Adaptations in Protein Expression and Regulated Activity of Pyruvate Dehydrogenase Multienzyme Complex in Human Systolic Heart Failure

Pyruvate dehydrogenase (PDH) complex, a multienzyme complex at the nexus of glycolytic and Krebs cycles, provides acetyl-CoA to the Krebs cycle and NADH to complex I thus supporting a critical role in mitochondrial energy production and cellular survival. PDH activity is regulated by pyruvate dehydrogenase phosphatases (PDP1, PDP2), pyruvate dehydrogenase kinases (PDK 1-4), and mitochondrial pyruvate carriers (MPC1, MPC2). As NADH-dependent oxidative phosphorylation is diminished in systolic heart failure, we tested whether the left ventricular myocardium (LV) from end-stage systolic adult heart failure patients (n=26) exhibits altered expression of PDH complex subunits, PDK, MPC, PDP, and PDH complex activity, compared to LV from nonfailing donor hearts (n=21). Compared to nonfailing LV, PDH activity and relative expression levels of E2, E3bp, E1α, and E1β subunits were greater in LV failure. PDK4, MPC1, and MPC2 expressions were decreased in failing LV, whereas PDP1, PDP2, PDK1, and PDK2 expressions did not differ between nonfailing and failing LV. In order to examine PDK4 further, donor human LV cardiomyocytes were induced in culture to hypertrophy with 0.1 μM angiotensin II and treated with PDK inhibitors (0.2 mM dichloroacetate, or 5 mM pyruvate) or activators (0.6 mM NADH plus 50 μM acetyl CoA). In isolated hypertrophic cardiomyocytes in vitro, PDK activators and inhibitors increased and decreased PDK4, respectively. In conclusion, in end-stage failing hearts, greater expression of PDH proteins and decreased expression of PDK4, MPC1, and MPC2 were evident with higher rates of PDH activity. These adaptations support sustained capacity for PDH to facilitate glucose metabolism in the face of other failing bioenergetic pathways

Postnatal shifts in ischemic tolerance and cell survival signaling in murine myocardium

The immature heart is known to be resistant to ischemia-reperfusion (I/R) injury; however, key proteins engaged in phospho-dependent signaling pathways crucial to cell survival are not yet defined. Our goal was to determine the postnatal changes in myocardial tolerance to I/R, including baseline expression of key proteins governing I/R tolerance and their phosphorylation during I/R. Hearts from male C57Bl/6 mice (neonates, 2, 4, 8, and 12 wk of age, n = 6/group) were assayed for survival signaling/effectors [Akt, p38MAPK, glycogen synthase kinase-3 beta (GSK-3 beta), heat shock protein 27 (HSP27), connexin-43, hypoxiainducible factor-1 alpha (HIF-1 alpha), and caveolin-3] and regulators of apoptosis (Bax and Bcl-2) and autophagy (LC3B, Parkin, and Beclin1). The effect of I/R on ventricular function was measured in isolated perfused hearts from immature (4 wk) and adult (12 wk) mice. The neonatal myocardium exhibits a large pool of inactive Akt; high phospho-activation of p38MAPK, HSP27 and connexin-43; phosphoinhibition of GSK-3 beta; and high expression of caveolin-3, HIF-1 alpha, LC3B, Beclin1, Bax, and Bcl-2. Immature hearts sustained less dysfunction and infarction following I/R than adults. Emergence of I/R intolerance in adult vs. immature hearts was associated with complex proteomic changes: decreased expression of Akt, Bax, and Bcl-2; increased GSK-3 beta, connexin-43, HIF-1 alpha, LC3B, and Bax: Bcl-2; enhanced postischemic HIF-1 alpha, caveolin-3, Bax, and Bcl-2; and greater postischemic GSK-3 beta and HSP27 phosphorylation. Neonatal myocardial stress resistance reflects high expression of prosurvival and autophagy proteins and apoptotic regulators. Notably, there is high phosphorylation of GSK-3 beta, p38MAPK, and HSP27 and low phosphorylation of Akt (high Akt "reserve"). Subsequent maturation-related reductions in I/R tolerance are associated with reductions in Akt, Bcl-2, LC3B, and Beclin1, despite increased expression and reduced phospho-inhibition of GSK-3 bet