Phenotype And Mechanism Of Ventricular Arrhythmia Caused By The E1766K Variant In The C-terminal Domain Of Ankyrin-B Gene

Background:It is known that most sudden cardiac deaths(SCD)occur in young and middle-aged people with normal cardiac structure.These patients usually look like healthy people,but a high incidence of malignant arrhythmia leads to SCD and has family inheritance.The pathogenesis of most hereditary arrhythmias is related to ion channels,and some are caused by mutations in non-ion channel protein genes,such as the ANK2 gene encoding ankyrin B(AnkB).As a universal"adapter" protein,ankyrin can connect the inner membrane protein of the cell to the cytoskeletal protein network(such as spectrin and actin)and participate in the maintenance of normal life activities of a variety of cells.The classic ankyrin includes membrane protein binding domain(MBD),spectrin binding domain(SBD),death domain(DD)and C-terminal domain(CTD),the latter two constitute the regulatory domain(RD).In this study,the location of the mutation is located in CTD,which is the most common area of mutation,which mainly plays a role in regulating the function of MBD and SBD area.In addition,the CTD region can also directly bind a variety of proteins,such as molecular chaperone Hdj 1/Hsp40,obscuration protein Obscurin,Fas protein and so on.At present,among the multiple mutation sites detected in the CTD region,only p.R1788W can cause "serious"functional changes,while other mutation sites,such as p.T1626N,p.L1622I,p.E1813K,p.T1726N,The results of functional analysis of p.T1552N,p.V1777M,and p.I3285T showed "very slight" changes,and molecular mechanism studies of related gene mutations are rare.There are no reports on the mutation sites and mechanisms of the C-terminal related genes of Ankyrin B gene in China.At present,most of the research on the mutation mechanism of AnkB in foreign countries is based on in vitro cell experiments,while in vivo animal experiments are rare.In recent years,our team has reported the molecular mechanism of mutations in the SBD region.Follow-up studies have found another mutation in a group of patients with ventricular tachycardia(ventricular tachycardia),a new mutation site p.E1766K.Therefore,this experiment will explore the molecular mechanism of p.E1766K mutation triggering ventricular arrhythmia and screen effective therapeutic drugs.Objective:The team has successfully constructed the ANK2 p.E1766K heterozygous mutation(ANK2+/E1766K)knock-in mouse model(KI)in the early stage and conducted in vivo functional research on it(the specific research results have been shown in the master’s thesis during the same period);Therefore,this topic mainly explores the electrophysiological and related molecular mechanisms of arrhythmia triggered by stress in KI mice.Finally,ANK2 p.E1766K mutation-induced arrhythmia model mice are used to screen individualized therapeutic drugs.Methods:1.Apply bioinformatics and high-throughput sequencing technology to screen for possible genetic mutations in patients with ventricular tachycardia;2.Use Langendorff isolated heart perfusion technique to acutely separate WT and KI mouse cardiomyocytes,record the electrophysiological parameters of mouse cardiomyocytes,calcium wave,calcium wave,and stress state(2mg/kg,adrenaline,intraperitoneal injection),respectively.Frequency of calcium sparks;3.Use plasmid recombination and co-transformation,immunoblotting,immunoprecipitation,immunofluorescence,GST-pull down technology and other methods to explore the molecular mechanism of p.E1766K-induced arrhythmia;4.Use ANK2 p.E1766K mutation-induced arrhythmia model mice for individualized drug screening.First,use the ECG telemetry sensor(TA10-ETA-F10)subcutaneous implantation technology to record the β-receptor blocker metoprolol(4mg/kg,intraperitoneal injection)and class Ic anti arrhythmic drug fluoride ECG changes after treatment with Carney(15mg/kg,intraperitoneal injection)and class IV anti arrhythmic drug verapamil(2.5mg/kg,intraperitoneal injection);then observe the incidence of triggering events in mouse cardiomyocytes after the above-mentioned drug treatment,And the regulation of calcium in myocardial cells.Result:1.Information on patients with ANK2 p.E1766K mutation and "Next-generation"sequencing resultsThrough "Next-generation" sequencing technology combined with bioinformatics analysis,we detected a total of 2 gene mutations in the patient’s peripheral blood DNA samples,namely SCN5A p.Q998K and ANK2 p.E1766K,through FATHMM,Mutation Assessor,SIFT,M-CAP,Mutation Taster,PolyPhen-2,PRO VEAN,CADD and other pathogenicity analysis software,we found that ANK2 p.E1766K is more pathogenic than SCN5A p.Q998K,which was verified by first-generation sequencing.After comparing with 1000Genomes,ESP6500,ExAC database,it is found that ANK2 p.E1766K has a lower mutation frequency in the population,and the mutation site is highly conserved in various species.2.Study on Electrophysiological and Molecular Mechanisms of Ventricular Arrhythmia Caused by ANK2 p.E1766K Mutation(1)The incidence of spontaneous diastolic calcium waves in cardiomyocytes in the basal state was not different between the WT and KI mouse groups(p>0.05);when the cardiomyocytes were under stress(luM,epinephrine),the KI group The incidence of spontaneous calcium waves in mouse cardiomyocytes is increased(**p<0.01).(2)There was no difference in the frequency of calcium sparks in cardiomyocytes of WT and KI mice under basal conditions(p>0.05);but under stress,the frequency of calcium sparks in cardiomyocytes of KI mice was higher than that of WT group.High(**p<0.01).(4)There was no difference in calcium transient amplitude,attenuation time course,and calcium capacity(both p>0.05)in cardiomyocytes of WT and KI mice in the basal state;under stress,calcium in cardiomyocytes of WT and KI mice There was still no difference in transient amplitude and decay duration(all p>0.05),but the calcium capacity of the KI group was higher than that of the WT group(**p<0.01).(4)The RYR2 channel inhibitor Ryanodine was given to block the channel,and it was found that Ryanodine can inhibit the incidence of spontaneous calcium waves(**p<0.01),but cannot reduce the calcium capacity(p>0.05);give IP3R2 channel inhibitor Xestospongin C.It was found that it increased the incidence of spontaneous calcium waves in the cardiomyocytes of KI mice(**p<0.05),and the calcium volume also increased(*p<0.05);when the IP3R2 agonist Thimerosal was used to treat the myocardium of KI mice After cells,it was found that the drug reduced the incidence of spontaneous calcium waves in cardiomyocytes(**p<0.01),and the calcium capacity also decreased(**p<0.01).(5)It was observed by ELISA experiment that the synthesis of IP3 in the heart tissues of mice in the WT and KI groups under stress was higher than that in the basal state,but there was no difference between the groups;the immunoprecipitation results showed that AnkB is full-length,Hsp40,Hsp70 There is an interaction relationship between the two pairs of IP3R2,and compared with the WT group,the binding relationship between the full length of AnkB and IP3R2,and the full length of AnkB and Hsp40 are reduced in the KI group;the results of the GST-pull down experiment further confirm that AnkB-CTD domain and Hsp40,Hsp70 and IP3R2 all have a direct relationship,and compared with the WT group,the binding of AnkB-CTD domain and Hsp40 in the KI group is reduced.(6)By constructing recombinant plasmids,using cell co-transfection and co-immunoprecipitation technology,the binding region of AnkB-CTD domain and Hsp40 is clarified,located in the α helix of AnkB-CTD domain and CTD1/CTD2 domain of Hsp40.3.Therapeutic drug exploration of ANK2 p.E1766K mutation-induced ventricular arrhythmia model(1)After treatment with metoprolol,flecainide and verapamil,the incidence of ventricular arrhythmia in the administration group under stress was reduced compared with the mice in the non-administered KI group(**p<0.05).(2)The electrophysiological results suggest that after treatment with metoprolol,flecainide and verapamil,compared with the mice in the non-administered KI group,the cardiomyocytes of the mice in the administration group under stress spontaneously The incidence of calcium waves is reduced(**p<0.01).Conclusion:1.The team used bioinformatics and next-generation sequencing technology to detect the mutation E1766K at the C-terminus of the ANK2 gene for the first time.2.The mechanism of ANK2 p.E1766K mutation-induced ventricular arrhythmia is related to abnormal sarcoplasmic reticulum calcium release caused by the decrease of AnkB anchoring IP3R2 in the KI group;the specific mechanism is that ANK2 p.E1766K inhibits the anchoring effect of AnkB protein on Hsp40,Thereby reducing the effect of Hsp40 on the co-chaperone protein Hsp70,and ultimately reducing its binding to the substrate IP3R2.3.Metoprolol,verapamil,and flecainide may be effective treatment drugs for patients with ANK2 p.E1766 mutation.