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 Ca(2+) regulation; however, the exact mechanism of these effects remains unclear. Thus, we examined Ca(2+)-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 Ca(2+) concentrations were measured using a live-cell fluorescence imaging system. Protein kinase C (PKC) isoforms and Ca(2+)-handling proteins were analysed using immunofluorescence and western blotting. RESULTS: After IH exposure for 4 days, the rate of Ca(2+) extrusion from the cytosol to the extracellular milieu during 40-mM KCl-induced Ca(2+) mobilization increased significantly, whereas ROS levels increased mildly. IH activated PKC isoforms, which translocated to the membrane from the cytosol, and Na(+)/Ca(2+) exchanger-1, leading to enhanced Ca(2+) efflux capacity. Simultaneously, IH increased sarcoplasmic reticulum (SR) Ca(2+)-ATPase and ryanodine receptor 2 (RyR-2) activities and RyR-2 expression, resulting in improved Ca(2+) uptake and release capacity of SR in cardiomyocytes. CONCLUSIONS: IH-induced mild elevations in ROS generation can enhance Ca(2+) efflux from the cytosol to the extracellular milieu and Ca(2+)-mediated SR regulation in cardiomyocytes, resulting in enhanced Ca(2+)-handling ability.