Abstract 13700: High Affinity CaM, but Not Normal Affinity CaM Can Prevent Arrhythmias in CPVT Which Mutation is Located at CaM-like Domain of RyR2

Abstract

Introduction: We have been previously reported that calmodulin (CaM) binding affinity to RyR2 decreases upon beta stimulation in catecholaminergic polymorphic ventricular tachycardia (CPVT) type knock-in mice with mutation at central domain (R2474S-KI). Meanwhile, amino acid residues from 4026 to 4172 of RyR2 have EF hand motifs and are called CaM-like domain (CaMLD). Some of the human CPVT mutations were reported in this CaMLD. In the present study we investigated the molecular mechanism of arrhythmogenesis of CPVT with mutation at CaMLD using N4103K KI mice. Methods and Results: VT and/or Vf were frequently observed after 120 mg/kg caffeine plus 1 mg/kg epinephrine infusion in KI mice, but not in WT mice (WT: 0%, KI: 43%). CaM binding affinity to RyR2 was assessed by incorporation of exogenous CaM labeled with HiLyte Fluor® in isolated saponin-permeabilized cardiomyocytes. The binding affinity of CaM decreased in KI cells (Kd: 161 nM) compared with WT cells (Kd: 89 nM). Addition of 1μM cAMP did not change the affinity of CaM to RyR2 in both KI and WT cells, while it decreased the affinity in R2474S-KI. Frequency of the Ca 2+ sparks was measured in isolated saponin-permeabilized cells. The Ca 2+ spark frequency (SpF: s -1 100μm -1 ) was much higher in KI than in WT cells {KI (n=79): 21.5±6.3; WT (n=34): 10.6±4.1; p<0.01}. Addition of 100 nM CaM to KI cells did not decrease SpF {(n=87): 22.4±6.5}, however, significant decrease was observed by HA-CaM (modified CaM which has higher affinity to RyR2). 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 and 5 Hz pacing, whereas that was observed in 33% of KI cells. Incorporation of CaM to intact KI cells using protein delivery kit (BioPORTER®) did not decrease incidence of SCaT (34% of KI cells), however, significantly decreased by HA-CaM incorporation. Conclusions: Different from R2474S-KI mice, CaM binding affinity to RyR2 is decreased without beta stimulation in KI mice with CaMLD mutation. Conformational change of CaMLD might affect CaM-binding domain (CaMBD) and decrease the affinity of CaM to CaMBD. The CaMLD-CaMBD interaction may be a novel therapeutic target of lethal arrhythmias.