Intracellular Na+ Overload of Cardiomyocytes Causes ROS Induced CaMKII Activation, Leading to RYR Dysfunction and Diastolic Ca2+ Mishandling

Abstract

Introduction: Intracellular sodium (Na+i) overload of cardiomyocytes is associated with abnormal calcium (Ca2+i) homeostasis and cell dysfunction. We hypothesized that the impaired Ca2+ handling is triggered by reactive oxygen species (ROS) -activated Ca2+/Calmodulin-dependent protein kinase II (CaMKII) which in turn on modulation of ryanodine receptor (RyR) activity, leading to Ca2+ leak from the sarcoplasmic reticulum (SR).Methods: We used confocal fluorescent imaging of intact and membrane-permeabilized rabbit ventricular cardiomyocytes to study Na+i overload, Ca2+i transients, and ROS production. Na+i overload in intact cardiomyocytes was induced by anemone toxin-II (ATX-II), a late Na+ current (INaL) enhancer. CaMKII oxidation and phosphorylation were measured using Western Blot analysis.Results: Rabbit ventricular myocytes treated with ATX-II (5 nM) exhibited Na+i overload, aberrant Ca2+i transients, and enhanced ROS production, accompanied by CaMKII oxidation and phosphorylation. CaMKII activation was reduced by the INaL inhibitor ranolazine (10 μM), by antioxidants (5 mM DDT or 10 μM Q10co), or by the CaMKII inhibitor KN93 (10 μM). These findings indicate a pathogenic cascade of events from Na+i overload to ROS production and to CaMKII activation. To determine how ROS and CaMKII contribute to the Ca2+i mishandling caused by Na+i overload, we studied spontaneous Ca2+ waves in membrane-permeabilized myocytes. Superfusion with high Na+ solutions elevated diastolic Ca2+i and accelerated Ca2+ waves frequency. These effects indicated Ca2+ leak through RyRs. These Ca2+ changes were dependent from ROS and CaMKII inhibition.Conclusions: The data show that Na+i overload causes Ca2+i mishandling through increased ROS production and CaMKII activation. Na+i overload enhances ROS production, which activates CaMKII, leading to the phosphorylation of RyRs, resulting in SR Ca2+ leak and increase in diastolic Ca2+.