Ros and CaMKII Modulate Sodium Overload-Induced Calcium Leak from Ryanodine Receptors in Ventricular Myocytes

Abstract

Introduction: Elevation of the diastolic free calcium concentration ([Ca2+]cyt) alters excitation-contraction coupling and contractility in cardiac myocytes. It is known that Na+ overload increases the levels of diastolic [Ca2+]cyt and induces arrhythmias via reverse mode of Na+/ Ca2+ exchanger. We hypothesized that Ca2+ leak induced by oxidation/phosphorylation of ryanodine receptors (RyRs) contributes to the rise in [Ca2+]cyt during Na+ overload. Methods: We used confocal fluorescence imaging of intact and membrane-permeabilized isolated rabbit and rat ventricular myocytes to study Ca2+ transients, cytosolic sodium ([Na+]cyt ) and intracellular reactive oxygen species (ROS) levels. Cellular Na+ overload was induced using 5 nM anemone toxin-II (ATX-II), a late Na+ current (INaL) enhancer. Results: ATX-II-induced [Na+]cyt overload caused abnormal Ca2+ transients in intact rabbit and rat myocytes. Additionally, high cytosolic Na+ increased ROS levels and activated CaMKII. These effects were abolished by the INa inhibition with ranolazine and TTX, CaMKII inhibition with KN93 and AIP or by the application of antioxidants such as DTT and coenzyme Q10. In the experiments with membrane-permeabilized myocytes, rapid elevation of [Na+]cyt from 5 mM to 20 mM resulted in an increase of ROS production in mitochondria and Ca2+ leak from RyRs by measuring the frequency of spontaneous Ca2+ waves. The CaMKII inhibitors, KN-93 and AIP, attenuated the effects of high [Na+]cyt on Ca2+ signaling. Conclusions: These data suggest that [Na+]cyt regulates RyRs activity. An increase in [Na+]cyt induces ROS production; ROS activates CaMKII leading to altered Ca2+ transients and abnormal Ca2+ handling.