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Short-coupled polymorphic ventricular tachycardia at rest linked to a novel ryanodine receptor (RyR2) mutation: leaky RyR2 channels under non-stress conditions.

ABSTRACT: Ryanodine receptor (RyR2) mutations have largely been associated with catecholaminergic polymorphic ventricular tachycardia (PMVT). The role of RyR2 mutations in the pathogenesis of arrhythmias and syncope at rest is unknown. We sought to characterize the clinical and functional characteristics associated with a novel RyR2 mutation found in a mother and daughter with PMVT at rest.A 31-year-old female with syncope at rest and recurrent short-coupled premature ventricular contractions (PVCs) initiating PMVT was found to be heterozygous for a novel RyR2-H29D mutation. Her mother, who also had syncope at rest and short-coupled PMVT, was found to harbor the same mutation. Human RyR2-H29D mutant channels were generated using site-directed mutagenesis and heterologously expressed in HEK293 cells together with the stabilizing protein calstabin2 (FKPB12.6). Single channel measurements of RyR2-H29D mutant channels and wild type (WT) RyR2 channels were compared at varying concentrations of cytosolic Ca(2+). Binding affinities of the RyR2-H29D channels and RyR2-WT channels to calstabin2 were compared. Functional characterization of the RyR2-H29D mutant channel revealed significantly higher open probability and opening frequency at diastolic levels of cytosolic Ca(2+) under non-stress conditions without protein kinase A treatment. This was associated with a modest depletion of calstabin2 binding under resting conditions.The RyR2-H29D mutation is associated with a clinical phenotype of short-coupled PMVT at rest. In contrast to catecholaminergic PMVT-associated RyR2 mutations, RyR2-H29D causes a leaky channel at diastolic levels of Ca(2+) under non-stress conditions. Leaky RyR2 may be an under-recognized mechanism for idiopathic PMVT at rest.

SUBMITTER: Cheung JW

PROVIDER: S-EPMC4281514 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Short-coupled polymorphic ventricular tachycardia at rest linked to a novel ryanodine receptor (RyR2) mutation: leaky RyR2 channels under non-stress conditions.

Cheung Jim W JW Meli Albano C AC Xie Wenjun W Mittal Suneet S Reiken Steven S Wronska Anetta A Xu Linna L Steinberg Jonathan S JS Markowitz Steven M SM Iwai Sei S Lacampagne Alain A Lerman Bruce B BB Marks Andrew R AR

International journal of cardiology 20141125

<h4>Background</h4>Ryanodine receptor (RyR2) mutations have largely been associated with catecholaminergic polymorphic ventricular tachycardia (PMVT). The role of RyR2 mutations in the pathogenesis of arrhythmias and syncope at rest is unknown. We sought to characterize the clinical and functional characteristics associated with a novel RyR2 mutation found in a mother and daughter with PMVT at rest.<h4>Methods and results</h4>A 31-year-old female with syncope at rest and recurrent short-coupled ...[more]

PMID: 25463374

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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: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.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.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.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. ?-adrenergic stimulation breaks the delicate Ca(2+) equilibrium of RyR2-V2475F(+/-) hearts and triggers life-threatening arrhythmias.

Dataset Information

Short-coupled polymorphic ventricular tachycardia at rest linked to a novel ryanodine receptor (RyR2) mutation: leaky RyR2 channels under non-stress conditions.

Publications

Short-coupled polymorphic ventricular tachycardia at rest linked to a novel ryanodine receptor (RyR2) mutation: leaky RyR2 channels under non-stress conditions.

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