Reperfusion of rat ventricular myocytes following a simulated hypoxia is associated with the production of reactive oxygen species (ROS) (King et al. 2001). The origin of these ROS is controversial (Duranteau et al. 1998), although in isolated hepatocytes ROS are generated at complex III (Coenzyme Q cycle) of the respiratory chain in the mitochondria (Caraceni et al. 1995). In this study we have investigated whether complex III is also the source of the ROS produced by ventricular myocytes. To achieve this, we have used the complex III inhibitors, myxothiazol (increases the lifespan of ubiquinol) and antimycin A (increases the lifespan of ubisemiquinone).

Male Wistar rats were humanely killed by cervical dislocation and the hearts dissected. Ventricular myocytes were isolated, loaded with 10 µM 5- (and 6-)-chloromethyl-2Ì,7Ì-dichlorodihydrofluorescein diacetate (CM-H2DCFda), and used to measure the fluorescence of CM-H2DCF as described previously (King et al. 2001). Initially, all cells were perfused with normal glucose-containing Tyrode solution. After 10 min, test cells were exposed to simulated hypoxia by switching the Tyrode to a solution containing 2.5 mM NaCN, no glucose and either 1 µM myxothiazol or antimycin A. Ten minutes after the onset of rigor, the test cells were reperfused with the normal Tyrode solution. All experiments were of 60 min duration. Fluorescence data are presented as a percentages of the initial value.

Control cells perfused continuously with normal glucose-containing Tyrode solution showed a small but insignificant rise in fluorescence (120 ± 12 to 160 ± 25 %, means ± S.E.M., n = 4). During reperfusion, the fluorescence of cells previously perfused with NaCN and myxothiazol significantly decreased from 103 ± 5 to 82 ± 5 % (P < 0.01, ANOVA repeated measures, n = 9), suggesting a reduction in ROS generation. In contrast, during reperfusion, the fluorescence of cells previously perfused with NaCN and antimycin A significantly increased from 149 ± 14 to 436 ± 78 % (P < 0.05, ANOVA repeated measures, n = 11), suggesting that ROS production had been enhanced. This suggests that one origin of the ROS produced during reperfusion following a simulated hypoxia is complex III of the respiratory chain in the mitochondria.This work was supported by the British Heart Foundation.

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