Abstract 15138: Dantrolene Inhibits Aberrant Ca2+ Release and Arrhythmogenesis via Enhancing Binding of Calmodulin to RyR2 in CPVT-Associated Mutation
Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated the pathogenic role of CaM in the channel disorder in CPVT, using knock-in (KI) mouse model with CPVT-associated RyR2 mutation (R2474S).
Methods and results: Transmembrane potentials were recorded in whole cell current mode before and after pacing (1 to 5 Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with Alexa Fluor® in saponin-permeabilised myocytes. In the presence of cAMP (1 μM) the binding affinity of CaM decreased in KI cells (Kd: 160 to 300 nM), but not in WT cells (Kd: 170 to 200 nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5 Hz pacing, whereas both DAD and TA were observed in 20% and 12% of KI cells, respectively. In response to 10 nM isoproterenol, only DAD (but not TA) was observed in 11% of WT cells, whereas in KI cells the incidence of DAD and TA further increased to 64% and 32% of cells, respectively. Addition of dantrolene (1 uM) decreased both DADs and TA. Addition of dantrolene to saponin-permeabilized KI cells decreased Ca2+ spark frequency (+6% of WT cells), which otherwise markedly increased without dantrolene (+50% of WT cells). To assess the effect of dantrolene on CaM binding to RyR2, fluorescently labeled CaM binding to RyR2 in the presence of dantrolene was examined. Dantrolene significantly increase the CaM binding (+52% of without dantrolene) to RyR2 even in the presence of the cAMP.
Conclusions: In CPVT-associated mutation of RyR2 CaM binding affinity is decreased upon PKA phosphorylation. Dantrolene normally restored CaM binding to the mutant RyR2 and inhibits aberrant Ca2+ release, DADs, and triggered activity.
- © 2013 by American Heart Association, Inc.