In the present study, we observed that old dogs with myocardial insulin resistance developed an accelerated course of dilated cardiomyopathy in response to rapid ventricular pacing compared to Young-Control. Treatment with a continuous infusion of GLP-1(7–36) amide improved myocardial insulin sensitivity, reduced myocardial mitochondrial oxidative stress, preserved expression of key mitochondrial proteins associated with oxidative balance (UCP-3) and mitochondrial respiration (MCO), and preserved coronary flow reserve culminating in an improved clinical course and reduced mortality following rapid pacing. These findings underscore the role of mitochondrial dysfunction and associated impaired myocardial insulin responsiveness in the progression of heart failure in advanced age.
Aging has been associated with altered mitochondrial protein expression and reduced energetic reserve in studies of skeletal muscle ,. These alterations have been linked to the cellular mechanisms of insulin resistance, mediated through increased accumulation of NEFA , features which have been associated with increased reactive oxygen species production in senescent mitochondria . Under conditions in which ROS production exceeds anti-oxidative capacity, increased UCP-3 activity dissipates the electromotive force and reduces mitochondrial ROS production . A consequence of the decreased transmenbrane potential (∆φ) is reduced electromotive force for ATP generation, which may become relevant under circumstances of increased energetic demand, as occurs with pacing induced heart failure ,. When these physiological adaptive responses are further compromised by alterations in components of the electron transfer chain, such as MCO or decreased expression or function of UCP-3, further ROS generation leads to lipid peroxidation and eventually mitochondrial permeability transition, and ultimately necrotic cell death ,.
In the present study we observed myocardial insulin resistance and altered mitochondrial ROS generation in association with decreased MCO, and UCP-3 expression in intrafibrillar myocardial mitochondria in older beagles. We focused on IFM as the most relevant component given their role in myocardial contractility and because age-associated changes seem to affect IFM preferentially . Decreased UCP-3 expression would be expected to further impair the mitochondrial capacity to mitigate ROS leading to lipid peroxidation and further mitochondrial dysfunction .
We have shown previously that GLP-1 infusion improves cardiovascular function when administered for 48 hours to young dogs with severe dilated cardiomyopathy . We have also shown that the hemodynamic improvement was associated with both increased myocardial glucose uptake and increased expression of key components of the mitochondrial electron transport chain (ETC.), specifically MCO, the expression of which was significantly reduced in dilated cardiomyopathy. Furthermore, GLP-1 treatment was associated with reduced mitochondrial ROS production and increased UCP-3 expression. UCP-3 has been shown to be an important buffer against excess free radical accumulation within the mitochondrial matrix (29). We have also shown that normal aging in older beagles is associated with similar changes in the absence of heart failure (4). Here, we observe a similar effect of GLP-1 infusion on UCP-3 and MCO and also observe better preservation of these components following rapid pacing in old animals without baseline heart failure. Together, these salutary changes in mitochondrial protein expression were associated with slowing the progression of heart failure in the GLP-1 treated animals. Thus, both aging and heart failure appear to be associated with impaired mitochondrial function and myocardial glucose utilization and the effects appear to be synergistic.
We have also previously shown that the development of advanced heart failure in conscious dogs is associated with impaired coronary flow and vasodilator reserve in response to coronary vasodilators . Here we demonstrate that aging alone in the presence of myocardial insulin resistance is associated with impaired coronary flow reserve in response to sub-maximal doses of adenosine. Importantly, these data are the first to confirm that GLP-1 treatment improves coronary flow responses to insulin and adenosine. Furthermore, coronary flow reserve was better preserved in advanced heart failure in the Old + GLP-1 compared to Old-Control. This is the first demonstration of a vascular protective effect of GLP-1 on coronary flow reserve in a model of non-ischemic DCM.
There is an extensive literature that has demonstrated the salutary effects of both DPP-4 inhibitors  and DPP-4 resistant long acting receptor analogs  in mitigating ischemia- reperfusion injury and reducing infarct size in rodent models through a PKA dependent mechanism. These findings underscore the importance of GLP-1 receptor medicated signaling in ischemia-reperfusion syndromes. Our findings are the first to document significant salutary effects of GLP-1 (7–36) amide in a large animal model of aging and heart failure. The findings are notable in light of the recent clinical trials data that have suggested that DPP-4 inhibitors increase the risk of heart failure hospitalizations in patients with Type 2 diabetes . A critical difference is the fact that in our study, the native peptide is metabolized to the active metabolite, GLP-1 (9–36), which we  have shown to be biologically active, particularly vasoactive. These additive effects of the metabolite are not present with DPP-4 inhibitors or DPP-4 resistant GLP-1 receptor agonists. Secondly, the continuous infusion of the native peptide resulted in steady state pharmacological concentrations of total GLP-1 (~100 pM) while DPP-4 inhibitors result in physiological levels (20 pM). These differences may not only be relevant to our findings in older beagles but also explain why clinical studies of heart failure patients  and post-operative patients  have shown benefits with continuous infusions of the native peptide GLP-1 (7–36) amide.
The findings of increased mitochondrial ROS, reduced mitochondrial protein expression, and impaired myocardial insulin resistance were associations that do not prove causality in terms of the accelerated course of DCM in Old-Control. Other cellular process may also be deranged which could contribute to the response. Nonetheless, GLP-1 treatment mitigated the mitochondrial and metabolic abnormalities and protected Old-GLP-1against an accelerated course. Secondly, we measured mitochondrial protein expression not function. Additional studies detailing mitochondria abundance and morphology may help to further understand the role of mitochondrial dysfunction in this model and the extent to which GLP-1 may influence mitochondrial biogenesis and autophagy. Thirdly, it took several years to complete this study as the availability of senescent beagles was rate limiting. We began using MDA as a method to assess lipid peroxidation and continued to do so for the purposes of consistency over time. We recognize that currently there may be better quantitative methods of lipid peroxidation quantification. Finally, GLP-1 treatment was associated with improved coronary flow responses, suggesting vascular protective effects of GLP-1. While we cannot ascertain the relative contribution of these favorable changes to the improved clinical outcome in Old + GLP-1 dogs, these data demonstrate that GLP-1 treatment has multiple salutary cardiovascular effects in heart failure beyond glycemic control.