Non-Lethal Levels of Oxidative Stress in Response to Short-Term Intermittent Hypoxia Enhance Ca2+ Handling in Neonatal Rat Cardiomyocytes

Abstract

Background/Aims: Intermittent hypoxia (IH) may exert pre-conditioning-like cardioprotective effects and alter Ca²⁺ regulation; however, the exact mechanism of these effects remains unclear. Thus, we examined Ca²⁺-handling mechanisms induced by IH in rat neonatal cardiomyocytes. Methods: Cardiomyocytes were exposed to repetitive hypoxia-re-oxygenation cycles for 1-4 days. Mitochondrial reactive oxygen species (ROS) generation was determined by flow cytometry, and intracellular Ca²⁺ concentrations were measured using a live-cell fluorescence imaging system. Protein kinase C (PKC) isoforms and Ca²⁺-handling proteins were analysed using immunofluorescence and western blotting. Results: After IH exposure for 4 days, the rate of Ca²⁺ extrusion from the cytosol to the extracellular milieu during 40-mM KCl-induced Ca²⁺ mobilization increased significantly, whereas ROS levels increased mildly. IH activated PKC isoforms, which translocated to the membrane from the cytosol, and Na⁺/Ca²⁺ exchanger-1, leading to enhanced Ca²⁺ efflux capacity. Simultaneously, IH increased sarcoplasmic reticulum (SR) Ca²⁺-ATPase and ryanodine receptor 2 (RyR-2) activities and RyR-2 expression, resulting in improved Ca²⁺ uptake and release capacity of SR in cardiomyocytes. Conclusions: IH-induced mild elevations in ROS generation can enhance Ca²⁺ efflux from the cytosol to the extracellular milieu and Ca²⁺-mediated SR regulation in cardiomyocytes, resulting in enhanced Ca²⁺-handling ability.