The Clinical Significance Of Anti-m2R Autoantibody And Anti-cardiac Mysosin Heavy Chain Autoantibody In Hypertensive Patients With Atrial Fibrillation And Their Effects On Electrophysiological Property Of Human Atrial Myocyte

Part I The detection and clinical significance of anti-M2R autoantibody and anti-cardiac mysosin heavy chain autoantibody in hypertensive patients with atrial fibrillationObjective:To investigate the change of anti-M2R autoantibody and anti-cardiac mysosin heavy chain autoantibody in hypertensive patients with atrial fibrillation and elucidate the relationship between both autoantibodies and atrial fibrillation.Methods:The selected295hypertensive patients were divided into two groups:group one consisting of145cases with atrial fibrillation, and group two consisting of150cases without atrial fibrillation used as control group. Peripheral blood samples were collected, and then tested for the detection of four autoantibodies (AAM2R, AAMHC, AAβ1R and AAANT) by using enzyme-linked immunoabsorption assays (ELISA). At last, the relationships between autoantibodies and atrial fibrillation were analyzed through univariate and multivariate logistic regression analysis. We also selected80subjects for health examination as normal control group, in order to observe the differences of autoantibodies detection with the above groups.Results:Compared with group two (control group), the positive rate of both autoantibodies (AAM2R and AAMHC) significantly increased in hypertensive patients with atrial fibrillation (AAM2R:44.8%vs13.3%; AAMHC:27.6%vs14.7%; P<0.01). The incidence of atrial fibrillation in autoantibody-positive hypertension patients remarkable increased in contrast with autoantibody-negative patients(AAM2R:76.5%vs38.1%, P<0.001; AAMHC:64.5%vs45.1%, P<0.01). Autoantibodies both AAM2R and AAMHC were significant risk factors and strong predictors (AAM2R:OR=5.251; AAMHC:OR=2.696). The positive rate of normal control group was significantly lower than that of the two groups of hypertensive patients.Conclusion:The present study indicated that autoantibodies both AAM2R and AAMHC were closely associated with atrial fibrillation in hypertensive patients, suggesting that autoimmune mechanism might play a crucial role in the initiation and development of atrial fibrillation. Part Ⅱ The extraction, purification and identification of anti-M2receptor and anti-cardiac mysosin heavy chain autoantibodies from hypertensive patients with atrial fibrillationObjective:There were highly detection of autoantibodies both AAM2R and AAMHC in the sera from hypertensive patients with atrial fibrillation. The goal of this study was to determine the binding specificity of both autoantibodies with corresponding antigen respectively. Methods:The right atrial appendages from patients underwent cardiac open-chest surgery were extracted for whole proteins and enzymatically isolated for single atrial myocytes. By using salt precipitation in (NH4)2SO4and affinity chromatography, the autoantibodies were purified from the sera of hypertensive patients.The binding specificities of both autoantibodies with corresponding antigen were confirmed through immunoblotting and cellular immunofluorescence techniquesResults:AAM2R specially reacted with M2receptors of human atrial tissue and recombinant human M2receptor expressed in CHO cells. By using laser confocal immunofluorescence, AAM2R specially reacted with human atrial myocytes. The negative sera and autoantibody absorbed by corresponding antigen failed to do so. There were similar results of AAMHC by immunoblotting and immunofluorescence. Furthermore, AAMHC could cross-reacted with human β1receptor.Conclusion:There were special autoantibodies of both AAM2R and AAMHC in the sera from hypertensive patients with atrial fibrillation. They could specially be bound with human atrial M2receptor and myosin heavy chain respectively. AAMHC could cross-reacted with human β1receptor. Part III The effects of anti-M2receptor and anti-cardiac mysosin heavy chain autoantibodies on repolarization currents and action potential of human atrial myocytesObjective:Autoantibodies both AAM2R and AAMHC were closely associated with atrial fibrillation in hypertensive patients, which were important risk factors and strong predictors of atrial fibrillation. the aim of the present study is to explore the influence of autoantibodies on the repolarization current and action potential of huaman atrial myocytes, through which the electrophysiological mechanism of arrhythmogenesis of autoantibodies could be elucidated.Methods:Right atrial appendages were obtained from cardiac surgical patients with sinus rhythm, which were enzymatically isolated for single atrial myocytes. The effects of autoantibodies on a variety of repolarization currents of human atrial myocytes were recorded by using whole-cell and perforated patch clamp techniques. The effects of autoantibodies on action potential of atrial myocytes were observed by current-clamp mode with perforated patch clamp technique.Results:In contrast with the autoantibody-negative sera, AAM2R produced muscarinic agonist-like actions, such as the open of Ikach channel, the reduction of ICa-L current, the shortening of action potential duration, which were abolished upon autoantibody absorption using corresponding antigenic peptide. However, AAM2R failed to change Itol and Ikur currents. The effects of AAMHC on the above currents and action potential of atrial myocyte were not observed.Conclusion:the alteration of electrophysiological properties of human atrial myocyte ion channels by AAM2R could occurred, which was contributed to atrial electrical remodeling, leading to the initiation and perpetuation of atrial fibrillation. However, AAMHC failed to do so and perhaps influence atrial fibrillation via other mechanism such as inflammation. Our study suggested autoimmune might took part in the initiation and development of atrial fibrillation.