Abstract 12935: Enhanced Binding of Calmodulin to RyR2 Inhibits Aberrant Ca2+ Release and Arrhythmogenesis 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). Gly-Ser-His-CaM (GSH-CaM) showed higher binding affinity than CaM in cAMP-treated KI cells (70 nM), although the ability of GSH-CaM to increase CaMKII activity was similar to CaM. 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 GSH-CaM (200 nM) decreased both DADs and TA (DAD: 20% of cells; TA: 10% of cells), whereas CaM (200 nM) did not. Addition of GSH-CaM to saponin-permeabilized KI cells decreased Ca2+ spark frequency (+6% of WT cells), which otherwise markedly increased without GSH-CaM (+50% of WT cells), whereas CaM revealed much less effect on the Ca2+ spark frequency (+23% of WT cells). Then, by incorporation of CaM or GSH-CaM to intact cells (by protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM]=∼240 nM, cytosolic [GSH-CaM]=∼230 nM) on the frequency of spontaneous Ca2+ transient (sCaT, % of total cells). Addition of 10 nM isoproterenol to KI cells increased sCaT after transient 5 Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) (p Conclusions. In CPVT-associated mutation of RyR2 CaM binding affinity is decreased upon PKA phosphorylation, whereas normally restored CaM binding to the mutant RyR2 inhibits aberrant Ca2+ release, DADs, and triggered activity.