Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation.
Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, and the combination of both attenuated the hypoxia-reoxygenation-induced reduction in Delta(psi)m, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.
Sharikabad
M N
MN
Division of Clinical Pharmacology and Toxicology, Clinical Chemistry Department, Ullevaal University Hospital, N-0407 Oslo, Norway. m.n.sharikabad@ioks.uio.no
Ostbye
K M
KM
Brørs
O
O
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology; D048429:Cell Size; D002998:Clonazepam / Q000494:pharmacology; D005434:Flow Cytometry; D018757:GABA Modulators / Q000494:pharmacology; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D017202:Myocardial Ischemia / Q000378:metabolism; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000166:cytology*; D051381:Rats; D017208:Rats, Wistar
D018757:GABA Modulators; D002998:Clonazepam; D008274:Magnesium; D002118:Calcium