Analysis Of MiRNA Expression Profile In Mouse Atrial Fibrosis Model And The Role Of MiR-483-5p In Fibrosis And Prognosis Analysis Of Patients With Atrial Fibrillation After Bioprosthetic Valve Replacement

Part ?.Differentially expressed profiles of microRNA in mouse atrial fibrosis modelObjective:Atrial fibrillation(AF)is the most common arrhythmia during daily clinical practice.The microRNA(miRNA)signature between atrial tissue samples from patients with AF versus sinus rhythm was significantly different,indicating that miRNA might participate in AF's pathological progression.The mouse atrial fibrosis model induced by angiotensin ?has been widely used for mechanism research related to AF.However,no literature depicted the miRNA expression profile of the atria in this model.Therefore,this study established angiotensin ?-induced mice model and performed transcriptome sequencing to draw the miRNA and mRNA differential expression profile and provided some basic information for the experiments that intended to use this model for further investigation.This study will also try to select some miRNAs related to atrial fibrosis based on the study results and previously published articles.Methods:Micro-osmotic pumps were used for continuous subcutaneously delivery of 2 mg/kg/day angiotensin II for 14 days in male C57BL/6N wild-type mice aged 10-12 weeks to induce atrial fibrosis.Atrial dilatation was confirmed by echocardiography,and atrial fibrosis was verified by pathological methods,and the susceptibility to atrial fibrillation was tested by burst electrical stimulation in the right atria.Then transcriptome sequencing was conducted using the mice atria(n=3 in each group)to detect the differential expression in miRNA and mRNA.MiRanda,TargetScan,and RNAhybrid were used for target prediction,and the overlapped predicted target genes in any two of the three databases were then selected for further analysis.The G0,KEGG,Reactome databases and GESA calculation method were used to make a functional annotation for the intersection of differentially expressed mRNA and selected predicted target genes as well as differentially expressed mRNA.In addition,referring to the previously published sequencing results from human-derived atrial specimens,candidate miRNAs suitable for the application of this model for molecular mechanism research were screened out.Results:In this study,atrial fibrosis mice models induced by angiotensin? were successfully constructed,in which the atrial enlargement and atrial fibrosis were confirmed,and the susceptibility to atrial fibrillation also increased.According to functional annotation analysis,differentially expressed mRNAs were mainly concentrated in the extracellular matrix and inflammatory activation related pathways;differentially expressed miRNA target genes were concentrated in the Ras pathway,TGF-? signaling pathway,and participate in the formation of extracellular matrix space,calcium ion binding,potassium ion channel activation.By comparison with published literature,6 miRNAs might involve in the development of AF:miR-21a-3p,miR-24-5p,miR-30c-5p,miR-421-5p,miR-483,miR-212.Moreover,according to the functional annotation analysis,miR-483 might play a role in regulating the fibrosis process.Conclusion:This study successfully established an angiotensin ?-induced atrial fibrosis mice model,mapped the miRNA and mRNA expression profiles in this model.The study might provide some miRNA and mRNA data for researches that also used the same mice model.Meanwhile,6 miRNAs were considered to be involved in the development of AF according to our study.Part ?.Overexpression of miR-483-5p reduces type ? collagen secretion in cardiac fibroblastsObjective:Compared with sinus rhythm patients,miR-483-5p is differentially expressed in both atrium and plasma of patients with AF,suggesting that it might be involved in the development of AF.Based on the results of previous animal experiments,miR-483-5p might play a role in regulating the fibrosis process.Therefore,this study aimed to explore its effect on fibrosis.Methods:This study firstly used RT-qPCR to explore the expression of miR-483-5p in primary mouse cardiac fibroblasts treated with angiotensin? in vitro.MiR-483-5p mimics and inhibitors were transfected into cardiac fibroblasts to overexpress or knock down the molecules.The expression of collagen COL1A1 and COL3A1 was determined by Western blot.In addition,the CCK-8 method was used to detect the effect of overexpression or inhibition of miR-483-5p on cell proliferation.Results:The expression of miR-483-5p was down-regulated after angiotensin ? stimulation.Overexpression of miR-483-5p in primary fibroblasts reduced the expression of COL3A1 protein,but the expression of COL1A1 protein was unchanged.Downregulation of miR-483-5p promoted the protein production of COL3A1 significantly,while the COL1A1 protein expression level remained unchanged.Overexpressed miR-483-5p could inhibit cell proliferation,while miR-483-5p inhibitor increased the cell proliferation ability.Conclusion:MiR-483-5p decreased in cardiac fibroblasts stimulated by angiotensin ? in vitro,overexpression of miR-483-5p showed an anti-fibrotic effect but inhibited cell proliferation.Part ?.The characteristics and long-term outcomes in patients with atrial fibrillation and bioprosthetic valvesObjective:With the progress of aging,patients with atrial fibrillation with bioprosthetic valves have been increasing.However,previous studies focused on AF included few such patients,so clinical data in this population was scarce.Therefore,this study reviewed data to investigate the clinical characteristics,long-term prognosis,risk factors and dynamic changes and anticoagulation status.Methods:The retrospective study enrolled 903 patients with bioprosthetic valve(s)replacement at our hospital and discharged them with a diagnosis of atrial fibrillation from January 2010 to December 2018.Baseline data were collected by reviewed the hospital' s electronic medical record system.The information during the follow-up period was collected by reviewing hospital records and telephone visit and completed by May 2020.Categorical variables were expressed as frequencies and percentages,and continuous variables were expressed as means with standard deviations or medians with quartiles,depending on the distribution characteristics of variables.Cox regression model was used to analyze the risk factors of all-cause death and thromboembolic events;C-statistics was calculated to evaluate the predictive ability of CHA2DS2-VASc score in nonanticoagulated population;logistic regression analysis was performed to identify factors related to oral anticoagulant use during follow-up.Results:The median age was 65.6(61.9-69.1)years old,and 548(60.7%)patients were women.Patients had a high prevalence of rheumatic heart disease(63%),and 642(71.1%)patients underwent mitral bioprosthetic valve replacement.The percentage of surgical ablation and surgical left atrial appendage occlusion or exclusion was 31.8%and 14.5%,respectively.The current proportion of oral anticoagulants was 52.3%,59%and 63.2%,respectively in the CHA2DS2-VASc score 0 in male or 1 in female,1 in male or 2 in female and>2 in male or 3 in female groups according to baseline CHA2DS2-VASc score.Age,left atrial size,coronary heart disease and rheumatic heart disease were positively associated with the use of oral anticoagulants.The history of chronic kidney dysfunction,surgical ablation,and antiplatelet agent use was inversely related to the use of oral anticoagulants.During a follow-up period of 3.84(2.64-5.51)years,68(1.8%/year)patients died,81(2.1%/year)patients developed thromboembolism,23(0.6%/year)patients experienced major bleeding.Admission estimated glomerular filtration rate(HR:0.966,95%CI:0.943-0.99,P=0.005),left ventricular ejection fraction(HR:0.962,95%CI:0.931-0.993,P=0.016),concomitant surgical ablation(HR:0.348,95%CI:0.171-0.711,P=0.004)or rheumatic heart disease history(HR:0.516,95%CI:0.311-0.854,P=0.01)were associated with low risk of death.Surgical ablation(HR:0.263,95%CI:0.133-0.519,P<0.001)and oral anticoagulants(HR:0.587,95%CI:0.375-0.918,P=0.019)were related to low risk of thromboembolism.The CHA2DS2-VASc score,as a categorical variable(three categories),predicted the risk of thromboembolism with the C-statistic of 0.6(95%CI:0.511-0.689,P=0.046).The incidence of CHA2DS2-VASc score increment was 11.6 per 100 person-years,.Conclusion:Chinese patients with AF and bioprosthetic valve(s)were relatively young and had a high prevalence of rheumatic heart disease with few comorbidities.The percentage of mitral bioprosthetic valve replacement was high;some patients underwent concomitant surgical ablation or surgical left atrial appendage occlusion or exclusion.The thromboembolic events were the major long-term adverse events and needed more attention.The anticoagulation therapy was underused in patients with CHA2DS2-VASc score?1 in male or 2 in female.The CHA2DS2-VASc score was also verified for predicting the thromboembolic risk in this population.Also,the risk factor of thromboembolism dynamically changed,it needed to be re-estimated once the risk factor changed.