Identifying the site of the source of reactive oxygen species within the mitochondria after transient exposure of cardiac myocytes to hydrogen peroxide

H.M. Viola,¹ E. Ingley,² P.G. Arthur¹ and L.C. Hool,¹ ¹School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA 6009, Australia and ²Western Australian Institute for Medical Research, Perth, WA 6000, Australia.

Oxidative stress is a feature of cardiovascular disease and hydrogen peroxide (H₂O₂) can act as a signaling molecule to mediate cardiovascular pathology. We have previously demonstrated that transient exposure of ventricular myocytes to H₂O₂ leads to a further increase in reactive oxygen species (ROS) from the mitochondria (supporting the “ROS-induced ROS-release” hypothesis). Exposure of cardiac myocytes to 30μM H₂O₂ for 5 min followed by 10U/ml catalase for 5 min to degrade the H₂O₂ caused a 65.4 ± 8.4% further increase in superoxide by the mitochondria (n = 47) (Viola et al., 2007). NADPH-oxidase, xanthine oxidase and nitric oxide did not contribute to the increase in superoxide. We tested whether a transient exposure to H₂O₂ altered protein synthesis in the myocytes. Ventricular myocytes were isolated from hearts excised from anesthetised guinea pigs. We found that 5 min exposure to 30μM H₂O₂ followed by 10U/ml catalase for 5 min caused a two fold increase in protein synthesis measured as ³H-Leucine incorporation (n = 10). This suggests that a transient exposure to H₂O₂ may be sufficient to induce cardiac hypertrophy. We wished to identify the site of the source of ROS in the mitochondria. Previous studies have shown the main source of ROS production by the mitochondria occurs via complex III although complex I may also be a source of ROS production (Turrens, 1997; St-Pierre et al., 2002; Muller et al., 2003; Turrens, 2003). We exposed myocytes to 1μM DPI, which binds just prior to the ROS generation site of complex I, followed by 30μM H₂O₂ for 5 min and 10U/ml catalase for 5 min. Superoxide was assessed with the fluorescent indicator dihydroethidium (DHE). The presence of DPI completely attenuated the increase in DHE after exposure to H₂O₂. We also exposed guinea pig cardiac myocytes to 1μM rotenone, which binds just after the ROS generation site of complex I, followed by 30μM H₂O₂ for 5 min and 10U/ml catalase for 5 min. The presence of rotenone attenuated the increase in DHE after exposure to H₂O₂ by 45%. These data suggest that the source of production of ROS is distal to complex I. Identifying the site of production of ROS may represent a possible therapeutic target to prevent the development of cardiac hypertrophy associated with a transient exposure to H₂O₂.

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