A gain of function ryanodine receptor 2 mutation (R1760W-RyR2) in catecholaminergic polymorphic ventricular...

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with Ca²⁺ leak predominantly caused by ryanodine receptor 2 (RyR2) mutations. We identified a R1760W-RyR2 mutation located between the N-terminal domain and the central domain of RyR2 in a CPVT patient by DNA sequencing. Recombinant mutant RyR2_-2801mcherry plasmid generated by the overlap extension polymerase chain reaction and seamless cloning was transfected in HEK293 cells for the cell model. Single-cell luminal and cytosolic Ca²⁺ imaging was measured by endoplasmic reticulum (ER) luminal Ca²⁺ -sensitive protein D1ER and Fura-2 AM on a confocal laser scanning microscope, respectively. We found that in RyR2 mutant cells, the propensity for store-overload-induced Ca²⁺ release (SOICR) was enhanced representing increased Ca²⁺ oscillations, reduced activation and termination thresholds of spontaneous Ca²⁺ release; and the sensitivity to cytosolic Ca²⁺ activation was increased manifesting reduced steady state ER Ca²⁺ levels. Our results indicated that R1760W-RyR2 mutation induced calcium leak, representing a gain of function. Further, antiarrhythmic drugs propafenone and flecainide significantly suppressed SOICR caused by the R1760W-RyR2 mutation at a concentration of 20 μM, which was lower than the concentration at which carvedilol suppressed SOICR.