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Phospholamban knockout breaks arrhythmogenic Ca²? waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

ABSTRACT: 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.

SUBMITTER: Bai Y

PROVIDER: S-EPMC3864692 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Phospholamban knockout breaks arrhythmogenic Ca²⁺ waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

Bai Yunlong Y Jones Peter P PP Guo Jiqing J Zhong Xiaowei X Clark Robert B RB Zhou Qiang Q Wang Ruiwu R Vallmitjana Alexander A Benitez Raul R Hove-Madsen Leif L Semeniuk Lisa L Guo Ang A Song Long-Sheng LS Duff Henry J HJ Chen S R Wayne SR

Circulation research 20130715 5

<h4>Rationale</h4>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.<h4>Objective</h4>We investigate the impact of PLN-KO on SCWs, triggere ...[more]

PMID: 23856523

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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.

Dataset Information

Phospholamban knockout breaks arrhythmogenic Ca²? waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

Publications

Phospholamban knockout breaks arrhythmogenic Ca²⁺ waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

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