Effects of energy deprivation and hydrogen peroxide on contraction and myoplasmic free calcium concentrations in isolated myocardial muscle cells

The effect of energy deprivation and H₂O₂ on the contraction, shape, and intracellular free Ca²⁺ concentration of myocardial muscle cells was investigated using suspensions of freshly isolated, electrically stimulated rat ventricle heart cells. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to decrease the rate of ATP synthesis. At 0.9 mm extracellular Ca²⁺, CCCP (0.25 μm) reduced the number of contracting cells by 50% after 5 min, and the number of rod-shaped cells by 40% after 10 min. The effects of CCCP were associated with a substantial decrease in measured cellular ATP concentrations. The deleterious effect of exposure of myocytes to CCCP for periods of up to 5 min was enhanced by an increase in the extracellular Ca²⁺ concentration, but markedly reduced in the absence of electrical stimulation. Verapamil protected myocytes from the deleterious effects of CCCP during the first 5 min but not at later times. In the presence of 46 mm extracellular K⁺, CCCP caused a marked increase in the myoplasmic free Ca²⁺ concentration (measured using quin2). This effect was inhibited by verapamil and was not observed in the absence of K⁺-induced depolarization. Exposure of myocytes to H₂O₂ (0.5 mm) caused a substantial decrease both in the number of cells which exhibited normal end-to-end synchronous contraction and in the total number of cells which contracted either partially or fully. The effects of H₂O₂ were more pronounced at higher concentrations of the peroxide, with longer times of exposure to the agent, and at higher concentrations of extracellular Ca²⁺, and were partially reversed by dimethyl sulfoxide. The results indicate that both ATP deprivation and H₂O₂, possibly through the generation of free radicals, cause substantial and rapid damage to cardiac myocytes and induce the movement of additional Ca²⁺ across the sarcolemma to the myoplasm. In the case of ATP deprivation, this initially occurs through voltage-operated channels.