Reduced Tyrosine Nitration of VDAC and Decreased Apoptosis by Mitochondria-Directed Therapy After Cardiac Ischemia Reperfusion in Isolated Hearts

Abstract

Superoxide (O2•-) produced during cardiac ischemia-reperfusion (IR) injury reacts with nitric oxide to form peroxynitrite (ONOO-). ONOO- induces protein tyrosine nitration (tyrN) that causes protein structural alteration and dysfunction. The mitochondrial voltage-dependent anion channel (VDAC) plays an important role in regulating the metabolic and energetic functions of mitochondria and contributes to mitochondrial-mediated apoptosis. It is not known if VDAC is nitrated by ONOO- during IR or how this modification might compromise cardiac function after IR. Because of the importance of VDAC modification, we hypothesized that the clinically used anti-anginal drug ranolazine (RAN), which also reduces cardiac IR injury, does so via a mitochondrial mechanism, i.e., in part by decreasing VDAC tyrN. To test this, isolated guinea pig hearts were perfused with KR buffer for 40 min (time control, TC), or for 30 min of ischemia plus 10 min of reperfusion, with or without 10 µM RAN infused before ischemia. Mitochondria were isolated at the end of each treatment. VDAC tyrN was determined by IP with anti-nitrotyrosine antibody (NTab), followed by Western blotting (WB) with anti-VDAC antibody. The effect of RAN on VDAC tyrN was also examined. Cytochrome c release was checked as the marker for apoptosis. We found that enhanced VDAC tyrN was increased by 108% after IR vs. TC and cytochrome c was higher in the cytosol after IR than after TC. RAN treatment decreased VDAC tyrN by 31%, while decreasing cytochrome c release by 38%, compared to IR. These results indicate that VDAC tyrN and a concomitant increase in cytochrome c release occur during IR injury, and importantly, that cardioprotection by RAN occurs in part by reducing VDAC tyrN, which may impede activation of apoptotic pathways during IR injury.