Peroxynitrite induced nitration and inactivation of myofibrillar creatine kinase in experimental heart failure

Objective: Oxidative stress is implicated in the initiation and progression of congestive heart failure, but the putative reactive species and cellula r targets involved remain undefined. We have previously shown that peroxyni trite (ONOO-, an aggressive biological oxidant and nitrating agent) potentl y inhibits myofibrillar creatine kinase (MM-CK), a critical controller of c ontractility known to be impaired during heart failure. Here we hypothesize d that nitration and inhibition of MM-CK participate in cardiac failure in vivo. Methods: Heart failure was induced in rats by myocardial infarction ( left coronary artery ligation) and confirmed by histological analysis at 8 weeks postinfarct (1.3 +/- 1.4 vs. 37.7 +/- 3.2% left ventricular circumfer ence; sham control vs. CHF, n = 10 each). Results: Immunohistochemistry dem onstrated significantly increased protein nitration in failing myocardium c ompared to control (optical density: 0.5 +/- 0.06 vs. 0.93 +/- 0.09, sham v s. CHF, P < 0.05). Significant decreases in MM-CK activity and content were observed in failing hearts (MM-CK k(cat): 6.0 +/- 0.4 vs. 3.0 +/- 0.3 <mu> mol/nM M-CK/min, P < 0.05; 6.8 +/- 1.3 vs. 4.7 +/- 1.2% myofibrillar protei n, P < 0.05), with no change in myosin ATPase activity. In separate experim ents, isolated rat cardiac myofibrils were exposed to ONOO- (2-250 muM) and enzyme studies were conducted. Identical to in vivo studies, selective red uctions in MM-CK were observed at ONOO- concentrations as low as 2 muM (IC5 0=92.5 +/- 6.0 muM); myosin ATPase was unaffected with ONOO- concentrations as high as 250 muM. Concentration dependent nitration of MM-CK occurred an d extent of nitration was statistically correlated to extent of CK inhibiti on (P < 0.001). Immunoprecipitation of MM-CK from failing left ventricle yi elded significant evidence of tyrosine nitration. Conclusion: These data de monstrate that cardiac ONOO- formation and perturbation of myofibrillar ene rgetic controllers occur during experimental heart failure; MM-CR may be a critical cellular target in this setting. (C) 2001 Elsevier Science B.V. Al l rights reserved.