Abstract P1083: Calcium-dependent Proteolysis Of Mitochondrial Opa1 By Calpain Contributes To Arrhythmogenesis In Catecholaminergic Polymorphic Ventricular Tachycardia

Abstract

Intracellular Ca 2+ cycling in ventricular myocytes (VMs) is tightly linked to mitochondrial metabolic output, and disturbances in this coupling contribute to cardiac arrhythmias. We recently demonstrated in the heritable arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT), the arrhythmogenic phenotype is revealed by oxidation of the cardiac ryanodine receptor (RyR2) calcium release channel by mitochondrial reactive oxygen species (mito-ROS), secondary to causative genetic mutation. However, exact mechanisms linking RyR2 dysfunction to mitochondrial injury remain unclear. We hypothesized that RyR2 hyperactivity activates the intermembrane space (IMS)-residing mitochondrial Ca 2+ -dependent protease , driving proteolysis of OPA1, a protein responsible for cristae flattening. This results in altered mitochondrial cristae diameter and mito-ROS production, exacerbating the arrhythmogenic CPVT phenotype. To test this, we created a unique CPVT RyR2-S2222L (+/-) rat model. Electron microscopy studies revealed a significant increase in cristae diameters in Langendorff-perfused CPVT rat hearts. Western blot analysis showed significantly reduced OPA1 expression in isolated CPVT VMs, suggesting that Ca 2+ -dependent proteolysis of OPA1 could underlie these ultrastructural changes. Pilot experiments suggest specific inhibition of via adeno-associated viral injection of IMS-targeted calpastatin (CAST, 10 12 VP, 4 weeks) can reduce ventricular tachycardia incidence of CPVT rats in vivo. Adenoviral expression of IMS-CAST in isolated VMs restored OPA1 expression levels and significantly improved intracellular Ca 2+ cycling in CPVT, suggesting a marked improvement in RyR2 function. Furthermore, genetic probes mtRCamp1h and HyPer-7 revealed that IMS- inhibition significantly improves mitochondrial matrix Ca 2+ homeostasis and mito-ROS in CPVT VMs, respectively. Importantly, these beneficial effects were lost in VMs with shRNA-mediated knockdown of OPA1. Our data suggest that enhanced activity of RyR2 promotes IMS- -mediated degradation of OPA1, resulting in mitochondrial structural damage that contributes to proarrhythmic remodeling in CPVT.