Analysis of Mitochondrial Proteins in the Surviving Myocardium after Ischemia Identifies Mitochondrial Pyruvate Carrier Expression as Possible Mediator of Tissue Viability

Abstract

The endogenous mechanisms contributing to tissue survival following myocardial infarction are not fully understood. We investigated the alterations in the mitochondrial proteome after ischemia-reperfusion (I/R) and its possible implications on cell survival. Mitochondrial proteomic analysis of cardiac tissue from an in vivo porcine I/R model found that surviving tissue in the peri-infarct border zone showed increased expression of several proteins. Notably, these included subunits of the mitochondrial pyruvate carrier (MPC), namely MPC1 and MPC2. Western blot, immunohistochemistry, and mRNA analysis corroborated the elevated expression of MPC in the surviving tissue. Furthermore, MPC1 and MPC2 protein levels were found to be markedly elevated in the myocardium of ischemic cardiomyopathy patients. These findings led to the hypothesis that increased MPC expression is cardioprotective due to enhancement of mitochondrial pyruvate uptake in the energy-starved heart following I/R. To test this, isolated mouse hearts perfused with a modified Krebs buffer (containing glucose, pyruvate, and octanoate as metabolic substrates) were subjected to I/R with or without the MPC transport inhibitor UK5099. UK5099 increased myocardial infarction and attenuated post-ischemic recovery of left ventricular end-diastolic pressure. However, aerobically perfused control hearts that were exposed to UK5099 did not modulate contractile function, although pyruvate uptake was blocked as evidenced by increased cytosolic lactate and pyruvate levels. Our findings indicate that increased expression of MPC leads to enhanced uptake and utilization of pyruvate during I/R. We propose this as a putative endogenous mechanism that promotes myocardial survival to limit infarct size. Ischemic heart disease resulting from the obstruction of coronary arteries commonly culminates in myocardial infarction. Although restoring blood flow is necessary to re-establish oxygen and nutrients to the ischemic myocardium, reperfusion itself may promote damage due to exacerbated oxidative stress and inflammation (1). Even when patients survive, the damage caused to the heart typically triggers events leading to heart failure. Unfortunately, there are still no effective interventions for limiting injury after ischemia despite the potential of such therapeutic options to reduce morbidity and mortality.Laboratory and clinical studies suggest that the post-infarction failing heart is characterized by a diminished capacity to convert chemical energy into mechanical work due to mitochondrial imbalances (2). The primary function of mitochondria is to generate ATP via oxidative phosphorylation, which is used by the heart to meet its significant energy demands. In the well perfused healthy heart, ϳ70 -90% of ATP is via ␤-oxidation of fatty acids, with most of the remaining amount coming from the oxidation of glucose and lactate (3, 4). However, when oxygen is restricted as occurs during ischemia or sub-optimal reperfusion, there is a metabolic shift with glycolysis becoming the ...

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“…However, studies regarding the importance of MPC activity in cardiac function or development of heart failure have been quite limited. Expression of MPC1 and MPC2 was shown to be an important marker of surviving myocardium near the border of infarct zones in a pig model, and this study also identified increased MPC expression in human hearts with ischemic heart failure 39 . While this current work was in preparation, another report showed that failing human hearts exhibited decreased expression of the MPC proteins 23 , which we have confirmed in this current study.…”

Pyruvate and Metabolic Flexibility: Illuminating a Path Toward Selective Cancer Therapies

“…Over the course of development, the heart transitions such that fatty acid β -oxidation largely fuels the adult heart, but the heart retains the flexibility to temporarily switch to a glycolytic phenotype depending on nutrient availability and energetic demand [10]. It is becoming clear that the development of heart failure is accompanied by a reversion back to the fetal glycolytic program and a loss of metabolic flexibility [11-14], which may be potentiated by decreased expression of the MPC [15,16]. This metabolic regime was first described over 100 years ago, when patients with various heart maladies temporarily improved after administration of cane sugar [17].…”

“…MCT-1 and MCT-4 have both been implicated in lactate export, although recent data suggest that MCT-4 plays the predominant role in many cancers. Much like LDH-A, several studies have demonstrated that MCT-4 expression, which is elevated in many cancers, is required for the full expression of the cancer phenotype and is associated with poor clinical outcomes [15,16,42-46]. …”

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“…Due to epitope masking, ECM proteins may not be detectable by antibodies. (ii) Antibodies are often not commercially developed to target proteins in species beyond the commonly used mice and rats such as canine and porcine models for myocardial infarction, 2 , 45 , 46 rabbit and goat models for studies involving atrial fibrillation 9 , 47–49 and sheep as models of dilated cardiomyopathy 50 . While some anti-human or anti-mouse antibodies will cross-react, many others will not recognize their target protein in different species or will display a high degree of non-specific binding.…”

“…For instance, cardiac ECM proteins are markedly less abundant than cytosolic and mitochondrial proteins 46 . Thus, studies that use whole tissue lysates for proteomic analysis inevitably mask the identification of the less abundant ECM constituents.…”