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Paradoxical effect of increased diastolic Ca(2+) release and decreased sinoatrial node activity in a mouse model of catecholaminergic polymorphic ventricular tachycardia.

ABSTRACT: Catecholaminergic polymorphic ventricular tachycardia is characterized by stress-triggered syncope and sudden death. Patients with catecholaminergic polymorphic ventricular tachycardia manifest sinoatrial node (SAN) dysfunction, the mechanisms of which remain unexplored.We investigated SAN [Ca(2+)](i) handling in mice carrying the catecholaminergic polymorphic ventricular tachycardia-linked mutation of ryanodine receptor (RyR2(R4496C)) and their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2(R4496C) mice after isoproterenol injection, analogous to what was observed in catecholaminergic polymorphic ventricular tachycardia patients after exercise. Pacemaker activity was explored by measuring spontaneous [Ca(2+)](i) transients in SAN cells within the intact SAN by confocal microscopy. RyR2(R4496C) SAN presented significantly slower pacemaker activity and impaired chronotropic response under ?-adrenergic stimulation, accompanied by the appearance of pauses (in spontaneous [Ca(2+)](i) transients and action potentials) in 75% of the cases. Ca(2+) spark frequency was increased by 2-fold in RyR2(R4496C) SAN. Whole-cell patch-clamp experiments performed on isolated RyR2(R4496C) SAN cells showed that L-type Ca(2+) current (I(Ca,L)) density was reduced by ?50%, an effect blunted by internal Ca(2+) buffering. Isoproterenol dramatically increased the frequency of Ca(2+) sparks and waves by ?5 and ?10-fold, respectively. Interestingly, the sarcoplasmic reticulum Ca(2+) content was significantly reduced in RyR2(R4496C) SAN cells in the presence of isoproterenol, which may contribute to stopping the "Ca(2+) clock" rhythm generation, originating SAN pauses.The increased activity of RyR2(R4496C) in SAN leads to an unanticipated decrease in SAN automaticity by a Ca(2+)-dependent decrease of I(Ca,L) and sarcoplasmic reticulum Ca(2+) depletion during diastole, identifying subcellular pathophysiological alterations contributing to the SAN dysfunction in catecholaminergic polymorphic ventricular tachycardia patients.

SUBMITTER: Neco P

PROVIDER: S-EPMC3434373 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Paradoxical effect of increased diastolic Ca(2+) release and decreased sinoatrial node activity in a mouse model of catecholaminergic polymorphic ventricular tachycardia.

Neco Patricia P Torrente Angelo G AG Mesirca Pietro P Zorio Esther E Liu Nian N Priori Silvia G SG Napolitano Carlo C Richard Sylvain S Benitah Jean-Pierre JP Mangoni Matteo E ME Gómez Ana María AM

Circulation 20120618 4

<h4>Background</h4>Catecholaminergic polymorphic ventricular tachycardia is characterized by stress-triggered syncope and sudden death. Patients with catecholaminergic polymorphic ventricular tachycardia manifest sinoatrial node (SAN) dysfunction, the mechanisms of which remain unexplored.<h4>Methods and results</h4>We investigated SAN [Ca(2+)](i) handling in mice carrying the catecholaminergic polymorphic ventricular tachycardia-linked mutation of ryanodine receptor (RyR2(R4496C)) and their wil ...[more]

PMID: 22711277

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Project description:Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca²? ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca²? load and Ca²? leak. Conversely, PLN-KO accelerates Ca²? sequestration and aborts arrhythmogenic spontaneous Ca²? waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca²?-triggered arrhythmias.We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT.We generated a PLN-deficient, RyR2-mutant mouse model (PLN-/-/RyR2-R4496C+/-) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT-associated RyR2-R4496C mutant mice. Ca²? imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/- ventricular myocytes during sarcoplasmic reticulum Ca²? overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca²? sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca²? overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca²? ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li? failed to convert mini-waves to cell-wide SCWs in PLN-/-/RyR2-R4496C+/- ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs.Our results demonstrate that despite severe sarcoplasmic reticulum Ca²? leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca²? sequestration represents an effective approach for suppressing Ca²?-triggered arrhythmias.

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.

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Paradoxical effect of increased diastolic Ca(2+) release and decreased sinoatrial node activity in a mouse model of catecholaminergic polymorphic ventricular tachycardia.

Publications

Paradoxical effect of increased diastolic Ca(2+) release and decreased sinoatrial node activity in a mouse model of catecholaminergic polymorphic ventricular tachycardia.

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