Oxidation of Ryanodine Receptor following Ischemia/Reperfusion Increases the Propensity of Ca Waves during Beta-Adrenergic Receptor Stimulation

Abstract

Beta-Adrenergic receptor (beta-AR) stimulation generates the main positive inotropic response in the heart. However, after ischemia/reperfusion (I/R), beta-AR stimulation can generate arrhythmias. I/R also increased intracellular reactive oxygen species (ROS) production. We investigated whether ryanodine receptor (RyR) oxidation by ROS contributes to the transition from positive inotropic to arrythmogenic effect in ventricular myocytes after I/R. Measurements of contractile and electrical activity from ex vivo rabbit hearts revealed that global I/R produces severe tachi-arrhythmias. Ventricular myocytes isolated from ischemic hearts were characterized by increased both SR Ca leak rates and fractional SR Ca release compared to cells from non-ischemic hearts. Furthermore, myocytes from ischemic hearts showed increased ROS production, decreased level of free thiols in RyRs (RyR oxidation), and increased level of oxidized glutathione (GSSG). Pretreatment of myocytes from ischemic hearts with the reducing agent mercaptopropionylglycine attenuated the oxidization of free thiols in RyRs and normalized systolic SR Ca release and diastolic Ca leak. In myocytes from ischemic hearts, isoproterenol (ISO; 10 nM) led to the occurrence of spontaneous Ca waves, whereas in cells from non-ischemic hearts the same dose of ISO only caused a positive inotropic effect. Treatment of myocytes from non-ischemic hearts with H2O2 (0.1 mM) increased SR Ca leak and fractional SR Ca release to similar levels observed in myocytes from ischemic hearts. Moreover, application of ISO (10 nM) to myocytes from non-ischemic hearts pretreated with H2O2 increased the propensity of Ca waves. These results indicate that augmentation of ROS production following I/R causes RyR oxidation. This post-translational modification of the RyR plays a critical role in the transition from positive inotropic to arrhythmogenic effect during beta-AR stimulation.