Ryanodine receptor–bound calmodulin is essential to protect against catecholaminergic polymorphic ventricular tachycardia
Yoshihide Nakamura, Takeshi Yamamoto, Shigeki Kobayashi, Masaki Tamitani, Yoriomi Hamada, Go Fukui, Xiaojuan Xu, Shigehiko Nishimura, Takayoshi Kato, Hitoshi Uchinoumi, Tetsuro Oda, Shinichi Okuda, Masafumi Yano
Yoshihide Nakamura, Takeshi Yamamoto, Shigeki Kobayashi, Masaki Tamitani, Yoriomi Hamada, Go Fukui, Xiaojuan Xu, Shigehiko Nishimura, Takayoshi Kato, Hitoshi Uchinoumi, Tetsuro Oda, Shinichi Okuda, Masafumi Yano
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Research Article Cardiology

Ryanodine receptor–bound calmodulin is essential to protect against catecholaminergic polymorphic ventricular tachycardia

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in the cardiac type 2 ryanodine receptor (RyR2). Using a knockin (KI) mouse model (R2474S/+), we previously reported that a single point mutation within the RyR2 sensitizes the channel to agonists, primarily mediated by defective interdomain interaction within the RyR2 and subsequent dissociation of calmodulin (CaM) from the RyR2. Here, we examined whether CPVT can be genetically rescued by enhancing the binding affinity of CaM to the RyR2. We first determined whether there is a possible amino acid substitution within the CaM-binding domain in the RyR2 (3584–3603 residues) that can enhance its binding affinity to CaM and found that V3599K substitution showed the highest binding affinity of CaM to the CaM-binding domain. Hence, we generated a heterozygous KI mouse model (V3599K/+) with a single amino acid substitution in the CaM-binding domain of the RyR2 and crossbred it with the heterozygous CPVT-associated R2474S/+-KI mouse to obtain a double-heterozygous R2474S/V3599K-KI mouse model. The CPVT phenotypes — bidirectional or polymorphic ventricular tachycardia, spontaneous Ca2+ transients, and Ca2+ sparks — were all inhibited in the R2474S/V3599K mice. Thus, enhancement of the CaM-binding affinity of the RyR2 is essential to prevent CPVT-associated arrhythmogenesis.

Authors

Yoshihide Nakamura, Takeshi Yamamoto, Shigeki Kobayashi, Masaki Tamitani, Yoriomi Hamada, Go Fukui, Xiaojuan Xu, Shigehiko Nishimura, Takayoshi Kato, Hitoshi Uchinoumi, Tetsuro Oda, Shinichi Okuda, Masafumi Yano

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Figure 11

CaM binding to the RyR2 in cardiac homogenates.

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CaM binding to the RyR2 in cardiac homogenates. (A) (Top) Representative...
(A) (Top) Representative immunoblots of the RyR2-bound CaM-SANPAH (a photoreactive cross-linker). The WT and KI cardiac homogenates were first diluted in binding buffer (150 mM KCl, 10 μM CaCl2, and 20 mM MES at pH 6.8) and then reacted with various concentrations of CaM-SANPAH (32–1024 nM) in the presence or absence of cAMP (1 μM) and okadaic acid (1 μM). CaM binding to the RyR2 was determined by immunoblotting with anti-CaM antibody to detect the RyR2-bound CaM. (Bottom) Summarized data of CaM binding to the RyR2 as a function of the concentration of CaM-SANPAH. CaM binding was expressed as the ratio to the maximum binding of CaM (at 1024 nM). Data are presented mean ± SEM of 3 hearts. (B) Effect of cAMP (1 μM) and suramin (10 μM) on CaM binding to the RyR2. The concentration of CaM-SANPAH was fixed at 128 nM. Data are presented as mean ± SEM of 3 hearts. *P < 0.05; NS, not significant (Student’s t test).