Abstract 4625: Dantrolene Inhibits Spontaneous Ca2+ Release as a Substrate for Catecholaminergic Polymorphic Ventricular Tachycardia (PSVT) by Correcting Defective Inter-Domain Interaction within the Ryanodine Receptor
We previously reported that dantrolene, a therapeutic agent for malignant hyperthermia (MH), prevented abnormal Ca2+ leak by correction of the defective inter-domain interaction between N-terminal (1– 600) and central (2000 –2500) domains in MH-type ryanodine receptor (RyR). These N-terminal and central domains of the RyR, harbor many mutations associated with MH in RyR1 and CPVT in RyR2. Here, we examined the effect of dantrolene on the Ca2+ release in CPVT-associated RyR2R2474S/+ knock-in (KI) mice model. In KI (but not in wild-type:WT) mice (n=6), ventricular tachycardia was observed during or after exercise with treadmill (6/6), which was prevented by pretreatment of dantrolene (20 mg−1kg−1day−1, for 7 days). Cardiac sarcoplasmic reticulum (SR) vesicles were isolated (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP2460 –2495 (DPc10), harboring the CPVT mutation site; R2474S, as a site-directing carrier. Only in KI (but not WT) SR, cAMP (1 μM) reduced stabilizing interactions between N-terminal and central domains (viz. domain unzipping), as assessed by the quenching of the MCA fluorescence by a large-size fluorescence quencher. Dantrolene (1 μM) inhibited the cAMP-induced domain unzipping (in KI) without effect on the cAMP-induced increase in Ser2808 phosphorylation that showed no difference between WT and KI. Using a quartz crystal microbalance technique (a highly sensitive mass-measuring technique), dantrolene was found to specifically bind to the domain 601– 620 of RyR2. In isolated, saponin-permeabilized cardiomyocytes, local Ca2+ release events were measured 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· 100 μm−1) was much more increased in response to 1μM cAMP (9.7±0.4 in WT; 14.5±0.4 in KI, p<0.01). In the co-presence of dantrolene (1 μM), the increase in SpF in KI was markedly inhibited. In conclusion, dantrolene, by normally restoring the defective inter-domain interaction, seems to correct the hyper-activated channel gating caused by RyR2 mutation, thereby inhibiting spontaneous Ca2+ release leading to lethal arrhythmia.