Mechanisms of Ca2+ overload induced by extracellular H2O2 in quiescent isolated rat cardiomyocytes.

Rat cardiomyocytes were exposed to H2O2 (1-100 micromol/L) for 10 min with washout for 10 min. Intracellular Ca2+ concentration ([Ca2+]i) was measured using fluo-3. [Ca2+]i increased with 100 micromol/L H2O2 and further increased during washout, causing irreversible contracture in one-half of the cells. The increase in [Ca2+]i with 10 micromol/L H2O2 was modest with few cells showing irreversible contracture and attenuated by caffeine, and [Ca2+]i gradually decreased during washout and this decrease was accelerated by a calcium-free solution, while 1 micromol/L H2O2 did not have any effects on [Ca2+]i or cell viability. Ca2+ overload caused during exposure to 100 micromol/L H2O2 was attenuated by caffeine with improved cellular viability but not by chelerythrine, KB-R7943 or nifedipine. With 100 micromol/L H2O2 calcium-free solution attenuated the increase during exposure and washout while KB-R7943 or chelerythrine partly attenuated further increase during washout but not improved cell viability, but chelerythrine did not have additional effect on calcium-free treatment. Catalase abolished the effects of H2O2. We concluded that the increased [Ca2+]i during exposure to 100 micromol/L H2O2 was caused both by release of Ca2+ from the intracellular store sites including the sarcoplasmic reticulum and by influx through route(s) other than the voltage-dependent Ca2+ channels or Na+/Ca2+ exchanger, although the Na+/Ca2+ exchanger or protein kinase C-mediated mechanism was partly responsible for a further increase during washout.