Abstract
Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Recent studies suggest that mitochondria function as a protective Ca buffer to absorb RyR2-mediated aberrant Ca release in CPVT. However, the molecular mechanism underlying the protective Ca buffering function of CPVT mitochondria remains unclear. Hypothesis: We hypothesize that the tethering between SR and mitochondria, also known as mitochondria-associated-membranes (MAMs) are promoted in CPVT to facilitate SR-mitochondria Ca transfer. Moreover, manipulating SR-mitochondria tethering or MAMs impacts arrhythmogenesis in CPVT. Methods: Ventricular myocytes were isolated from a CPVT model of CASQ2 knockout (Cnull) mouse. Cellular immunofluorescence assays and western blots were employed to detect MAMs remodeling in CPVT cells. Pharmacological approach was employed to disrupt MAMs and examine its effect on cellular arrhythmogenesis using live-cell imaging. Results: SR-mitochondria tethering was assessed by examining the interaction between RyR2 and Voltage-dependent anion channel (VDAC), a protein localized in the mitochondrial-outer-membrane. As compared with wild-type (WT), we detected a higher degree of colocalization of immunofluorescence between RyR2 and VDAC in Cnull cells, as well as increased RyR2-VDAC interactions by proximity ligation assay. The expression of SR-mitochondria tethering protein Mitofusin2 was increased in MAMs isolated from CPVT hearts. Colchicine was employed to induce a partial disruption of MAMs in Cnull cells. In intact Cnull cells perfused with β agonist isoproterenol, colchicine exacerbated arrhythmogenic Ca waves. In permeabilized cells, colchicine increased the frequency of arrhythmogenic Ca waves and reduced mitochondrial Ca uptake. Conclusions: Our results support that MAMs are promoted in CPVT cells to facilitate SR-mitochondria Ca transfer so mitochondria can function as a protective Ca buffer. Pharmacological disruption of SR-mitochondria tethering limits SR-mitochondria Ca transfer, thus exacerbating cellular arrhythmic burden.
Published Version
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