This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.

This study was undertaken to investigate the effects of oxygen free radicals on myofibrillar creatine kinase activity. Isolated rat heart myofibrils were incubated with xanthine+xanthine oxidase (a superoxide anion radical-generating system) or hydrogen peroxide and assayed for creatine kinase activity. To clarify the involvement of changes in sulfhydryl groups in causing alterations in myofibrillar creatine kinase activity, 1) effects of N-ethylmaleimide (sulfhydryl groups reagent) on myofibrillar creatine kinase activity, 2) effects of oxygen free radicals on myofibrillar sulfhydryl groups content, and 3) protective effects of dithiothreitol (sulfhydryl groups-reducing agent) on the changes in myofibrillar creatine kinase activity due to oxygen free radicals were also studied. Xanthine+xanthine oxidase inhibited creatine kinase activity both in a time- and a concentration-dependent manner. Superoxide dismutase (SOD) showed a protective effect on the depression in creatine kinase activity caused by xanthine+xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a concentration-dependent manner; this inhibition was prevented by the addition of catalase. N-ethylmaleimide reduced creatine kinase activity in a dose-dependent manner. The content of myofibrillar sulfhydryl groups was decreased by xanthine+xanthine oxidase; this reduction was prevented by SOD. Furthermore, the depression in myofibrillar creatine kinase activity by xanthine+xanthine oxidase was protected by the addition of dithiothreitol. Oxygen free radicals may inhibit myofibrillar creatine kinase activity by modifying sulfhydryl groups in the enzyme protein. The reduction of myofibrillar creatine kinase activity may lead to a disturbance of energy utilization in the heart and may contribute to cardiac dysfunction due to oxygen free radicals.

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The Bio 14.6 cardiomyopathic Syrian hamster is an animal model of human idiopathic cardiomyopathy. The pathogenesis of the disease in this animal has not yet been clearly elucidated. It is well known that alpha- and beta-adrenergic receptors are increased in the myocardium of this animal, but that isoprenaline does not produce an augmented response. We examined the activity of cardiac stimulatory GTP-binding protein (Gs), which couple with beta-adrenergic receptors to stimulate adenylate cyclase, in Bio 14.6 cardiomyopathic hamsters at 90 and 160 days of age. The cardiac norepinephrine concentration was significantly increased in Bio 14.6 hamsters compared with control hamsters (F1B) at 90 days of age (1,739 +/- 120 vs 1,470 +/- 161 ng/g wet tissue weight, p less than 0.05). Cardiac forskolin-stimulated adenylate cyclase activities at 90 and 160 days of age were lower in the cardiomyopathic hamsters than in the F1B controls (90 days old: 98 +/- 24 vs 122 +/- 29 pmol/min/mg protein, p less than 0.05; 160 days old: 74 +/- 13 vs 124 +/- 28 pmol/min/mg protein, p less than 0.01). Cardiac Gs activities at 90 and 160 days of age were significantly lower in Bio 14.6 hamsters than those in F1B hamsters (90 days old: 204 +/- 42 vs 259 +/- 49 pmol/min/mg protein, p less than 0.05; 160 days old: 156 +/- 39 vs 211 +/- 60 pmol/min/mg protein, p less than 0.05). We thus demonstrated functional defects in cardiac Gs protein and adenylate cyclase activity in the Bio 14.6 cardiomyopathic hamsters at 90 to 160 days of age (the hypertrophic stage of cardiomyopathy).(ABSTRACT TRUNCATED AT 250 WORDS)