Generation of peroxynitrite contributes to ischemia-reperfusion injury in isolated rat hearts

OBJECTIVE: The acute release of radicals upon reperfusion following myocardial ischemia may include both nitric oxide (NO) and superoxide anion (O2-.). The generation of peroxynitrite (ONOO-) from these radicals may contribute to ischemia-reperfusion injury. Our objective was to measure the generation of ONOO- during reperfusion of isolated hearts subjected to ischemia and to determine the effects of inhibition of NO synthase with NG-monomethyl-L-arginine (L-NMMA), or supplementation of NO with S-nitroso-N-acetyl-D,L-penicillamine (SNAP), on ONOO- generation and on the recovery of mechanical function. METHODS AND RESULTS: Isolated rat hearts were perfused at constant pressure with Krebs' buffer containing L-tyrosine, which reacts with ONOO- to form dityrosine, a fluorescent product. Dityrosine was detected in the coronary effluent of hearts infused with synthetic ONOO-. In hearts subjected to 20 min of global, no-flow ischemia there was a marked rise in endogenous ONOO- formation which peaked at 30 s of reperfusion. Formation of ONOO- was dependent upon synthesis of both NO and O2-., as dityrosine release was abolished by L-NMMA or superoxide dismutase, respectively. L-NMMA caused a concentration-dependent improvement in the recovery of mechanical function during reperfusion. Infusion of SNAP also abolished dityrosine release at reperfusion and improved the recovery of post-ischemic function. CONCLUSIONS: Our results show for the first time that reperfusion of the ischemic heart causes the acute production of ONOO-. Inhibiting the biosynthesis of ONOO- with L-NMMA or antagonizing its oxidant actions with SNAP are possible strategies to protect the heart from ischemia-reperfusion injury.