The Effect And Mechanisms Of Atrial Calcium Mishandling And Atrial Fibrosis Mediated By The Increased β1 Adrenergic Receptor Autoantibodies In Atrial Fibrillation

Background:Autoimmune diseases are the emerging mechanism of atrial fibrillation（AF）.β1 adrenergic receptor antibody（β1AAb）is associated with the progression of atrial fibrillation.In the model of atrial fibrillation susceptibility with overexpression ofβ1AAbs,we aim to（1）clarify the characterization alterations of atrial remodeling in the active immunization models;（2）based on the in vitro optical mapping technology to study the electrophysiological mechanism of atrial fibrillation induced byβ1AAbs;（3）based on high-throughput phosphorylation quantitative proteomics to explore the molecular mechanism of atrial fibrillation mediated byβ1AAbs in order to expand the understanding of the intricate pathogenesis of atrial fibrillation from the perspective of autoimmune.Methods:Part I:Active immunization models were established via subcutaneous injection of the second extracellular loop（ECL2）peptide forβ1 adrenergic receptor（β1AR）,which is injected once a week with an interval of two weeks.The reverse group took orally bisoprolol（a commonβ1AR blocker）for treatment and was taken orally for two consecutive weeks from the eighth week.The resting heart rate in awake state was recorded by Powerlab instruments to evaluate the autonomic nervous tone,the atrial electrophysiological characteristics were evaluated by intracardiac electrophysiological measurement,and the atrial structural remodeling was studied by cardiac ultrasound and pathological detection.Part II:Under Langendorff perfusion,the atrial action potential duration（APD）and calcium transient duration（Ca D）under sinus condition were evaluated by optical mapping technique.The atrial conduction properties and atrial Ry R2refractoriness were evaluated by burst pacing and the arrhythmogenic alternations and reentry were evaluated under S1-S1 pacing mode.Part III:Quantitative phosphorylation proteomics are utilized to identify the differential phosphorylation sites under the condition ofβ1AAb overexpression correspond to the biological process of differential protein enrichment,and identify the differential phosphorylated site changes of calcium regulation related molecules under the condition ofβ1AAb overexpression,and the prediction of downstream potential kinase regulatory molecules.According to the results of high-throughput data,we search the molecular target and regulatory mechanism of atrial fibrillation and has been verified molecules expression at the tissue and cell levels.Exogenousβ1AAbs treatment was given into the atrial myocytes and atrial fibroblasts to ensure the calcium regulatory signal changes at the cellular level and induces the arrhythmogenic cellular phenotypes.Results:Part I:Theβ1AAb titers increased significantly from two weeks after the first active immunization to the 8th week,and the increased trend of c AMP in tissues is consistent with the serumβ1AAb titers,indicating that biological activity ofβ1AAb in the atrium.Suchβ1AAb overexpression increased heart rate and P-wave dispersion,but did not seem to affect frequency-dominated heart rate variability.Electrophysiological results show that the increase ofβ1AAbs aggravate the atria electrical instability,which is manifested by the shortening of AERP,the enlargement of vulnerable window（WOV）,the increase of atrial fibrillation inducibility and the prolongation of atrial fibrillation duration,suggesting thatβ1AAb is involved in autoimmune atrial electrical remodeling.ECG result display that the overexpression ofβ1AAbs expand the inner diameter of the atrium and aggravate the cardiac dysfunction.This significant atrial remodeling was in direct proportion to the degree of atrial interstitial fibrosis.Bisoprolol is effective in alleviatingβ1AAb-induced atrial electrical and structural remodeling has certain effects.Part II:In sinus rhythm mode,compared with the control group,β1AAbs significantly prolonged the time course from APD90 to APD20,and increased the repolarization heterogeneity of APD among the four different regions of the atrium,mainly with the increase of APD dispersion and coefficient of variation（COV）of APD.β1AAbs significantly prolonged the dispersion of atrial Ca D and calcium transients in different regions.Vm channel displays thatβ1AAbs slows down the rate of atrial depolarization and prolongs the time required for atrial Ca T to reach the maximum amplitude.Overexpressionβ1AAbs significantly reduced the degree of calcium transients in various regions of the atrium,and these effects could be reversed by bisoprolol to a certain extent.Overexpression in S1-S2 mode,β1AAbs shorten the ERP of isolated atrium and hinders the conduction performance of atrium,which is mainly manifested by the decrease of CV and the increase of conduction heterogeneity.The results of Ca²⁺channel recording reveal that,β1AAbs overexpression shorten the atrial Ry R2 refractoriness and increases the heterogeneity of the recovery of atrial calcium release between different regions,mainly with the decrease of Ca T recovery ratio and the increase of COV-Ca T.Based on burst stimulation mode,β1AAbs overexpression significantly increased the induction rate of atrial arrhythmias and the incidence of spatial SDA,and dual-channel recording showed thatβ1AAbs overexpression lead to the synchronization of arrhythmogenic AP alternations and calcium alternations.These effects can be prevented by bisoprolol.The synchronization phase reveals that AP alternation is coupled with re-entry circuits.Part III:Quantitative phosphorylation proteomics display that the molecules corresponding to the differential modification sites challenged byβ1AAbs are mainly enriched in the biological processes associated with excitation-contraction coupling,such as cardiac muscle hypertrophy,cardiac muscle contraction,etc.The enrichment of KEGG pathway suggests that hypertrophic cardiomyopathy pathway and Apelin pathway are the signals related to calcium regulation in top15.Kinase GSEA analysis identifies multiple potential regulatory kinases downstream ofβ1AAbs including MAPKAPK2,MAPK1,CDK18,RPS6KA1,PRKCA,PAK1,LRRK2 and GSK3B.The kinase activity analysis further shows that the functional potential regulatory molecules was affected byβ1AAbs including:BCKDK,PDK4,ROCK1,PRKAA1 and CAMK1D.The heat map shows that Ry R2 is modified at 5 differential phosphorylation sites（S1341,S1398,S1400,Y2822 and S1402）inβ1AAb group.β1AAbs activate the Ca MKII（Thr286）/Ry R2（S2814）axis and suppress the PLB phosphorylation at Ser16site.Consistent with the results at the tissue level,exogenous intervention byβ1AAbs（1μM）identically induce the Ca MKII/Ry R2signal activation and PLB inactivation,which can be partially rescued by blockingβ1AR.More than this,β1AAbs overexpression induce the transformation of fibroblasts into myofibroblasts,specifically represented by the increased levels ofα-SMA and Col-1.Conclusion:Suppressingβ1-AAbs enable to protect the atrial vulnerable substrate by ameliorating intracellular Ca²⁺mishandling and atrial fibrosis,preventing the development of the autoimmune associated AF.Bisoprolol performs a potential to prevent the AF related to autoimmune.