A novel de novo calmodulin mutation in a 6-year-old boy who experienced an aborted cardiac arrest
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Abstract:
Mutations in the human calmodulin genes (CALM1, CALM2, and CALM3) are associated with life-threatening conditions in childhood, such as idiopathic ventricular fibrillation (VF) and long QT syndrome (LQTS).1–3 Furthermore, CALM1 mutations were described in a catecholaminergic polymorphic ventricular tachycardia (CPVT)-like phenotype.4 Sudden unexplained death in the young can be the first clinical manifestation of an underlying arrhythmogenic disorder such as idiopathic VF.5,6 After an aborted cardiac arrest, determining the diagnosis begins with a systematic clinical evaluation.Keywords:
Sudden cardiac arrest
Sudden Death
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Ryanodine receptor 2 (RyR2) is the major Ca2+ release channel on the sarcoplasmic reticulum (SR) and plays a central role in EC-coupling in the heart. Abnormal activation of RyR2 by gain-of-function mutations such as catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked mutations is known to increase propensity of spontaneous Ca2+ release from SR and causes ventricular arrythmia. In such cases, drugs that suppress RyR2 activity are expected to have anti-arrhythmic effects. However, this hypothesis has not been directly tested since no specific RyR2 inhibitors had been identified yet. We recently searched for RyR2 inhibitors utilizing a high-throughput screening procedure with HEK293 cells expressing RyR2 and an ER Ca2+ indicator (Murayama et al., Mol Pharmacol, 2018), and found several compounds that inhibited RyR2 but not RyR1. Furthermore, we successfully synthesized structural analogs from one of hit compounds with much higher affinity. In this study, we examined the effects of these newly found RyR2 inhibitors on arrhythmia in CPVT model mice which have a gain-of-function mutation in RyR2 and exhibit abnormal Ca2+ and membrane potential signals in their cardiomyocytes. Our results suggest that RyR2 inhibitors are promising antiarrhythmic drug candidates for arrhythmia in CPVT patients.
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Rationale: Most cardiac ryanodine receptor (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) are postulated to cause a distinctive form of Ca 2+ release dysfunction. Considering the spread distribution of CPVT mutations, we hypothesized that dysfunctional heterogeneity also was feasible. Objective: To determine the molecular and cellular mechanisms by which a novel RyR2-V2475F mutation associated with CPVT in humans triggers Ca 2+ -dependent arrhythmias in whole hearts and intact mice. Methods and Results: Recombinant channels harboring CPVT-linked RyR2 mutations were functionally characterized using tritiated ryanodine binding and single-channel recordings. Homologous recombination was used to generate a knock-in mouse bearing the RyR2-V2475F mutation. Ventricular myocytes from mice heterozygous for the mutation (RyR2-V2475F +/− ) and their wild-type littermates were Ca 2+ -imaged by confocal microscopy under conditions that mimic stress. The propensity of wild-type and RyR2-V2475F +/− mice to have development of arrhythmias was tested at the whole heart level and in intact animals. Recombinant RyR2-V2475F channels displayed increased cytosolic Ca 2+ activation, abnormal protein kinase A phosphorylation, and increased activation by luminal Ca 2+ . The RyR2-V2475F mutation appears embryonic-lethal in homozygous mice, but heterozygous mice have no alterations at baseline. Spontaneous Ca 2+ release events were more frequent and had shorter latency in isoproterenol-stimulated cardiomyocytes from RyR2-V2475F +/− hearts, but their threshold was unchanged with respect to wild-type. Adrenergically triggered tachyarrhythmias were more frequent in RyR2-V2475F +/− mice. Conclusions: The mutation RyR2-V2475F is phenotypically strong among other CPVT mutations and produces heterogeneous mechanisms of RyR2 dysfunction. In living mice, this mutation appears too severe to be harbored in all RyR2 channels but remains undetected under basal conditions if expressed at relatively low levels. β-adrenergic stimulation breaks the delicate Ca 2+ equilibrium of RyR2-V2475F +/− hearts and triggers life-threatening arrhythmias.
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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.
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Background: Ryanodine receptor 2 (RYR2) gene mutation causing catecholaminergic polymorphic ventricular tachycardia (CPVT) is one of the identified causes of sudden death in adults and children.
Sudden Death
Sudden cardiac arrest
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Sudden Death
Fibrillation
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Objective Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal inherited disease characterized by ventricular arrhythmias induced by physical exercise or emotional stress. The major cause of CPVT is mutations in RYR2, which encodes the cardiac ryanodine receptor channel. Recent advances in sequencing technology have yielded incidental findings of RYR2 variants in other cardiac diseases. Analyzing the characteristics of RYR2 variants related to CPVT will be useful for differentiation from those related to other cardiac diseases. We examined the phenotypic characteristics of patients with RYR2 variants. Methods Seventy-nine probands carrying RYR2 variants whose diagnoses were either CPVT (n=68) or long QT syndrome (LQTS; n=11) were enrolled. We compared the characteristics of the electrocardiogram (ECG) and the location of the RYR2 mutations-N-terminal (NT), central region (CR) or C-terminal (CT)-between the two patient groups. Results Using the ECGs available from 53 probands before β-blocker therapies, we analyzed the heart rates (HRs). CPVT probands showed bradycardia more frequently (25/44; 57%) than LQTS probands (1/9; 11%; p=0.024). In CPVT patients, 20 mutations were located in NT, 25 in CR and 23 in CT. In LQTS patients, 5 mutations were located in NT, 2 in CR and 4 in CT. There were no significant differences in the locations of the RYR2 mutations between the phenotypes. Conclusion Bradycardia was highly correlated with the phenotype of CPVT. When a clinically-diagnosed LQTS patient with bradycardia carries an RYR2 mutation, we should be careful to avoid making a misdiagnosis, as the patient may actually have CPVT.
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