Abstract 3702: Correction of Defective Inter-Domain Interactions within Ryanodine Receptor by Dantrolene may be a Potent Therapeutic Strategy Against Lethal Arrhythmia
Catecholaminergic polymorphic ventricular tachycardia(CPVT) is linked with mutations in cardiac rhyanodine receptor (RyR2). DPc10, a synthetic peptide (Gly2460-Pro2495) of RyR2 (one of the common mutable domains in CPVT and malignant hyperthermia:MH), mimics channel dysfunction in human CPVT, by acting competitively to reduce stabilizing interactions between the N-terminal1− 600 and central2000 −2500 domains of RyR2 (viz. domain unzipping). Dantrolene, a specific drug for the treatment of MH, was recently found to bind the N-terminal domain of RyR1 and RyR2. Here, we examined the effect of dantrolene on the inter-domain interaction within RyR2 and Ca2+ release function.
Methods and Results: Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (normal: n=6, pacing-induced heart failure: n=6), then RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) using DPc10 as a site-directing carrier. DPc10 mediated a specific MCA fluorescence labeling of RyR2 among many other SR proteins. Addition of DPc10 to the MCA-labeled SR competitively induced the domain unzipping, as confirmed by a marked quenching of the MCA fluorescence by a large-size fluorescence quencher. Dantrolene inhibited both DPc10-induced domain unzipping and Ca2+ leak with the same IC50 (0.3 μM). In DPc10-incoporated cardiomyocytes (by protein delivery kit), both Ca2+ spark (CaS; s−1.100μm−1: 3.8±1.4, p<0.01) and delayed afterdepolarization-mediated Ca2+ transient (DAD-CaT) were frequently seen, compared to normal ones (CaS:1.4±0.4), even though SR Ca2+ content, assessed by 20 mM caffeine application, was markedly decreased. Both CaS (1.5±0.5, p<0.01) and DAD-CaT were inhibited by 1.0 μM dantrolene, concurrently with an increase in SR Ca2+ content by 45%. In failing cardiomyocytes, in which domain unzipping, prominent CaS (4.0±1.5, p<0.01), and DAD-CaT have already occurred like DPc10-incorporated normal ones, dantrolene normally restored all of these abnormalities.
Conclusions: Dantrolene seems to correct the defective inter-domain interaction between N-terminal and central domains within RyR2, thereby inhibiting diastolic Ca2+ sparks and DAD-CT that triggers lethal arrhythmia in CPVT/ heart failure.