Parallel activation of Ca²⁺-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress

Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca²⁺ signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)-1] elicited large and oscillatory intracellular Ca²⁺ concentration increases. These Ca²⁺ signals were inhibited by nifedipine, Cd ²⁺, U73122, xestospongin C and ryanodine, suggesting contributions from both Ca²⁺ influx through voltage dependent L-type Ca ²⁺ channels plus Ca²⁺ release from intracellular stores mediated by IP₃ receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca²⁺ levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca²⁺] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca²⁺ influx and raising intracellular Ca²⁺ concentration, activates Ca²⁺ release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca²⁺ increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca²⁺-induced survival and death pathways.