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Prevention of ventricular arrhythmia and calcium dysregulation in a catecholaminergic polymorphic ventricular tachycardia mouse model carrying calsequestrin-2 mutation.

ABSTRACT: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium-regulation proteins cardiac ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca(2+) overload and increased Ca(2+) leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT.CPVT mouse models that lack CASQ2 were treated with Ca(2+) -channel inhibitors, ?-adrenergic inhibitors, or Mg(2+) . Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca(2+) transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress-induced ventricular arrhythmia in mutant mice. The Ca(2+) channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca(2+) content in mutant myocytes, diminished diastolic Ca(2+) overload, increased systolic Ca(2+) amplitude, and prevented Ca(2+) oscillations in stressed mutant myocytes.Ca(2+) channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil-induced partial normalization of SR Ca(2+) content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca(2+) buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT.

SUBMITTER: Alcalai R

PROVIDER: S-EPMC3053436 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Prevention of ventricular arrhythmia and calcium dysregulation in a catecholaminergic polymorphic ventricular tachycardia mouse model carrying calsequestrin-2 mutation.

Alcalai Ronny R Wakimoto Hiroko H Arad Michael M Planer David D Konno Tetsuo T Wang Libin L Seidman Jon G JG Seidman Christine E CE Berul Charles I CI

Journal of cardiovascular electrophysiology 20100830 3

<h4>Background</h4>Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium-regulation proteins cardiac ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca(2+) overload and increased Ca(2+) leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT.<h4>Method and results</h4>CPVT mouse models ...[more]

PMID: 20807279

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Project description:AimsCatecholaminergic 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 resultsSixteen 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), β-adrenoceptor (βAR) 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 βAR stimulation.ConclusionIn 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 βAR stimulation in CPVT1, but also identify an independent and synergistic contribution from effects of high heart rate.

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.

Dataset Information

Prevention of ventricular arrhythmia and calcium dysregulation in a catecholaminergic polymorphic ventricular tachycardia mouse model carrying calsequestrin-2 mutation.

Publications

Prevention of ventricular arrhythmia and calcium dysregulation in a catecholaminergic polymorphic ventricular tachycardia mouse model carrying calsequestrin-2 mutation.

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