Effects of oxygen free radicals on isolated cardiac myocytes from guinea-pig ventricle: electrophysiological studies.

Free oxygen radicals are formed during early reperfusion and are thought to contribute to some types of reperfusion abnormalities, including arrhythmias and myocardial stunning. The purpose of this study was to investigate electrophysiological effects of oxygen free radicals using voltage clamped single ventricular myocytes from guinea-pig hearts. Oxygen free radicals were produced enzymatically by the direct addition of xanthine oxidase (XOD, 0.04 U/ml) in the experimental chamber to a solution containing hypoxanthine (0.96 mM). The generation of oxygen radicals was confirmed by the formation of adrenochrome from adrenaline. Oxygen radicals caused automaticity of isolated myocytes within 20-30 min, followed by later hypercontracture. The percentage of rod-shaped cells declined sigmoidally as a function of time, with a half maximal value at 40.9 +/- 1.6 min, and a Hill slope of -0.10 +/- 0.01 (n = 26). These effects were prevented by a combination of superoxide dismutase (10(5) U/L) plus catalase (10(6) U/L). The rate at which cells underwent morphological shape changes was unchanged by ryanodine (0.5 microM) which is thought to act on the sarcoplasmic reticulum or by the Ca2+ channel blockers nisoldipine (1 microM) or Cd2+ (30 microM). Cellular automaticity and hypercontracture were delayed by variable degrees, and sometimes completely prevented, by zero (1 mM EGTA) extracellular Ca2+, MnCl2 (2 mM) and LaCl3 (50 microM), and amiloride (1 mM). On the other hand, in the presence of a low extracellular Na+ (30 mM) or caffeine (10 mM), hypercontracture occurred at a faster time scale. Whole cell voltage clamping revealed a decrease of the inward rectifying K+ current (IK1), and a decrease of the peak of the L-type Ca2+ current (ICa,L). The total ICa,L during the clamp step was increased, mainly because of an increased time constant of inactivation (47.6 +/- 4.7 ms to 72.7 +/- 15.5 ms after 30 min, n = 4, P less than 0.05). We conclude that oxygen radicals cause automaticity and hypercontracture of isolated myocytes, that these effects may be due to an increased intracellular Ca2+ concentration ([Ca2+]i), and despite an increased ICa,L, that the enhanced Ca2+ influx may occur predominantly via the Na/Ca exchange.