Abstract 5303: Decreased Affinity of Calmodulin Binding to RyR2 May Cause Leaky Channel in CPVT-Associated Mutation: Insight from RyR2 R2474s/+ Knock-In Mouse Model
Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), has been shown to play a significant role in regulating the channel gating in RyR2. However, the role of calmodulin on the channel gating in diseased hearts is unclear. Here, using knock-in (KI) mice model with CPVT-associated RyR2 mutation (R2474S), we investigated whether CaM plays a role in the pathogenesis of CPVT. Sarcoplasmic reticulum (SR) vesicles were isolated from mouse hearts (n=4). To evaluate the characteristics of CaM binding to RyR2, SR was mixed with various concentrations of CaM-SANPAH conjugate (16nM-256nM) , followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. There was no significant difference in the affinity of CaM binding to RyR2 at baseline between KI (Kd=26nM) and WT (Kd=16nM). However, compared to WT, CaM binding was markedly decreased in response to cAMP (1 μM) in KI (18±1.9 % decrease, at 128nM CaM), although 2808Ser phosphorylation in RyR2 was similarly increased by cAMP in WT and KI. This decrease of CaM binding in KI was, however, markedly diminished by co-addition of dantrolene which was previously found to prevent the abnormal Ca2+ leak by the correction of the defective inter-domain interaction between N-terminal (1–600) and central (2000–2500) domains in both MH-type and CPVT-type RyR2. Moreover, in the presence of FK506 (10μM), which was found to dissociate FKBP12.6 from RyR2 and subsequently to induce domain unzipping, CaM binding was indeed decreased in WT, whereas unchanged by FK506 in KI. In saponin-permeabilized, isolated cardiomyocytes, line scan images in cardiomyocytes were obtained to measure local Ca2+ release events using a confocal microscopy with Rhod-2 as a Ca2+ indicator; [Ca2+] was buffered at 30 nM by 0.5 mM EGTA. In the KI cardiomyocytes, the frequency of Ca2+ sparks (SpF:s−1·100μm−1) was more increased than in WT, in response to cAMP (at 1μM; +33%, p<0.01 vs WT). In conclusion, CaM binding ability to RyR2 seems to be markedly decreased in response to PKA phosphorylation in KI mice, perhaps via defective inter-domain interaction, causing spontaneous diastolic Ca2+ sparks that may lead to triggered activity and hence lethal arrhythmia.