Pathogenic Mechanism Of Disease-causing Mutation In RYR2 In Catecholaminergic Polymorphous Ventricular Tachycardia

Background:Catecholaminergic polymorphic ventricular tachycardia(CPVT)is a malignant arrhythmia induced by exercise or emotional stimulation.CPVT is a genetic disease,and more than two-thirds of patients have been detected mutations in the RYR2 gene,which encodes a calciun release channel in cardiomyocytes.There are many theories about the pathogenesis of CPVT caused by calcium channel mutation,but there are also a lot of scientific controversies.Objective:Using RyR2 as a molecular target,we aim to provide a new theoretical basis for the pathogenesis of CPVT,and inspire new ideas for clinical treatment of CPVT.Methods:We screened and identified a novel mutant A690E in the RYR2 gene in a CPVT patient.We have generated transgenic mice with knock-in A690E mutation,and demonstrated that A690E is a CPVT disease-causing mutation.We analyzed the three dimensional(3D)localization of the A690E mutation in the high-resolution cryo-electron microscopy structure of RyR2,and used the knock-in mouse models,we also performed RyR2 biochemical and functional experiments in the isolated cardiomyocytes.Taken together,we have characterized mutant RyR2 and CPVT phenotypes at molecular,cellular,and animal levels,and provided a deeper pathological and physiological mechanisms of the A690E CPVT mutation.Results:Structural biology experiments demonstrated that the A690E mutation is located in the domain of a segment named SPRY1,and we docked the SPRY1 domain into the 3D cryo-EM electron density map of the full-length of RyR2.The docking model revealed that the SPRY)domain is located at the FKBP binding site.FKBP is an important RyR2 functional modular protein,which enhances the calcium channel stability by tightly binding to RyR2.Dissociation of FKBP from RyR2 by the A690E mutation causes RyR2 channel instability,and prone to leak calcium ion,results in disorders in the cardiac muscle contraction.Based on this,we proposed that the mutation of A690E resulted in the molecular mechanism of CPVT.This study used abundant clinical resources to screen for mutations in CPVT disease-related genes to identify pathogenic mutations.The A690E mutation RYR2 gene is a novel mutation which has never been reported previously and we demonstrated it is a CPVT causing mutation.Biochemical and cardiomyocyte function experiments have shown that this mutation enhanced sarcoplasmic reticulum Ca2+ release and resulted in RyR2 channel open abnormality;and excluded the possibility that other Regulatory protein causing the opening abnormality of RyR2 channel,eventually we have further explained the pathogenic molecular mechanism of the CPVT-causing mutations.Figure 14 table 3 reference 52...