Effects And Mechanisms Of The P.Q1283H Variant In The Spectrin-binding Domain Of Ankyrin-B Gene On Ventricular Arrhythmias

Background:Sudden cardiac death（SCD）is a major problem of global public health,accounting for an estimated 15%-20%of all deaths.With the explosive advancement of medical technologies,especially for the wide application of implantable cardioverter defibrillators（ICDs）,the resuscitation rates have been generally improved throughout the world.However,effective treatments and preventive measures for SCD are still limited.Therefore,the potential etiology and molecular mechanism of SCD should be further clarified,providing valuable theoretical basis for the development of new therapeutic drugs that are effective against cardiac diseases.Ischemic heart disease is one of the main causes of SCD,whereas only a small number of SCD occur in patients with inherited arrhythmias,where the heart is structurally normal.Nevertheless,inherited arrhythmias have always been a difficult scientific problem in the field of cardiovascular disease diagnosis and treatment because they are common in children and young adults.Such cases appear to be healthy but are difficult to diagnose and treat,have a high rate of SCD,and have a familial genetic basis.Inherited arrhythmias are associated with genetic variants of ion channel genes that cause abnormalities in cardiac electrical function;therefore,such diseases are also called“ion channelopathies”.With the rapid development of molecular biology and molecular genetics,recent studies have proposed that non-ion channel gene variants,such as the ANK2 gene,which encodes the protein of ankyrin-B,may also cause inherited arrhythmias.These gene variants cause the electrical instability of the heart and thus arrhythmias mainly via affecting the activity of ion channels,.Human“loss-of-function”ANK2 variants as well as variants that decrease the expression of ankyrin-B have been demonstrated to cause a dominantly inherited condition,which involves a wide spectrum of arrhythmias that include long QT syndrome type 4,ventricular tachycardia（VT）,idiopathic ventricular fibrillation,catecholaminergic polymorphic ventricular tachycardia,sinus node dysfunction,atrial fibrillation,and even SCD.Such diseases are also termed“ankyrin-B syndrome”.In vitro studies have proposed that most ANK2 variants could decrease the expression of ankyrin-B and its binding partners(Na⁺/Ca²⁺exchanger,Na⁺/K⁺ATPase and inositol 1,4,5-trisphosphate receptor),which lead to abnormalities in cellular Ca²⁺cycling and triggered arrhythmias.Several ANK2 variants have been reported throughout the world,but most of them are mainly found in Caucasians.To date,no ANK2 variants have been reported in Chinese patients with ventricular arrhythmias.In our previous study,a rare missense ANK2 variant（p.Q1283H,rs755373114）was identified in a patient with recurrent VT.This variant showed a high pathogenicity index based on the assessment by using different in silico predictive algorithms.However,there have yet to be comprehensive studies that perform an in vivo functional analysis of the ANK2 p.Q1283H variant,and its specific arrhythmogenic mechanisms have not been elucidated.Objectives:This study engineered a knock-in（KI）mouse model with the ANK2p.Q1283H variant to perform an in vivo functional analysis;explore the electrophysiological and molecular mechanisms of p.Q1283H-induced arrhythmias;and further screen drug therapies that are effective for individuals with the p.Q1283H variant by using the KI model.Methods:KI mice with the ANK2 p.Q1283H variant were engineered through homologous recombination.Young adult mice（8 to 16 weeks）were used in our experimental design.Age-and sex-matched wild-type（WT）littermates were used as controls.Electrocardiographs were obtained from conscious WT and KI mice both at rest and under stress（2 mg/kg epinephrine,intraperitoneally）by using real-time telemetry monitoring（Data Sciences International,USA）.Myocytes from WT and KI hearts dissociated via the Langendorff perfusion system were used to measure the incidence rate of delayed afterdepolarizations（DADs）and triggered activity,spontaneous Ca²⁺release events and Ca²⁺sparks both at rest and under stress（1μmol/L isoproterenol,ISO）.The potential molecular mechanisms of arrhythmogenesis were explored by using a series of experiments,such as quantitative real-time polymerase chain reaction（PCR）,Western bolts,coimmunoprecipitation,immunofluorescence,GST-pull down assays,BIAcore analysis,analytical gel filtration chromatography,and isothermal titration calorimetry.Finally,we tested the inhibitory action of aβ1-adrenoceptor blocker（100 mg/kg/day metoprolol）or I_C antiarrhythmic drug（15 mg/kg flecainide）on the development of arrhythmias in KI mice.Results:1.In vivo functional analysis and cell electrophysiological study（1）After an intraperitoneal injection of epinephrine,KI mice bearing the ANK2p.Q1283H variant conferred an increased susceptibility to ventricular arrhythmias（e.g.,premature ventricular contractions,trigeminy,and nonsustained or sustained VT）,exhibiting some resemblance to the human p.Q1283H carrier’s phenotype.（2）In the presence of ISO plus 2 Hz pacing,KI cardiomyocytes showed an increased frequency of DADs and trigger activity compared with those of WT controls.（3）In the presence of ISO plus 2 Hz pacing,KI cardiomyocytes showed an increased frequency of spontaneous Ca²⁺release events compared with those of WT controls.（4）Compared with those of WT controls,the frequency of Ca²⁺sparks was significantly increased in KI cardiomyocytes under basal conditions;and was further enhanced in the presence of ISO.2.Molecular biology mechanisms（1）The phosphorylation of ryanodine receptor type 2（RyR2）Ser2814 was significantly increased in KI hearts under basal conditions,and was further enhanced in the presence of ISO.However,the phosphorylation of RyR2 Ser2808 was comparable between WT and KI hearts.（2）In KI hearts,KN93,a potent inhibitor of calcium/calmodulin-dependent protein kinase II（CaMKII）,significantly blunted the ISO-induced increase in the phosphorylation of RyR2 Ser2814.In KI cardiomyocytes,KN93 significantly decreased the frequency of DADs,spontaneous Ca²⁺release events and Ca²⁺sparks that were induced by ISO.In KI mice,KN93 inhibited the incidence of ventricular arrhythmias induced by epinephrine.（3）Coimmunoprecipitation assays showed that,compared with WT controls,the interaction of protein phosphatase 1 catalytic subunit（PP1c）with RyR2 in KI hearts was not affected,while KI hearts had a significantly reduced amount of protein phosphatase 2A catalytic subunit（PP2Ac）associated with RyR2.Consistently,immunofluorescence assays showed that,compared with WT controls,KI hearts had a decreased co-localization of PP2Ac and RyR2.（4）Coimmunoprecipitation,GST-pull down assays,and BIAcore analysis consistently suggested that the ANK2 p.Q1283H variant could lead to a reduced binding of ankyrin-B to B56α.3.Pharmacological interventions（1）In KI mice,metoprolol or flecainide reduced the incidence of ventricular arrhythmias induced by epinephrine.（2）The antiarrhythmic action of flecainide was not directly mediated through the inhibition of RyR2.Conclusions:1.ANK2 p.Q1283H is a“disease-associated”variant that confers susceptibility to ventricular arrhythmias,which may be associated with hyperphosphorylation of RyR2 and DAD-mediated trigger activity.2.The loss of PP2A activity from RyR2 is likely associated with reduced ankyrin-B/B56αbinding,which may be the molecular basis of hyperphosphor-ylation of RyR2 in KI hearts.3.Metoprolol or flecainide may be potential drugs for treating ANK2p.Q1283H-associated arrhythmias.4.Our study is the first to suggest that dysfunction of the ZU5^C domain may be responsible for developing ventricular arrhythmias.