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 cellular targets involved remain undefined. We have previously shown that peroxynitrite (ONOO^-, an aggressive biological oxidant and nitrating agent) potently inhibits myofibrillar creatine kinase (MM-CK), a critical controller of contractility known to be impaired during heart failure. Here we hypothesized 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 circumference; sham control vs. CHF, n=10 each). Results: Immunohistochemistry demonstrated significantly increased protein nitration in failing myocardium compared to control (optical density: 0.58±0.06 vs. 0.93±0.09, sham vs. 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 μmol/nM M-CK/min, P<0.05; 6.8±1.3 vs. 4.7±1.2% myofibrillar protein, P<0.05), with no change in myosin ATPase activity. In separate experiments, isolated rat cardiac myofibrils were exposed to ONOO^- (2-250 μM) and enzyme studies were conducted. Identical to in vivo studies, selective reductions in MM-CK were observed at ONOO^- concentrations as low as 2 μM (IC₅₀=92.5±6.0 μM); myosin ATPase was unaffected with ONOO^- concentrations as high as 250 μM. Concentration dependent nitration of MM-CK occurred and extent of nitration was statistically correlated to extent of CK inhibition (P<0.001). Immunoprecipitation of MM-CK from failing left ventricle yielded significant evidence of tyrosine nitration. Conclusion: These data demonstrate that cardiac ONOO^- formation and perturbation of myofibrillar energetic controllers occur during experimental heart failure; MM-CK may be a critical cellular target in this setting.