Contractile and morphological impairment of cultured fetal mouse myocytes induced by oxygen radicals and oxidants. Correlation with intracellular Ca2+ concentration.

There is evidence that reperfusion injury of cardiac tissue may be caused by the generation of oxygen-derived free radicals and oxidants and by the induction of intracellular calcium overload, although the relation between these two mechanisms of injury is uncertain. In addition, the relation between the types of cellular injury and specific active species is unclear. In an attempt to resolve these problems, we investigated the effects of oxygen radicals and oxidants, which are purportedly generated during reperfusion after prolonged ischemia, and various antioxidants on contractility and morphology of cultured fetal mouse cardiac myocytes. Xanthine oxidase in the presence of xanthine, H2O2, HOCl, and NH2Cl induced cessation of spontaneous beating followed by cessation of electrical stimulation-elicited beating but did not induce an increase in [Ca2+]i. After prolonged incubation with xanthine oxidase + xanthine and H2O2, the cardiac myocytes showed morphological degeneration (at least 80% of the cells developed hypercontraction) with a concomitant increase in [Ca2+]i. These observations suggest that contractile impairment does not result in an increase of [Ca2+]i, but hypercontraction does. Catalase, but not superoxide dismutase, protected the cultured cardiac myocytes against xanthine oxidase + xanthine- and H2O2-induced contractile and morphological impairment. In the light of this observation, we hypothesize that the superoxide anion is not responsible for these types of impairment. Addition of dimethylthiourea (an .OH scavenger) and intracellular preloading with deferoxamine (an iron chelator) protected the myocytes against H2O2-induced contractile and morphological damage, but intracellular preloading with iron enhanced it. These observations led us to hypothesize that intracellularly generated .OH may be a mediator of H2O2-induced injury to cultured cardiac myocytes. In addition, we observed that H2O2 itself induced cessation of spontaneous but not electrical stimulation-elicited beating.