Abstract 5289: Abnormally Tight Domain-Domain Interaction at Mutation Site Could be a Primary Cause of Catecholaminergic Polymorphic Ventricular Tachycardia: Insight from RyR2 S2246L/+ Knock-In Mouse Model
Mutations in cardiac ryanodine receptor 2 (RyR2) have been shown to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of this lethal arrhythmia, we developed knock-in (KI:RyR2S2246L/+) mice model with the human CPVT-associated RyR2 mutation (S2246L). 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 isolated cardiomyocytes, line scan images were obtained to measure local Ca2+ release events using a confocal microscopy with fluo-4 AM as a Ca2+ indicator. In the KI cardiomyocytes, the frequency of Ca2+ sparks (SpF: s−1·100μm−1) was much more increased in response to 100 nM isoproterenol than in WT cardiomyocytes (KI:4.7±0.5 vs WT:1.9±0.2, p<0.01). Using the canine cardiac SR, we fluorescently labeled RyR2 with methylcoumarin acetamido (MCA), using either DP2232–2266 or DP2232–2266mut; Ser is mutated to Leu (S2246L), as a carrier; DP2232–2266 harbors the same CPVT mutation site as KI mice (S2246L). The binding affinity of DP2232–2266mut to RyR2 (Kd=0.08μM) was higher than that of DP2232–2266 (Kd=0.32μM), suggesting that abnormally tight interaction of the domain pair (between the domain2232–2266 and another putative domain) may be formed by the S2246L mutation. Interestingly, addition of DP2232–2266 to the SR dose-dependently inhibited the cAMP (30μM)-induced Ca2+ leak; IC50=0.1μM, although DP2232–2266 had no effect on the cAMP-induced increase in 2808Ser phosphorylation. In conclusion, some type of RyR2 mutation in CPVT may causatively induce hyper-activated channel gating by forming abnormally tight domain-domain interaction, triggering diastolic Ca2+ release and hence lethal arrhythmia. Interruption of such abnormal domain-domain interaction (by competing with native domain) may lead to a new therapeutic strategy against CPVT.