the end-systolic stiffness (i.e., contractility) was increased through AM-EPI interaction. As shown in Fig. 3, AM-EPI produced a leftward shift of the ESPVR together with increased Ees, thereby indicating an improved contractile performance within the measured pressure range. The functional parameters dP/dtmax, PRSW, and ESV120 were unaltered by AM, thereby indicating that the interaction effect is synergistic in nature. AM is metabolically active and confers lipolytic properties in vitro (13), and catecholamines and sympathomimetic drugs are known to increase free fatty acids in plasma (27). Com- AM-EPI PVA (J•beat-1•100 g-1) AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00887.2011 • www.ajpheart.org PVA (J•beat-1•100 g-1) HEMODYNAMIC INTERACTIONS WITH AM-EPI COTREATMENT mediates coronary vasodilation through nitric oxide (NO) synthase (NOS) and EDHF pathways. In the isolated rat aorta, AM causes endothelium-dependent vasodilation by Ca2⫹-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which, in turn, activates endothelial NOS (eNOS) and NO production (32). In endothelial cells, cAMP accumulation and mobilization of intracellular Ca2⫹ stores via phospholipase C and inositol 1,4,5-trisphosphate formation have been observed after AM exposure (38). In addition to PI3K activation, increased levels of intracellular Ca2⫹ in endothelial cells can also produce vasodilation through the EDHF pathway with opening of endothelial small- and intermediate-conductance Ca2⫹-activated K⫹ channels inducing hyperpolarization of vascular smooth muscle (8). Interestingly, NO production in canine myocardial microvessels after exposure to AM or isoproterenol is substantially enhanced in subjects with heart failure despite eNOS downregulation, an effect abolished by NOS, PKA, and PI3K inhibition (45). Thus, cAMP and PI3K appear to serve as compensatory pathways that can sustain or augment NO production in situations with eNOS downregulation and endothelial dysfunction. Furthermore, as both pathways have been implicated in AM and -adrenergically induced vasorelaxation (45), they could potentially serve to explain the substantial augmentation of CBF observed with AM-EPI cotreatment. Conclusions. The main observations presented in this study is that low-dose AM is inotropically neutral and increases CO primarily through vasodilation and chronotropy, that low-dose AM-EPI cotreatment has the hemodynamic profile of an inodilator with CO and LV function augmented beyond individual drug effects, and that this functional enhancement is not associated with disproportionate increases in energetic expenditure. Thus, low-dose AM-EPI cotreatment has a hemodynamic profile that is attractive in ischemic heart failure and circulatory compromise with cardiac unloading, increased CO and inotropy, maintained systemic perfusion pressure, and mechanoenergetic neutrality. In addition, AM is potentially attractive in settings of ischemia and heart failure due to its effect on infarct size, arrhythmias, and mortality. In combination with an inotrope, this can take the inodilator treatment strategy beyond purely hemodynamic goals by exploiting the pleiotropic effects while supporting the circulation with an inotrope. The chronotropic effect of AM-EPI, however, is a concern in acute heart failure and should be evaluated in future studies. ACKNOWLEDGMENTS The authors are indebted to the staff at the Surgical Research Laboratory for providing excellent technical assistance. GRANTS This work was financially supported by the Northern Norway Regional Health Authority. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). 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