Oxidative stress-induced perturbations of calcium homeostasis and cell death in cultured myocytes: role of extracellular calcium.

Abstract

The role of extracellular calcium in the process of oxidative stress-induced calcium overload and cell death was investigated in cultured neonatal rat myocytes. Oxidative stress was induced by addition of cumene hydroperoxide (CHPO), a toxic organic hydroperoxide, in combination with varying extracellular calcium concentrations (1. normal calcium buffer: 2.5 mM Ca2+, 2. low calcium buffer: 5 microM Ca2+, 3. zero calcium buffer: 2.5 mM EGTA, no CaC12). Intracellular free calcium ion concentration ([Ca2+]i) was measured with fura-2 using a spectrofluorometer. To study the toxicant-induced changes in [Ca2+]i in more detail, single cell imaging was performed using digital imaging fluorescence microscopy (DIFM). In control experiments (in the absence of CHPO, but under different extracellular Ca2+ conditions) the [Ca2+]i remained at the basal level and cell viability was preserved. Administration of CHPO (50 microM) to the myocyte cultures generated a sustained increase in [Ca2+]i followed by loss of cell viability. A low extracellular calcium concentration (5 microM), in the absence or presence of diltiazem (10 microM), induced a delay in the rise in [Ca2+]i but was not able to prevent the CHPO-induced calcium overload and cell necrosis. Addition of EGTA (2.5 mM) to the low calcium buffer resulted also in CHPO-induced cell death, although no increase in [Ca2+]i was observed. In normal and low calcium buffers, DIFM revealed that CHPO produced a temporally and spatially heterogeneous distribution of [Ca2+]i in a group of myocytes. So, in the presence of normal or low extracellular Ca2+, CHPO intoxication of cultured myocytes leads to an increase of [Ca2+]i prior to the onset of cell death. If extracellular Ca2+ is chelated by EGTA, CHPO also induces cell death which is not preceded by cellular calcium overload. Apparently a disturbance in the calcium homeostasis is not causally related with oxidative stress-induced myocardial cell death.