Mechanisms of reoxygenation-induced calcium overload in cardiac myocytes: Dependence on pH(i)

This study investigated the selective effects of intracellular (pH(i)) or extracellular change in pH on reoxygenation-induced Ca2+ overload in simulated myocardial hypoxia. Experiments were performed in cultured cardiomyocytes isolated from the ventricle of neonatal ICE mouse. A model of chemical hypoxia with 2 mM sodium cyanide was developed to mimic the ATP depletion of hypoxia. This chemical hypoxia was ''reoxygenated' and the dynamics in intracellular Ca2+ concentration ([Ca2+](i)) and pH(i) were monitored using the fluorescent dyes fura-2 and 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. During a 40-min chemical hypoxia, pH(i) progressively fell from 7.2 to 6.6. Reoxygenation with control solution caused rapid recovery of pH(i) and a marked increase in [Ca2+](i) (1884 +/- 136 nM). Intracellular acidotic reoxygenation produced by lactate apparently prolonged the time course of pH(i) recovery and significantly suppressed reoxygenation-induced Ca2+ overload (1170 +/- 118 nM, P = 0.008). Extracellular acidotic reoxygenation with 2-(N-morpholino) ethanesulfonic acid (pK = 5.96) buffer somewhat suppressed the Ca2+ overload; however, the maximal value of [Ca2+](i) was not reduced significantly compared with the control (1790 +/- 122 nM, P = 0.130). In addition, inhibition of Na+-H+ exchange by amiloride potentiated prolongation of intracellular acidosis during reoxygenation and resulted in a minimal increase in [Ca2+](i) (985 +/- 102 nM P = 0.004). These results suggest that reoxygenation-induced Ca2+ overload is closely correlated with intracellular pH in the initial phase of reoxygenation, and the protective effects of extracellular acidosis is principally mediated by intracellular acidification of reoxygenated cardiomyocytes. (C) 1995 Academic Press, Inc.