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The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool.

ABSTRACT: Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca²⁺ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (E _GSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline E _GSH increase. Importantly, β-adrenergic stimulation resulted in excessive E _GSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically β-adrenergic stimulation significantly increased mitochondrial E _GSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic E _GSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca²⁺ leak induce a strong, but dantrolene-inhibited mitochondrial E _GSH oxidization in RyR2-R2474S cardiomyocytes.

SUBMITTER: Wegener JW

PROVIDER: S-EPMC8696262 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:BACKGROUND:Catecholaminergic polymorphic ventricular tachycardia is an inherited disease presenting with arrhythmic events during physical exercise or emotional stress. If untreated, catecholaminergic polymorphic ventricular tachycardia is a highly lethal condition: About 80% of affected individuals experience recurrent syncope, and 30% experience cardiac arrest. Catecholaminergic polymorphic ventricular tachycardia is caused by mutations in genes encoding ryanodine receptor type 2 (RyR2) and cardiac calsequestrin (CASQ2). In cases of sympathoadrenergic activation, both mutations result in a spontaneous Ca2+ release in cardiac cells, facilitating ventricular arrhythmias. CASE PRESENTATION:We present a case of a 17-year-old Caucasian boy who survived sudden cardiac death caused by ventricular fibrillation while performing running exercise in a fitness center. The diagnostic workup included blood tests, coronary angiography, electrophysiological testing, and cardiac magnetic resonance imaging, but all results were normal. Because the patient's medical history included recurrent syncope during physical and emotional stress, we strongly suspected catecholaminergic polymorphic ventricular tachycardia as the underlying disease. Genetic screening was performed and confirmed the diagnosis, revealing a new heterozygous point mutation in the gene for RyR2, c.12520T>A (p.F4174 l, exon 90, RyR2 gene). The patient was discharged from our hospital after undergoing implantation of an implantable cardioverter defibrillator for secondary prevention. Shortly after implantation, the implantable cardioverter defibrillator terminated a sustaining ventricular tachycardia episode by antitachycardic pacing. This episode occurred early in the morning while the patient was asleep. CONCLUSIONS:We present a case of catecholaminergic polymorphic ventricular tachycardia associated with a novel single point mutation in the RyR2 gene, which, to the best of our knowledge, has not been described in the literature so far. Our patient experienced arrhythmic events under both resting conditions and physical activity, an uncommon finding in patients with catecholaminergic polymorphic ventricular tachycardia. This novel mutation may cause arrhythmias independent of sympathoadrenergic stimulation, but further evidence is needed to prove causality.

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The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool.

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