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 μM Ca²⁺, 3. zero calcium buffer: 2.5 mM EGTA, no CaCl₂). 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 μM) to the myocyte cultures generated a sustained increase in [Ca²⁺]_i followed by loss of cell 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.