Mechanisms of reoxygenation injury in cultured ventricular myocytes.
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.
Quaife
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RA
Cardiology Division, University of Utah Medical Center, Salt Lake City, Salt Lake City 84132.
Kohmoto
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O
Barry
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WH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D002863:Chromogenic Compounds / Q000494:pharmacology; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D005609:Free Radicals; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009200:Myocardial Contraction / Q000502:physiology*; D015428:Myocardial Reperfusion Injury / Q000209:etiology*; D009206:Myocardium / Q000473:pathology*; D013997:Time Factors
D002863:Chromogenic Compounds; D005609:Free Radicals; C004717:diacetylmonoxime; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D003931:Diacetyl; D002118:Calcium