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Peroxynitrite induced nitration and inactivation of myofibrillar creatine kinase in experimental heart failure
M J Mihm1, C M Coyle, B L Schanbacher
1Division of Pharmacology/College of Pharmacy and OSU Heart and Lung Research Institute The Ohio State University, 412 Riffe Building, 500 West Twelfth Street, Columbus, OH 43210, USA.
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 micromol/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 microM) and enzyme studies were conducted. Identical to in vivo studies, selective reductions in MM-CK were observed at ONOO(-) concentrations as low as 2 microM (IC(50)=92.5+/-6.0 microM); myosin ATPase was unaffected with ONOO(-) concentrations as high as 250 microM. 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.
Insights
Oxidative stress contributes to heart failure by nitrating and inhibiting myofibrillar creatine kinase (MM-CK). This study confirms MM-CK is a key target in experimental cardiac failure.
Area of Science:
- Cardiovascular Research
- Biochemistry
- Oxidative Stress Biology
Background:
- Oxidative stress is a known factor in heart failure development.
- The specific reactive species and cellular targets remain unclear.
- Peroxynitrite (ONOO(-)) inhibits myofibrillar creatine kinase (MM-CK), crucial for heart contractility.
Purpose of the Study:
- To investigate if peroxynitrite-induced nitration and inhibition of MM-CK contribute to in vivo cardiac failure.
- To identify MM-CK as a cellular target in experimental heart failure.
Main Methods:
- Heart failure was induced in rats via myocardial infarction.
- Immunohistochemistry and enzyme activity assays were used to assess protein nitration and MM-CK function.
- Isolated cardiac myofibrils were exposed to peroxynitrite in vitro.
Main Results:
- Failing rat hearts showed increased protein nitration and significantly reduced MM-CK activity and content.
- In vitro studies demonstrated peroxynitrite selectively inhibited and nitrated MM-CK.
- MM-CK nitration strongly correlated with its inhibition.
Conclusions:
- Cardiac peroxynitrite formation and impaired myofibrillar energetics occur in experimental heart failure.
- Myofibrillar creatine kinase (MM-CK) is identified as a critical cellular target in this condition.