P676Mechanisms of sinoatrial node dysfunction in RyR2(R420Q) mice model of catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) has been considered as one of the most important causes of children's sudden cardiac death. Mutations in the genes for RyR2 and CASQ2, two mainly subtypes of CPVT, have been identified. However, the mutation in the gene of TECRL was rarely reported, which could be another genetic cause of CPVT. We evaluated myocardial contractility, electrophysiology, calcium handling in Tecrl knockout (Tecrl KO) mice and human induced pluripotent stem cell-derived cardiomyocytes. Immediately after epinephrine plus caffeine injection, Tecrl KO mice showed much more multiple premature ventricular beats and ventricular tachycardia. The Tecrl KO mice demonstrate CPVT phenotypes. Mechanistically, intracellular calcium amplitude was reduced, while time to baseline of 50 was increased in acute isolated cardiomyocytes. RyR2 protein levels decreased significantly upon cycloheximide treatment in TECRL deficiency cardiomyocytes. Overexpression of TECRL and KN93 can partially reverse cardiomyocytes calcium dysfunction, and this is p-CaMKII/CaMKII dependent. Therefore, a new CPVT mouse model was constructed. We propose a previously unrecognized mechanism of TECRL and provide support for the therapeutic targeting of TECRL in treating CPVT

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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

Circulation ◽

10.1161/circ.118.suppl_18.s_520-b ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Takeshi Suetomi ◽

Masafumi Yano ◽

Xiaojuan Xu ◽

Makoto Ono ◽

Hitoshi Uchinoumi ◽

...

Keyword(s):

Ventricular Tachycardia ◽

Underlying Mechanism ◽

Catecholaminergic Polymorphic Ventricular Tachycardia ◽

Polymorphic Ventricular Tachycardia ◽

Domain Pair ◽

Mice Model ◽

Domain Interaction ◽

Mutation Site ◽

Lethal Arrhythmia ◽

Putative Domain

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:RyR2 S2246L/+ ) 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 Ca 2+ release events using a confocal microscopy with fluo-4 AM as a Ca 2+ indicator. In the KI cardiomyocytes, the frequency of Ca 2+ 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 DP 2232–2266 or DP 2232–2266 mut; Ser is mutated to Leu (S2246L), as a carrier; DP 2232–2266 harbors the same CPVT mutation site as KI mice (S2246L). The binding affinity of DP 2232–2266 mut to RyR2 (Kd=0.08μM) was higher than that of DP 2232–2266 (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 DP 2232–2266 to the SR dose-dependently inhibited the cAMP (30μM)-induced Ca 2+ leak; IC50=0.1μM, although DP 2232–2266 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 Ca 2+ 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.

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