Abstract 13816: Dantrolene Enhances Binding of Calmodulin to RyR2 in CPVT Which Mutation Located at Central Domain but Does Not Enhance That in CPVT With Mutation at CaM-like Domain

Abstract

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by heterozygous mutation in RyR2 gene. Most mutations are known to be located at N-terminal domain, central domain or C-terminal channel pore region. Meanwhile, amino acid residues from 4026 to 4172 of RyR2 have EF hand motifs and are called calmodulin (CaM)-like domain (CaMLD). Some of the human CPVT mutations were reported in this CaMLD. In this study we investigated the molecular mechanism of arrhythmogenesis of CPVT with mutation at CaMLD comparing R2474S and N4103K KI mice. Methods and Results: VT and/or Vf were frequently observed after caffeine plus epinephrine infusion in KI mice (R2474S: 100%, N4103K: 43%), but not in WT mice. CaM binding affinity to RyR2 was assessed by incorporation of exogenous CaM labeled with HiLyte Fluor® in isolated saponin-permeabilized cardiomyocytes. The CaM binding affinity was decreased in both R2474S-KI (Kd: 400 nM) and N4103K-KI cells (Kd: 165 nM) compared with WT cells (Kd: 90 nM) in the presence of cAMP. Addition of 1 μM dantrolene (DAN), the zipper of N-terminal and central domain of RyR2, increased the affinity of CaM to RyR2 in R2474S-KI cells, but did not increase that in N4103K-KI cells. Frequency of the Ca 2+ sparks (SpF) was measured in isolated saponin-permeabilized cardiomyocytes. SpF was much higher in KI cells than in WT cells (R2474S-KI: 17.2±6.7, N4103K-KI: 21.5±6.3, WT: 10.6±4.1). Addition of 1 μM DAN to R2474S-KI cells significantly decreases SpF (10.4±5.5), however, DAN did not decrease SpF in N4103K-KI (21.8±6.9). Spontaneous Ca 2+ transient (SCaT) was measured during sequential pacing from 1 to 5 Hz in isolated intact cardiomyocytes. The SCaT was rarely observed in WT cells even with 10 nM isoproterenol, whereas that was observed in 37 % of R2474S-KI and in 33 % of N4103K-KI cells. Addition of DAN to KI cells, decrease frequency of SCaT in R2474S-KI (13 %), however, did not decrease in N4103-KI cells (30 %). Conclusions: In CPVT with central domain mutation, domain zipper is effective to inhibit arrhythmias. In CaMLD mutation, structural abnormality might be in the downstream of the N-terminal and central domain switch, so that the DAN is not effective. CaMLD might be a novel therapeutic target of lethal arrhythmias.