Abstract 5314: Domain2114–2149 Within RyR2 Plays a Key Role for Correcting Channel Disorder in Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)
We recently identified the K201(JTV519)-binding site as domain2114–2149 of the ryanodine receptor (RyR2), and the binding of K201 to this domain corrects the defective inter-domain interaction between N-terminal (0–600) and central regions (2000–2500) of RyR2 in pacing-induced failing hearts. Here, we further investigated the role of the domain peptide2114–2149 (DP2114–2149) on the Ca2+ release in human CPVT-associated RyR2R2474S/+ knock-in (KI) mice model. The KI mice revealed no structural or histological abnormality in hearts, and also had no contractile or relaxation dysfunction at rest. In all KI mice (n= 6), however, bi-directional ventricular tachycardia (VT) was observed after exercise with treadmill (Ex:6/6), but not observed in wild-type (WT) mice (Ex:0/7 ). In saponin-permeabilized cardiomyocytes from WT and KI mice, line scan images in cardiomyocytes were obtained to measure local Ca2+ release events using a confocal microscopy with Rhod-2; [Ca2+] was buffered at 30 nM by 0.5 mM EGTA. In KI, the frequency of Ca2+ sparks (SpF: s−1·100μm−1) was much more increased in response to cAMP (0.1–1 μM) than in WT (KI:19.2±0.4 vs WT:14.9±0.4; at [cAMP]= 0.3 μM , p<0.01). DP2114–2149 abolished the cAMP-induced increase in SpF in KI. Sarcoplasmic reticulum (SR) vesicles were isolated from WT and KI hearts (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP2460–2495 (DPc10), which harbors the same mutation site in CPVT as KI mice;R2474S, as a site-directing carrier. Addition of cAMP (1 μM) to the SR from KI (but not WT) mice reduced stabilizing interactions between the N-terminal1–600 and central domains2000–2500 (i.e. domain unzipping), as assessed by the quenching of the MCA fluorescence by a large-size fluorescence quencher (Kq: 1.16 in KI: 1.10 in WT). Interestingly, DP2114–2149 corrected this domain unzipping in KI (Kq:1.13), although DP2114–2149 had no effect on the cAMP-induced increase in Ser2808 phosphorylation. In conclusion, RyR2 mutation seen in human CPVT may causatively induce hyper-activated channel gating in response to PKA phosphorylation, via defective inter-domain interaction. Correction of the defective inter-domain interaction may be a new therapeutic strategy against CPVT.