Mechanisms of reoxygenation injury in cultured ventricular myocytes.

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Abstract
To investigate factors contributing to reperfusion and reoxygenation myocardial injury, we exposed layers of cultured chick ventricular myocytes to severe hypoxia for up to 3 hours in the presence of 20 mM 2-deoxyglucose, zero glucose, and 5 mM pyruvate, and then exposed the myocytes to reoxygenation. Lactate dehydrogenase (LDH) release was moderately increased during 3 hours of hypoxia but was increased markedly during reoxygenation. Coincident changes in intracellular calcium concentration ([Ca2+]i) and cell motion were also measured during hypoxia and reoxygenation. During hypoxia, [Ca2+]i increased to more than 1 microM, and with reoxygenation, [Ca2+]i abruptly decreased slightly but remained elevated more than 1 microM. Cells developed a stable rigor after 30 minutes of hypoxia. Reoxygenation caused a marked hypercontracture within 5 minutes. Pretreatment of myocytes with either 2,3-butanedione monoxime, which inhibits Ca2(+)-dependent force development, or cyanide inhibited reoxygenation hypercontracture. LDH release after reoxygenation was also significantly reduced in the presence of 2,3-butanedione monoxime. Treatment of myocytes with superoxide dismutase and catalase during hypoxia also resulted in a decrease in LDH release during reoxygenation. We conclude that an abrupt increase in [Ca2+]i during reoxygenation does not account for reoxygenation injury. However, in the presence of elevated [Ca2+]i, reoxygenation and the resulting probable resynthesis of ATP causes [Ca2+]i-dependent myofilament crossbridge cycling, and the resulting hypercontracture contributes to myocyte damage. The generation of oxygen free radicals after reoxygenation also appears to contribute to cell injury in this system.

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