Ankyrin-B Q1283H Variant Linked to Arrhythmias Via Loss of Local Protein Phosphatase 2A Activity Causes Ryanodine Receptor Hyperphosphorylation

BACKGROUND: Human loss-of-function variants of ANK2 (ankyrin-B) are linked to arrhythmias and sudden cardiac death. However, their in vivo effects and specific arrhythmogenic pathways have not been fully elucidated. METHODS: We identified new ANK2 variants in 25 unrelated Han Chinese probands with ventricular tachycardia by whole-exome sequencing. The potential pathogenic variants were validated by Sanger sequencing. We performed functional and mechanistic experiments in ankyrin-B knockin (KI) mouse models and in single myocytes isolated from KI hearts. RESULTS: We detected a rare, heterozygous ANK2 variant (p.Q1283H) in a proband with recurrent ventricular tachycardia. This variant was localized to the ZU5(C) region of ANK2, where no variants have been previously reported. KI mice harboring the p.Q1283H variant exhibited an increased predisposition to ventricular arrhythmias after catecholaminergic stress in the absence of cardiac structural abnormalities. Functional studies illustrated an increased frequency of delayed afterdepolarizations and Ca(2+) waves and sparks accompanied by decreased sarcoplasmic reticulum Ca(2+) content in KI cardiomyocytes on isoproterenol stimulation. The immunoblotting results showed increased levels of phosphorylated ryanodine receptor Ser2814 in the KI hearts, which was further amplified on isoproterenol stimulation. Coimmunoprecipitation experiments demonstrated dissociation of protein phosphatase 2A from ryanodine receptor in the KI hearts, which was accompanied by a decreased binding of ankyrin-B to protein phosphatase 2A regulatory subunit B56α. Finally, the administration of metoprolol or flecainide decreased the incidence of stress-induced ventricular arrhythmias in the KI mice. CONCLUSIONS: ANK2 p.Q1283H is a disease-associated variant that confers susceptibility to stress-induced arrhythmias, which may be prevented by the administration of metoprolol or flecainide. This variant is associated with the loss of protein phosphatase 2A activity, increased phosphorylation of ryanodine receptor, exaggerated delayed afterdepolarization-mediated trigger activity, and arrhythmogenesis.