Mechanisms of Hydrogen Peroxide-Induced Increase in Intracellular Calcium in Cardiomyocytes.

BACKGROUND: Hydrogen peroxide (H(2)O(2)) in high concentrations has been implicated in heart dysfunction attributable to ischemia-reperfusion. Although H(2)O(2) is also known to increase the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in cardiomyocytes, the mechanisms 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 H(2)O(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. [Ca(2+)](i) was measured by employing cell suspension at room temperature and Fura-2 fluorescence technique. Incubation of cells with 0.25-l mmol/L H(2)O(2) increased [Ca(2+)](i) in a time- and concentration-dependent manner. Catalase attenuated the H(2)O(2)-induced increase in [Ca(2+)](i) significantly, whereas mannitol showed no effect. Neither the presence of verapamil, a sarcolemmal Ca(2+) channel blocker, nor the removal of Ca(2+) from the medium produced any significant reduction in the H(2)O(2)-induced increase in [Ca(2+)](i). Conversely, treatment of cardiomyoctes with staurosporin, a protein kinase C inhibitor, thapsigargin, a sarcoplasmic reticulum Ca(2+)-pump adenosine triphosphatase inhibitor, as well as ryanodine, a sarcoplasmic reticulum Ca(2+)-release channel blocker, markedly prevented the 0.5-mmol/L H(2)O(2)-induced increase in [Ca(2+)](i). The responses of cardiomyoctes to H(2)O(2) and other Ca(2+)-mobilizing agents, such as KCl or adenosine triphosphate, were additive. No changes in cardiomyocyte viability were seen on incubation with 0.5 and 1 mmol/L H(2)O(2). Perfusion of the isolated heart with H(2)O(2) (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 H(2)O(2) under pathophysiological conditions such as ischemic heart disease may induce changes in Ca(2+) homeostasis in cardiomyocytes 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(2)O(2) in cardiomyocytes.