GCN5L1 inhibits pyruvate dehydrogenase phosphorylation during cardiac ischemia-reperfusion injury

Abstract

Myocardial infarction remains one of the leading causes of mortality. Reperfusion of the infarcted myocardium restores blood flow and reduces primary ischemic injury. However, despite its protective function, reperfusion is also associated with several deleterious outcomes that can result in ischemia-reperfusion (I/R) injury to cardiac tissue. While negative outcomes such as reactive oxygen species generation are strongly associated with I/R injury, cardiac energy metabolism is also greatly disrupted. Furthermore, previous studies have shown that the restoration of normal fuel oxidation in the myocardium regulates the extent of contractile recovery. A better understanding of the pathophysiological mechanisms underlying I/R injury may allow us to develop new treatments that limit the negative aspects of the process. In this study, we examined the role played by GCN5L1, a protein implicated in the regulation of energy metabolism, in I/R injury. We demonstrate that cardiac-specific loss of GCN5L1 promotes the inhibitory phosphorylation of pyruvate dehydrogenase in vitro and in vivo, a process likely to inhibit glucose oxidation, and that this corresponds to increased myocardial damage following ischemia-reperfusion (I/R) injury.

Figure 1:. GCN5L1 abundance is inversely correlated with pyruvate dehydrogenase phosphorylation in cardiac AC16 cells.

A. Representative immunoblots from stable cardiac AC16 cell lines with overexpression (OE) or knockdown (KD) of GCN5L1. In cells overexpressing GCN5L1, the endogenous protein is largely absent, suggesting a homeostatic response to limit total GCN5L1 levels. B-G. Protein abundance of GCN5L1, PDH, p-PDH, PDK4, PDP1, and PDPR in GCN5L1 OE or KD cells. N = 3; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; one-way ANOVA with Tukey’s post-hoc test.

Figure 2:. GCN5L1 knockdown in AC16 cells leads to increased abundance of pyruvate dehydrogenase inhibitory proteins after hypoxia/reoxygenation stress.

A. Representative immunoblots from control or GCN5L1 KD AC16 cells after normoxia (Norm) or hypoxia/reoxygenation (H/R) treatment. B-G. Protein abundance of PDH, p-PDH, PDK4, PDP1, and PDPR in control and GCN5L1 KD AC16 cells after normoxia or hypoxia/reoxygenation. N = 3; * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001; one-way ANOVA with Tukey’s post-hoc test.

Figure 3:. Inhibitory pyruvate dehydrogenase phosphorylation is elevated in GCN5L1 cKO hearts following ischemia-reperfusion injury.

A. Representative immunoblots from wildtype (Con) and cardiac-specific GCN5L1 knockout (GCN5L1 cKO) mouse hearts after sham or ischemia-reperfusion (I/R) injury surgery. B-C. Protein abundance of GCN5L1 and p-PDH in wildtype or GCN5L1 cKO mouse hearts after sham or ischemia/reperfusion injury surgery. N = 4; ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001; one-way ANOVA with Tukey’s post-hoc test.

Figure 4:. Loss of cardiomyocyte GCN5L1 expression leads to elevated tissue damage markers in the absence of functional decline.

A-D. Echocardiographic analysis of left ventricle (LV) systolic function (EF, ejection fraction; FS, fractional shortening) and chamber volume at systole (Sys) and diastole (Dia) in wildtype and GCN5L1 cKO mice 24 h after sham or I/R surgery. N = 7–8. E-F. Serum troponin and lactate dehydrogenase (LDH) 24 h after sham or I/R surgery in wildtype and GCN5L1 cKO mice. N = 4; * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001; one-way ANOVA with Tukey’s post-hoc test.