The effects of genetic ablation of CaMKII oxidation on early ischemia-reperfusion arrhythmias in langendorff-perfused hearts

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority Introduction Ventricular arrhythmias are common during the early phase of reperfusion after an ischemic event. Spontaneous diastolic release of Ca2+ from the sarcoplasmic reticulum (SR) contribute to arrhythmias in this stage, and reactive oxygen species (ROS) have been implicated in the process, through destabilization of key Ca2+ handling proteins. Specifically, ROS has been shown to activate the Ca2+/calmodulin-dependent protein kinase II (CaMKII), by oxidation of two methionines at positions 281/282 in the regulatory domain. This activation pathway contributes to Ca2+-dependent arrhythmia mechanisms in atrial fibrillation (AF), heart failure (HF) and diabetes. However, the role of oxidized CaMKII (Ox-CaMKII) in ventricular arrhythmias during early reperfusion has not been directly tested. We aimed to investigate the importance of ox-CaMKII for diastolic Ca2+ release events and arrhythmias in the early phase of reperfusion. Methods We evaluated the propensity for early reperfusion arrhythmias in Langendorff-perfused hearts from mice carrying CaMKII resistant to oxidation (homozygous CaMKIIδ M281/282V mice) and wild type control mice (WT). The effect of beta-adrenoceptor stimulation, indirect inhibition of CaMKII via calmodulin antagonism, and antioxidant therapy was evaluated by the addition of isoprenaline, KN93 and N-acetylcysteine (NAC), respectively. Diastolic Ca2+ release events were recorded by confocal line scan imaging of isolated cardiomyocytes transiently exposed to hypoxia and ischemia-like conditions in the presence or absence of isoprenaline, and effects on Ca2+ handling proteins were assessed by immunoblotting. Results Langendorff-perfused hearts from CaMKIIδ M281/282V mice showed no significant difference in the frequency of ventricular arrhythmias compared to WT during the early reperfusion phase, neither in absence, nor presence of beta-adrenoceptor stimulation with isoprenaline. The incidence of ventricular arrhythmias increased significantly upon beta-adrenoceptor stimulation but was prevented by the antioxidative agent NAC and reduced with CaMKII-inhibition with KN93 when tested in WT hearts. Immunoblotting of left ventricular tissue from perfused hearts revealed no significant differences in the abundance or phosphorylation levels of Ca2+-handling proteins. Ca2+ imaging experiments