The present study demonstrates that 11 weeks of GLP-1 or AC3174 infusion to post-MI rats developing CHF can significantly improve multiple clinically-relevant parameters of cardiac function in a model of moderate, stable, compensated heart failure. In comparison with vehicle-treated rats, improvements were observed in LVEF, fractional shortening, transmitral flow ratio, E-wave velocity, cardiac wall-thinning ratio, LVEDP, dP/dT, and cardiac output. Measured parameters of cardiac morphology were also improved by GLP-1 or AC3174 infusion, including LV end systolic and end diastolic diameter, and left atrial volume. In contrast, heart rate and mean blood pressure in GLP-1R agonist-treated animals were not different from those of vehicle control animals. Left ventricular infarct size was not affected by GLP-1 or AC3174, and no differences in body weight-adjusted measures of heart weight were observed. However, unadjusted LV and RV weights were decreased. Furthermore, a statistically significant improvement in survival was observed with GLP-1 and AC3174 treatments at both low and high doses.
More detailed analyses of the physiological changes resulting from either GLP-1 or AC3174 infusion indicate that fluid balance, glucose metabolism, and respiratory efficiency improved compared with vehicle control animals with CHF. Differences in fluid balance were evidenced by reductions in lung weight (adjusted for body weight) and fluid mass compared with post-MI vehicle control animals. Improvements in glucose metabolism were evidenced by reductions in plasma glucose, plasma insulin, and insulin resistance. Improved exercise capacity in GLP-1 or AC3174-treated animals was associated with reduced peak oxygen consumption during exercise and lower basal lactate production, reflecting improved respiratory efficiency. Running distances in post-MI rats treated with GLP-1 or AC3174 were not significantly different from that of sham-operated animals and were double that of vehicle control animals with CHF.
Exploration of Possible Mechanisms
These studies did not identify the molecular mechanisms mediating the GLP-1 and AC3174 changes in physiological function. No differences in translocation of GLUT1 or GLUT4 to the plasma membrane were observed between GLP-1 and AC3174-treated rats and no statistically significant differences in expression of these proteins were identified. This contrasts with the results from chronically infarcted Wistar rat hearts where myocardial GLUT4 protein levels were 28% lower in infarcted hearts than in sham-operated hearts, and insulin-stimulated glucose uptake was 42% lower . No differences in the protein expression of AKT2, SERCA2a, or PI3Kbeta were observed, and the observed trend towards reduction in eNOS expression might be expected to diminish cardiac function rather than improve it. However, studies in normal primary human coronary artery endothelial cells in vitro have demonstrated a similar lack of overt exenatide effect on eNOS and AKT2 protein expression on a background of enhanced activation of both proteins . Indeed, endothelial cells had a substantial proliferative response to exenatide treatment and this response was mediated by activation of both the AKT2/eNOS and the PKA/PI3K signal transduction pathways. Furthermore, activation of the GLP-1 receptor was required upstream for stimulation of these pathways. Along the same lines of inquiry, treatment of diabetic post-MI mice with the DPP-4 inhibitor sitagliptin, which augments concentrations of endogenous full-length GLP-1, reduced mortality and improved cardiac function .
Role of Glucose Metabolism in CVD
This study demonstrated the development of insulin resistance and hyperglycemia in a MI-induced rat model of CHF, supporting the similarity of this model system with human CHF. Improvements in whole-body insulin sensitivity and glycemic control are closely associated with attenuation of cardiac insulin resistance and appear to protect the heart in both patients and animals with coronary heart disease [14, 41]. Several animal studies have shown that increasing glucose utilization not only improves cardiac function, but also attenuates cardiac remodeling during CHF [14, 35, 42]. Further, a role of GLP-1 in cardioprotection is supported by the cardiac phenotype of GLP-1R knockout mice where resting heart rate is reduced, LV wall thickness is increased, and LVEDP is elevated compare with wild-type mice . Although baseline hemodynamics are normal, after the administration of insulin or epinephrine LV contractility and diastolic function also show impairment.
The close relationship between the metabolic syndrome and cardiovascular diseases, including CHF, is well established [4, 11–13, 20, 25, 38]. In previous studies, exenatide progressively reduced body weight in obese animals and humans, and increased insulin sensitivity in obese animals [6, 44–47]. In the present study, the combined actions of the exenatide analog AC3174 to reduce body weight, fat mass, insulin resistance, cardiac remodeling, and improve glycemic control and cardiac function suggest the overall improvement in metabolic status observed with AC3174 treatment may contribute to its cardioprotective mechanisms. Further evidence was provided by the decreased rates of mortality in AC3174-treated MI rats compared with vehicle control rats. In patients with CHF and diabetes, but not in normoglycemic patients with CHF, a 5 week infusion of GLP-1 significantly reduced plasma glucose levels . However, cardiac function in both groups of patients was significantly and comparably improved by GLP-1. These results suggest that GLP-1 effects that are independent of whole body metabolic improvements contribute relatively more to GLP-1's cardioprotective effects, perhaps via direct myocardial actions.
In a global ischemia model in isolated rat hearts, GLP-1 treatment post-MI exhibited only a small tendency to increase mechanical (inotropic) performance . Rather, GLP-1's primary mechanism of action was cardioprotective in nature (39% reduction in infarct size) and mediated through the GLP-1 receptor. In isolated mouse hearts, GLP-1 increased functional recovery and cardiomyocyte viability after ischemia-reperfusion injury . In models of MI (ischemia with or without reperfusion) and heart failure, treatment with GLP-1 or exenatide treatment has generally been associated with improvements in post-ischemia cardiac function or infarct size. The most striking results were observed in studies with longer follow-up times. For example, in pigs treated for 2 days, exenatide reduced infarct area 33%, prevented deterioration of systolic and diastolic cardiac function, and decreased myocardial stiffness 54% when assessed on the third day after treatment initiation . At the molecular level, AKT activation increased in concert with increased expression of anti-apoptotic BCL-2 and decreased expression of pro-apoptotic caspase 3. In a second example, 7 days of pre-MI treatment with the GLP-1R agonist liraglutide reduced mouse cardiac infarct size, while improving cardiac output and survival . Four weeks post-MI, measures of systolic function (cardiac output, stroke volume) and mitral flow velocities (E/A ratio) were significantly improved compared with sham-operated mice, combined with reduced LV dilatation. Furthermore, all these effects were independent of liraglutide-induced weight loss. Ex vivo, liraglutide prevented ischemia-reperfusion injury in isolated, perfused mouse hearts and reduced apoptosis in neonatal mouse cardiomyocytes. In normal healthy mice (without MI), liraglutide increased AKT activation, a response that was absent in GLP-1R knockout mice. In the one study examining human patients with acute MI, 72-hours of GLP-1 infusion added to standard therapy was associated with significantly improved LVEF (29% to 39% compared with no change in the control group) and contractile function (-21% in regional wall motion score index versus no change in the control group) measured 6 to 12 hours after infusion . Moreover, in pigs and dogs GLP-1 improved myocardial glucose-uptake and metabolism [50, 51].
The ability of exenatide to reduce blood pressure in humans may contribute to the peptide's potential to play a cardioprotective role. In an open-label, 82-week study in patients with type 2 diabetes, exenatide reduced mean diastolic blood pressure and improved lipid profiles . In a 24-week, clinical trial in patients with type 2 diabetes, exenatide reduced mean systolic and diastolic blood pressure in contrast to non-significant changes in the placebo arm . The blood pressure effects of exenatide treatment lasting at least 6 months was also examined in pooled data from 6 trials including 2,171 subjects . Exenatide was associated with significantly decreased systolic BP compared with placebo or insulin in patients with elevated BP at baseline, with the greatest effects observed in subjects with baseline systolic BP ≥130 mmHg.
In another study, 12 weeks of exenatide treatment in patients with type 2 diabetes was associated with a trend towards lower 24-hr, daytime, and nighttime systolic blood pressure, but had no clinically meaningful effect on heart rate, compared with placebo . Further, using a well-established risk-assessment model, Sullivan et al.  projected substantial reductions in cardiovascular death rates and fewer cardiovascular events over 30 years in patients with diabetes treated with the GLP-1R agonist, liraglutide.
Exercise intolerance is a hallmark symptom of CHF regardless of disease etiology, and is closely related to increased insulin resistance . Agents that stimulate glucose oxidation (directly or indirectly) improve exercise capacity in humans [56–58]. In the present study, a reduction in basal plasma lactate and an increase in the ratio of exercise capacity to the lactate peak during exercise was observed with GLP-1 or AC3174 treatment, in parallel with increased insulin sensitivity. These data suggest whole body glucose utilization was improved in all treatment groups. Thus, it is possible that normalization of hyperglycemia and improvement in insulin sensitivity may have contributed to the enhancement of exercise performance, in addition to the benefits of improved cardiac function and remodeling. However, whether or not the improvements in insulin sensitivity associated with chronic GLP-1 or AC3174 treatment directly contributed to the cardioprotective effects of these peptides remains to be determined. Regarding the significantly lower VO2 levels observed in the 3174H group, reduced food intake/body weight may have contributed to this result. Previous studies have shown that equivalent doses of exenatide lower food intake in diet induced obese rats . However, the mechanism of action of AC3174 to change VO2 is not clear and is likely to be multifactorial.
Implications of GLP-R Activation for Survival after MI
Survival increased with GLP-1 or AC3174 treatment in the MI-induced CHF rat model. Although attenuation of insulin resistance by GLP-1 or AC3174 may contribute to this benefit, insulin-independent cardiac or extra-cardiac actions such as vasodilatation, renoprotection, and reduction of apoptosis [2, 59–61] may have also contributed to the reduction in mortality. Of mention, insulin sensitizers such as peroxisome proliferators-activated receptor γ (PPARγ) activators (e.g., thiazolidinediones) have cardioprotective effects similar to GLP-1. However, PPARγ activators are contraindicated in CHF due to their propensity to increase the incidence of fluid retention and edema in humans , and increase mortality in rats with MI-induced CHF .
While the mechanisms of the observed cardioprotective effects remain unclear, several likely mechanisms were explored. In a previous study of isolated perfused rat hearts subjected to ischemia and reperfusion, acute treatment with high concentrations of GLP-1 enhanced recovery of cardiac function by improving myocardial glucose uptake and translocation of the glucose transporters, GLUT-1 and GLUT-4, during reperfusion . Although the mechanism of translocation remains elusive, it appears the AKT-2 downstream signal transduction pathway contributes to the translocation of GLUT-4 . In the present study, long term treatment with GLP-1 or AC3174 did not significantly alter myocardial GLUT1 or GLUT4 translocation. These data suggest the observed cardioprotective effects may occur, at least in part, independent of specific cardiac metabolic improvements.
While GLP-1 and the exenatide analogue AC3174 exhibit comparable binding potency at the GLP-1 receptor , in this study AC3174 exhibited several distinct pharmacodynamic actions compared with GLP-1. For instance, treatment with AC3174 resulted in significant weight loss mediated by selective loss of body fat. Furthermore, the highest dose of AC3174 tested was associated with a relatively low PVO2. This could be due to relatively more potent and sustained inhibitory effects of exenatide on food intake and energy expenditure than observed with GLP-1 . However, the cardioprotective effects of the GLP-1R agonist liraglutide in a mouse MI model were found to be independent of weight reduction .
In an isolated rat heart model of MI, administration of GLP-1 during the first 15 minutes post-ischemia reperfusion reduced infarct size through a GLP-1 receptor-mediated pathway, but had no inotropic effects (mechanical performance) . In contrast, administration of the primary GLP-1 metabolite GLP-1(9-36) had no effect on infarct size, but did have a strong negative inotropic effect. Because GLP-1(9-36) has little or no binding affinity for the known GLP-1R, these data suggest the involvement of GLP-1R-independent effects on cardiac function post-MI. A more recent exploration of this hypothesis found that isolated mouse hearts rapidly convert infused GLP-1 to GLP-1(9-36) . After ischemia-reperfusion injury of isolated mouse hearts, administration of GLP-1(9-36) or exenatide improved functional recovery, reduced infarct size, improved cardiomyocyte viability, reduced lactate dehydrogenase release and decreased caspase-3 activation. Counter to expectations, the cardioprotective actions of GLP-1(9-36) were blocked by an antagonist of GLP-1R binding, yet preserved in cardiomyocytes from GLP-1R knockout mice. Overall, these data lend further support to a cardio-sparing signal transduction pathway distinct from that associated with the GLP-1 receptor.
One possible limitation of this study is that standard treatments for MI, e.g. ACE-inhibitors, were not co-administered with GLP-1 or AC3174. However, in a recent publication , the ACE inhibitor captopril had additive effects with AC3174 in reducing cardiac left ventricular mass and improving renal morphology in a rat model of hypertension characterized by profound hypertension, cardiac hypertrophy, insulin resistance, renal pathology, and early-onset mortality. AC3174 plus captopril lengthened survival and had anti-hypertensive, insulin-sensitizing, and renoprotective effects. Another possible limitation is that levels of catecholamines, cortisol, glucagon, free fatty acids, renin, and aldosterone were not measured in the MI rat model. Levels of these compounds can increase in heart failure patients and it is possible similar changes may have influenced the physiological responses to AC3174 or GLP-1 in the rat model.