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An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations.

ABSTRACT: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7?Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7?Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this 'worm arrhythmia' by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.

SUBMITTER: Fischer E

PROVIDER: S-EPMC5727474 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations.

Fischer Elisabeth E Gottschalk Alexander A Schüler Christina C

Scientific reports 20171213 1

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca<sup>2+</sup> release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca<sup>2+</sup> binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, ...[more]

PMID: 29235522

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Project description:AIMS:Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) predisposes to ventricular tachyarrhythmias (VTs) during high heart rates due to physical or psychological stress. The essential role of catecholaminergic effects on ventricular cardiomyocytes in this situation is well documented, but the importance of heart rate per se for arrhythmia initiation in CPVT1 is largely unexplored. METHODS AND RESULTS:Sixteen CPVT1 patients performed a bicycle stress-test. Occurrence of VT triggers, i.e. premature ventricular complexes (PVC), depended on high heart rate, with individual thresholds. Atrial pacing above the individual PVC threshold in three patients did not induce PVCs. The underlying mechanism for the clinical observation was explored using cardiomyocytes from mice with the RyR2-R2474S (RyR2-RS) mutation, which exhibit exercise-induced VTs. While rapid pacing increased the number of Ca2+ waves in both RyR2-RS and wild-type (p<0.05), ?-adrenoceptor (?AR) stimulation induced more Ca2+ waves in RyR2-RS (p<0.05). Notably, Ca2+ waves occurred despite decreased sarcoplasmic reticulum (SR) Ca2+ content in RyR2-RS (p<0.05), suggesting increased cytosolic RyR2 Ca2+ sensitivity. A computational model of mouse ventricular cardiomyocyte electrophysiology reproduced the cellular CPVT1 phenotype when RyR2 Ca2+ sensitivity was increased. Importantly, diastolic fluctuations in phosphorylation of RyR2 and SR Ca2+ content determined Ca2+ wave initiation. These factors were modulated towards increased propensity for arrhythmia initiation by increased pacing rates, but even more by ?AR stimulation. CONCLUSION:In CPVT1, VT propensity depends on individual heart rate thresholds for PVCs. Through converging data from clinical exercise stress-testing, cellular studies and computational modelling, we confirm the heart rate-independent pro-arrhythmic effects of ?AR stimulation in CPVT1, but also identify an independent and synergistic contribution from effects of high heart rate.

Project description:RATIONALE:Autosomal-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. OBJECTIVE:To 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 RESULTS:Guide 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. CONCLUSIONS:AAV 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.

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An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations.

Publications

An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations.

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