Effect of Calcium on Reactive Oxygen Species in Isolated Rat Cardiomyocytes During Hypoxia and Reoxygenation

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It has been suggested that calcium (Ca2+) overload and oxidative stress damage the myocardium during ischemia and reperfusion. We investigated the possible effect of varying extracellular Ca2+and total cell Ca2+on reactive oxygen species (ROS) levels in resting adult rat cardiomyocytes. Cardiomyocytes were isolated by trypsin/collagenase digestion and exposed to 1 h of hypoxia (H) (95% N2/5% CO2, no glucose) and 2 h of reoxygenation (R) (95% air/5% CO2, glucose 5.5 m M) in suspension. Cell Ca2+was measured by uptake of45Ca2+. ROS was measured by flow cytometry of ethidium's red fluorescence formed by oxidation of dihydroethidium mostly by superoxide anion. Cell viability decreased during H and R, expressed as uptake of trypan blue, loss of rod shape morphology and release of lactate dehydrogenase. Rapidly exchangeable cell Ca2+was closely correlated with extracellular Ca2+concentration. Cell Ca2+was unchanged during H but increased three to four times after R. This increase was attenuated by adding 3,4-dichlorobenzamil, 10 μ m at R, and amplified by adding ouabain 1 m M (from start), respectively. Levels of ROS in hypoxic cells were unchanged or slightly reduced at the end of H and increased significantly by 20% compared to control after R. Levels of ROS were significantly decreased by lowering total extracellular Ca2+from 1 m M to 0.1 m M or by decreasing free extracellular Ca2+with EGTA 0.9 m M at the onset of R. Keeping extracellular Ca2+constant, ROS levels were neither affected by attenuating the increase in cell Ca2+by DCB nor by amplifying the increase in cell Ca2+by ouabain. In conclusion, ROS (superoxide anion) levels increase rapidly after reoxygenation, are correlated with extracellular-free Ca2+and are reduced by lowering extracellular-free Ca2+. Levels of ROS are apparently not consistently correlated with total cell Ca2+.