Increased [Mg2+]oreduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation

Abstract

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 [Mg2+]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 (Δψm). 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 ∼40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]iwere 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 μM), 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 Δψm, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Δψm 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 Δψm 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.