Background: Hydrogen peroxide (H2O2) in high concentrations has been implicated in heart dysfunction attributable to ischemia-reperfusion. Although H 2O2 is also known to increase the intracellular concentration of Ca2+ ([Ca2+]i) in cardiomyocytes, the mecha nisms for such a change are not clear. In this study, the sources and mechanisms of increase in [Ca 2+]i caused by high concentrations of H2O 2 in cardiomyocytes were explored.
Methods and Results: Cardiomyocytes were isolated from adult male Sprague-Dawley rats. Cell viability was examined by trypan blue exclusion test. [Ca2+] i was measured by employ ing cell suspension at room temperature and Fura-2 fluorescence technique. Incubation of cells with 0.25-1 mmol/L H2O2 increased [Ca 2+]i in a time- and concentration-dependent manner. Catalase attenuated the H2O2-induced increase in [Ca2+] i significantly, whereas mannitol showed no effect. Neither the presence of verapamil, a sarcolemmal Ca2+ channel blocker, nor the removal of Ca2+ from the medium produced any significant reduction in the H2O2-induced increase in [Ca2+] i. Conversely, treatment of cardiomyoctes with stauro sporin, a protein kinase C inhibitor, thapsigargin, a sarcoplasmic reticulum Ca2+-pump aden osine triphosphatase inhibitor, as well as ryanodine, a sarcoplasmic reticulum Ca2+-release channel blocker, markedly prevented the 0.5-mmol/L H2O2-induced increase in [Ca 2+]i. The responses of cardiomyoctes to H2O 2 and other Ca2+-mobilizing agents, such as KCl or aden osine triphosphate, were additive. No changes in cardiomyocyte viability were seen on incu bation with 0.5 and 1 mmol/L H2O2. Perfusion of the isolated heart with H2O2 (0.1-0.5 mmol/L) depressed the left ventricular developed pressure, rate of contraction, and rate of relaxation, whereas the left ventricular end-diastolic pressure was increased.
Conclusions: These results indicate that formation of H2O2 under pathophysiological con ditions such as ischemic heart disease may induce changes in Ca2+ homeostasis in cardiomy ocytes and may induce contractile dysfunction. Furthermore, the sarcoplasmic reticulum involving a protein kinase C-mediated mechanism appears to be the main site of action of H 2O2 in cardiomyocytes.
