Abstract 795: Enhanced Sensitivity Of The Cardiac Ryanodine Receptor To Activation By Luminal Ca 2+ As A Primary Cause Of Catecholaminergic Polymorphic Ventricular Tachycardia

Abstract

Mutations in cardiac ryanodine receptor (RyR2) was found to be linked with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of CPVT, we developed knock-in mice harboring the Arg-to-Ser (R2474S) mutation. The RyR2 R2474S/+ knock-in (KI) mice revealed no structural or histological abnormality in hearts. Echocardiography showed no contractile or relaxation dysfunction at rest. In all KI mice (n=6), bidirectional ventricular tachycardia (VT) was observed during or after exercise with treadmill, but never observed in wild-type (WT) mice (n=6). In intact cardiomyocytes, the frequency of Ca 2+ sparks (SpF; s −1 ·100μm −1 ) was significantly increased in KI mice, but not in WT mice (at 2 mM [Ca 2+ ]; KI:6.4±0.7, WT:0.9±0.08, p<0.01). To investigate the sensitivity of the RyR2 channel to activation by luminal Ca 2+ {[Ca 2+ ] in sarcoplasmic reticulum (SR)}, we measured cytoplasmic [Ca 2+ ] ([Ca 2+ ] C ) and luminal [Ca 2+ ] ([Ca 2+ ] L ) simultaneously in saponin-permeabilized cardiomyocytes, using Rhod-2 and Fluo-5N AM as Ca 2+ indicators, respectively. When [Ca 2+ ] C was buffered at 100 nM (by 1 mM EGTA), the spontaneous Ca 2+ sparks were frequently observed both in KI and WT cardiomyocyts (SpF: KI:22.1±0.9, WT:22.0±0.8, p=ns). When we added thapsigargin (1 μM) to the cardiomyocytes under this condition ([Ca 2+ ] C =100 nM), both SpF and [Ca 2+ ] L gradually decreased due to a decrease in SR Ca 2+ content caused by an inhibition of SR Ca 2+ ATPase. The relationship curve between SpF and [Ca 2+ ] L (SpF -[Ca 2+ ] L ) during the addition of thapsigargin was markedly shifted to the left in KI cardiomyocytes compared to WT cardiomyocytes, thereby lowering the threshold of [Ca 2+ ] L to induce Ca 2+ sparks to approximately one-fifth in KI cardiomyocytes. In conclusion, the enhanced sensitivity of the RyR2 channel to activation by [Ca 2+ ] L : i.e. decreased threshold [Ca 2+ ] L to induce spontaneous Ca 2+ release, may be a primary cause of CPVT.