Interference with calcium-dependent mitochondrial bioenergetics in cardiac myocytes isolated from doxorubicin-treated...

In previous studies we showed that doxorubicin (DOX) interferes with mitochondrial calcium homeostasis and that cardiac mitochondria from DOX-treated rats express a dose-dependent, cumulative, and irreversible decrease in calcium-loading capacity. Associated with this is an increased sensitivity to calcium-induced cell killing of cardiac myocytes from DOX-treated rats. Because mitochondria play an important role in cytosolic calcium regulation, we questioned whether cardiac myocytes isolated from DOX-treated rats express an exaggerated response to interventions that increase cytosolic calcium. Adult male Sprague-Dawley rats received six weekly (sc) injections of either 2 mg/kg of doxorubicin or an equivalent volume of saline and were killed the following week. Cardiac myocytes were isolated and exposed in vitro to 25 microM of A23187, 20 mM of caffeine, or 500 microM of ouabain. A23187 and ouabain caused a dose-dependent increase in intracellular calcium, as measured fluorometrically with Fura-2AM. In the case of ouabain, the increase in cytosolic calcium was greater for myocytes from DOX-treated rats than for cells from control rats. With caffeine, however, the increase in intracellular calcium was not evident unless the mitochondria were depolarized. A23187 also caused depolarization of mitochondria, the extent of which was greater for cardiac myocytes from DOX-treated rats. All three agents caused depletion of cardiac myocyte ATP and cell killing, both of which were more profound in cells from DOX-treated rats than in controls. We suggest that by interfering with mitochondrial calcium regulation, long-term treatment with DOX renders myocytes susceptible to agents that increase cytosolic calcium, presumably by increasing the calcium-dependent disruption of mitochondrial function, leading to depletion of ATP and eventually cell death. This interference with mitochondrial calcium regulation may underlie the pathogenesis of DOX-induced cardiomyopathy.