Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging

Excess superoxide (O-2(center dot-)) and nitric oxide (NO center dot) forms peroxynitrite (ONOO-) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO-. Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O-2(center dot-)/ONOO- during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15 kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC-MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35 kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO-. We also found that ONOO- directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO- formed oligomers. Resveratrol (RES), a scavenger of O-2(center dot-)/ONOO-, reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO center dot generation, only reduced tyr-N levels of native VDAC. O-2(center dot-). and ONOO- levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO- during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function. (C) 2012 Published by Elsevier B.V.