Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging.
Excess superoxide (O(2)(-)) and nitric oxide (NO) 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)(-)/ONOO(-) during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15kDa 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 35kDa 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)(-)/ONOO(-), reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O(2)(-) 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.
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Meiying
M
Yang
Amadou K S
AK
Camara
Bassam T
BT
Wakim
Yifan
Y
Zhou
Ashish K
AK
Gadicherla
Wai-Meng
WM
Kwok
David F
DF
Stowe
D000818:Animals; D006168:Guinea Pigs; D013058:Mass Spectrometry; D024101:Mitochondrial Proteins / Q000378:metabolism*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis; D030421:Peroxynitrous Acid / Q000378:metabolism*; D000077185:Resveratrol; D013267:Stilbenes / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism*
D024101:Mitochondrial Proteins; D013267:Stilbenes; D050994:Voltage-Dependent Anion Channels; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D014443:Tyrosine; D000077185:Resveratrol; D019331:NG-Nitroarginine Methyl Ester