Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes

Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated (a) the pathogenic role of CaM in the channel disorder in CPVT and (b) the possibility of correcting the CPVT-linked channel disorder, using knock-in (KI) mouse model with CPVT-associated RyR2 mutation (R2474S).Transmembrane potentials were recorded in whole cell current mode before and after pacing (1–5Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with HiLyte Fluor® in saponin-permeabilized myocytes. In the presence of cAMP (1μM) the apparent affinity of CaM binding to the RyR decreased in KI cells (Kd: 140–400nM), but not in WT cells (Kd: 110–120nM). Gly-Ser-His-CaM (GSH-CaM that has much higher RyR-binding than CaM) restored normal binding to the RyR of cAMP-treated KI cells (140nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5Hz pacing, whereas both DAD and TA were observed in 20% and 12% of KI cells, respectively. In response to 10nM 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 60% and 38% of cells, respectively. Addition of GSH-CaM (100nM) to KI cells decreased both DADs and TA (DAD: 38% of cells; TA: 10% of cells), whereas CaM (100nM) had no appreciable effect. Addition of GSH-CaM to saponin-permeabilized KI cells decreased Ca2+ spark frequency (+33% of WT cells), which otherwise markedly increased without GSH-CaM (+100% of WT cells), whereas CaM revealed much less effect on the Ca2+ spark frequency (+76% of WT cells). Then, by incorporating CaM or GSH-CaM to intact cells (with protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM]=∼240nM, cytosolic [GSH-CaM]=∼230nM) on the frequency of spontaneous Ca2+ transient (sCaT, % of total cells). Addition of 10nM isoproterenol to KI cells increased sCaT after transient 5Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) (P<0.01 vs CaM).Several disorders in the RyR channel function characteristic of the CPVT-mutant cells (increased spontaneous Ca2+ leak, delayed afterdepolarization, triggered activity, Ca2+ spark frequency, spontaneous Ca2+ transients) can be corrected to a normal function by increasing the affinity of CaM binding to the RyR.