Effect of extracellular Mg2+ on ROS and Ca2+ accumulation during reoxygenation of rat cardiomyocytes

Abstract

The effects of Mg2+ on reactive oxygen species (ROS) and cell Ca2+ during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2′,7′-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H2O2 and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O2 −·), respectively. Rapidly exchangeable cell Ca2+ was determined by 45Ca2+uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100–130% during reoxygenation alone and further increased 60% by increasing extracellular Mg2+concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16–24% during reoxygenation but were not affected by Mg2+. Cell Ca2+ increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg2+, 57% by 10 μM 3,4-dichlorobenzamil (DCB) (inhibitor of Na+/Ca2+ exchange), and 75% by combining Mg2+ and DCB. H2O2 (25 and 500 μM) reduced Ca2+ accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso- N-acetyl-penicillamine (1 mM) had no effect. Mg2+ reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg2+ to 5 mM increased the fluorescence of the H2O2/NO-sensitive probe DCF without increasing that of the O2 −·-sensitive probe ETH, reduced Ca2+ accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg2+, may be linked to the effect of Mg2+ on Ca2+ accumulation and/or ROS levels.