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 culture d cardiomyocytes isolated from the ventricle of neonatal ICE mouse. A model of chemical hypoxia with 2 mM sodium cyanide was developed to mi mic the ATP depletion of hypoxia. This chemical hypoxia was ''reoxygen ated' 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 4 0-min chemical hypoxia, pH(i) progressively fell from 7.2 to 6.6. Reox ygenation with control solution caused rapid recovery of pH(i) and a m arked 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 reo xygenation with 2-(N-morpholino) ethanesulfonic acid (pK = 5.96) buffe r somewhat suppressed the Ca2+ overload; however, the maximal value of [Ca2+](i) was not reduced significantly compared with the control (17 90 +/- 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 initi al phase of reoxygenation, and the protective effects of extracellular acidosis is principally mediated by intracellular acidification of re oxygenated cardiomyocytes. (C) 1995 Academic Press, Inc.