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CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia.

ABSTRACT: Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro 'patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after ?-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), an antiarrhythmic drug that inhibits Ca(2+)/calmodulin-dependent serine-threonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies.

SUBMITTER: Di Pasquale E

PROVIDER: S-EPMC3824678 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia.

Di Pasquale E E Lodola F F Miragoli M M Denegri M M Avelino-Cruz J E JE Buonocore M M Nakahama H H Portararo P P Bloise R R Napolitano C C Condorelli G G Priori S G SG

Cell death & disease 20131010

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro 'patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the g ...[more]

PMID: 24113177

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Project description:RationaleAutosomal-dominant mutations in ryanodine receptor type 2 ( RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia.ObjectiveTo determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+).Methods and resultsGuide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 ( Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients.ConclusionsAAV serotype 9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.

Project description:IntroductionCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare inherited cardiac ion channelopathy. The present study aims to examine the clinical characteristics, genetic basis, and arrhythmic outcomes of CPVT patients from China to elucidate the difference between CPVT patients in Asia and Western countries.MethodsPubMed and Embase were systematically searched for case reports or series reporting on CPVT patients from China until 19 February 2022 using the keyword: "Catecholaminergic Polymorphic Ventricular Tachycardia" or "CPVT", with the location limited to: "China" or "Hong Kong" or "Macau" in Embase, with no language or publication-type restriction. Articles that did not state a definite diagnosis of CPVT and articles with duplicate cases found in larger cohorts were excluded. All the included publications in this review were critically appraised based on the Joanna Briggs Institute Critical Appraisal Checklist. Clinical characteristics, genetic findings, and the primary outcome of spontaneous ventricular tachycardia/ventricular fibrillation (VT/VF) were analyzed.ResultsA total of 58 unique cases from 15 studies (median presentation age: 8 (5.0-11.8) years old) were included. All patients, except one, presented at or before 19 years of age. There were 56 patients (96.6%) who were initially symptomatic. Premature ventricular complexes (PVCs) were present in 44 out of 51 patients (86.3%) and VT in 52 out of 58 patients (89.7%). Genetic tests were performed on 54 patients (93.1%) with a yield of 87%. RyR2, CASQ2, TERCL, and SCN10A mutations were found in 35 (71.4%), 12 (24.5%), 1 (0.02%) patient, and 1 patient (0.02%), respectively. There were 54 patients who were treated with beta-blockers, 8 received flecainide, 5 received amiodarone, 2 received verapamil and 2 received propafenone. Sympathectomy (n = 10), implantable cardioverter-defibrillator implantation (n = 8) and ablation (n = 1) were performed. On follow-up, 13 patients developed VT/VF.ConclusionThis was the first systematic review of CPVT patients from China. Most patients had symptoms on initial presentation, with syncope as the presenting complaint. RyR2 mutation accounts for more than half of the CPVT cases, followed by CASQ2, TERCL and SCN10A mutations.

Project description:BackgroundGenetic variants in calsequestrin-2 (CASQ2) cause an autosomal recessive form of catecholaminergic polymorphic ventricular tachycardia (CPVT), although isolated reports have identified arrhythmic phenotypes among heterozygotes. Improved insight into the inheritance patterns, arrhythmic risks, and molecular mechanisms of CASQ2-CPVT was sought through an international multicenter collaboration.MethodsGenotype-phenotype segregation in CASQ2-CPVT families was assessed, and the impact of genotype on arrhythmic risk was evaluated using Cox regression models. Putative dominant CASQ2 missense variants and the established recessive CASQ2-p.R33Q variant were evaluated using oligomerization assays and their locations mapped to a recent CASQ2 filament structure.ResultsA total of 112 individuals, including 36 CPVT probands (24 homozygotes/compound heterozygotes and 12 heterozygotes) and 76 family members possessing at least 1 presumed pathogenic CASQ2 variant, were identified. Among CASQ2 homozygotes and compound heterozygotes, clinical penetrance was 97.1% and 26 of 34 (76.5%) individuals had experienced a potentially fatal arrhythmic event with a median age of onset of 7 years (95% CI, 6-11). Fifty-one of 66 CASQ2 heterozygous family members had undergone clinical evaluation, and 17 of 51 (33.3%) met diagnostic criteria for CPVT. Relative to CASQ2 heterozygotes, CASQ2 homozygote/compound heterozygote genotype status in probands was associated with a 3.2-fold (95% CI, 1.3-8.0; P=0.013) increased hazard of a composite of cardiac syncope, aborted cardiac arrest, and sudden cardiac death, but a 38.8-fold (95% CI, 5.6-269.1; P<0.001) increased hazard in genotype-positive family members. In vitro turbidity assays revealed that p.R33Q and all 6 candidate dominant CASQ2 missense variants evaluated exhibited filamentation defects, but only p.R33Q convincingly failed to dimerize. Structural analysis revealed that 3 of these 6 putative dominant negative missense variants localized to an electronegative pocket considered critical for back-to-back binding of dimers.ConclusionsThis international multicenter study of CASQ2-CPVT redefines its heritability and confirms that pathogenic heterozygous CASQ2 variants may manifest with a CPVT phenotype, indicating a need to clinically screen these individuals. A dominant mode of inheritance appears intrinsic to certain missense variants because of their location and function within the CASQ2 filament structure.

Project description:BACKGROUND:Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current standard of care. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here, we tested the hypothesis that CaMKII inhibition with a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models. METHODS:We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide, autocamtide-2-related inhibitory peptide [AIP], is fused to green fluorescent protein (GFP) and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated with invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells derived from patients with CPVT with different disease-causing mutations to determine the effectiveness of our proposed therapy on human induced pluripotent stem cell-derived cardiomyocytes and different pathogenic genotypes. RESULTS:AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extracardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2R176Q/+) effectively suppressed ventricular arrhythmias induced by either ?-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ?50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. Induced pluripotent stem cell-derived cardiomyocytes derived from 2 different patients with CPVT with different pathogenic mutations demonstrated increased frequency of abnormal calcium release events, which was suppressed by a cell-permeable form of AIP. CONCLUSIONS:This proof-of-concept study showed that AAV-mediated delivery of a CaMKII peptide inhibitor to the heart was effective in suppressing arrhythmias in a murine model of CPVT. CaMKII inhibition also reversed the arrhythmia phenotype in human CPVT induced pluripotent stem cell-derived cardiomyocyte models with different pathogenic mutations.

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CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia.

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CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia.

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