Catecholaminergic Polymorphic Ventricular Tachycardia Is Caused by Mutation-Linked Defective Conformational Regulation of the Ryanodine Receptor

Abstract

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in a well-defined region of the cardiac type 2 ryanodine receptor (RyR)2. However, the underlying mechanism by which a single mutation in such a large molecule produces drastic effects on channel function remains unresolved. Objective: Using a knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2. Methods and Results: The R2474S/؉ KI mice showed no apparent structural or histological abnormalities in the heart, but they showed clear indications of other abnormalities. Bidirectional or polymorphic ventricular tachycardia was induced after exercise on a treadmill. The interaction between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains of the RyR2 (an intrinsic mechanism to close Ca 2؉ channels) was weakened (domain unzipping). On protein kinase A-mediated phosphorylation of the RyR2, this domain unzipping further increased, resulting in a significant increase in the frequency of spontaneous Ca Key Words: ryanodine receptor Ⅲ calcium Ⅲ ventricular tachycardia Ⅲ sarcoplasmic reticulum T o date, more than 70 cardiac ryanodine receptor (RyR)2 missense mutations have been identified that are linked with 2 inherited forms of sudden cardiac death: catecholaminergic polymorphic ventricular tachycardia (CPVT) 1 and arrhythmogenic right ventricular cardiomyopathy type 2. 1 These mutations cluster in 3 well-defined regions of the RyR2 that correspond to malignant hyperthermia or the central core disease mutable regions, designated as the N-terminal domain (amino acids 1 to 600), central domain (amino acids 2000 to 2500), and the C-terminal transmembrane channel domain of the skeletal muscle-type ryanodine receptor (RyR1). 1 This suggests that the RyR2 shares a common domain-mediated channel regulation mechanism with RyR1. Mutations at different positions in each of these domains result in the nearly identical phenotype of channel dysfunctions such as hyperactivation of the Ca 2ϩ channel and hypersensitization to agonists. To account for these phenomena, Ikemoto et al 2,3 proposed the so-called "domain switch hypothesis" and stated that in the resting or nonactivated state, the N-terminal domain and the central domain make close contact at several subdomains (domain zipping). Then, on physiological or pharmacological stimulation, these critical interdomain contacts are weakened, resulting in the loss of conformational constraints (domain unzipping), thus lowering the energy barrier for Ca 2ϩ channel opening. Consistent with this hypothesis, single particle analysis of the 3D structure of the RyR2 molecule revealed that the N-terminal and central domains (located in domains 5 and 6 of the so-called clamp Original received September 15, 2009; revision received February 9, 2010; accepted February 26, 2010 region, respectively) are in a close apposition ...

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

“…Hence, hyperactive RyR2 channels eager to release Ca 2+ on their own appear as essential components of this arrhythmogenic scheme. In fact, most RyR2-linked CPVT mutations characterized to date produce hyperactive RyR2 channels (9)(10)(11)(12). This scheme therefore appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations (13).…”

Dantrolene, a Therapeutic Agent for Malignant Hyperthermia, Inhibits Catecholaminergic Polymorphic Ventricular Tachycardia in a RyR2R2474S/+ Knock-In Mouse Model

“…ECG was monitored in R2474S/+ KI mice (n=6) and wildtype (WT) mice (n=6) in a conscious state by using ECG telemetry, as previously described, 13 with slight modification. Briefly, transmitters (Data Sciences International, St. Paul, MN, USA) were implanted in the back space with s.c. electrodes in a lead II configuration.…”

“…13 In brief, R2474S/+ KI and WT mice (2-3 months old) were anesthetized with pentobarbital sodium (70 mg/kg of body weight, i.p. ), intubated and ventilated with ambient air.…”

“…11 More recently, by using the knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we clarified that a single amino acid mutation within the RyR2 sensitizes the RyR2 channel to activation by luminal [Ca 2+ ] (i.e., a decreased threshold of luminal [Ca 2+ ] for channel activation), and in turn induces spontaneous Ca 2+ sparks and DAD, leading to CPVT, and that danrrolene stabilized the leaky RyR2 by correcting the defective inter-domain interaction. 13 Here, we investigated the in vivo anti-arrhythmic effect of dantrolene in the KI mice model. Background: Dantrolene, a specific agent for the treatment of malignant hyperthermia, was found to inhibit Ca 2+ leak through not only the skeletal ryanodine receptor (RyR1), but also the cardiac ryanodine receptor (RyR2) by correcting the defective inter-domain interaction between N-terminal (1-619 amino acid) and central (2,000-2,500 amino acid) domains of RyRs.…”

Mechanisms of Ca2+-Triggered Arrhythmias

“…On exposure to isoproterenol (-adrenergic agonist), this mouse model produced DADs and triggered activity underlying CPVT . Subsequent studies using other transgenic mouse models (Kannankeril et al, 2006;Lehnart et al, 2008;Uchinoumi et al, 2010) confirmed that RyR2 mutations modify intracellular Ca 2+ regulation through an increased SR Ca 2+ leak as a result of increased RyR2 open probability at resting conditions (Jiang et al, 2005). It is this increased SR Ca 2+ leak that accounts for the increased propensity of DAD-mediated triggered activity.…”