Effect Of Mirna-208a On Atrial Connexin40 Remodeling Of Human Chronic Atrial Fibrillation

Background and objective: Atrial fibrillation(AF)is one of the most common clinical arrhythmia,which contribute to the majority of global public health burden.The pathogenesis of atrial fibrillation is complicated.Especially,atrial electrical remodeling is central to AF.Atrial electrical remodeling leads to shortness of atrial effective refractory period and action potential duration and absence of physiological frequency adaptation of the refractory period,which reduces electrical conduction and increases heterogeneity of conduction.The shortening of atrial refractory period is disproportionately slower than the conduction velocity,and the wavelength of the reentrant wavelet is shortened,which contributes to reentry activity.The changes of density and(or)spatial distribution of Cx(connexin)in heart causes the change of atrial impulse spreading.Thus,to explore the mechanism of the change of the density or(and)the spatial distribution of Cx in the atrium and to reverse the atrial electrical remodeling will provide new ideas for the treatment of AF.Cx40 is mainly expressed in the atrial and ventricular conduction system and play an important role in cardiac conduction system.The density and spatial distribution of Cx40 in gap junction of cardiomyocytes is vital to cardiac conduction abnormalities.microRNAs(miRNAs)is a kind of endogenous,highly conserved and small non-encoding RNAs.They inhibit the translation of mRNA by binding to complementary sequences that are usually located in the 3'-untranslated region(UTR)of target mRNA and suppress the expression of proteins at the post transcriptional level.Studies have shown that mi RNA-208 a specifically expressed in the heart,and played an essential role in the pathogenesis of many cardiovascular diseases including myocardial infarction,dilated cardiomyopathy and arrhythmia.GJA5,which encodes Cx40,is the target gene of miRNA-208a-3p by bioinformatics prediction.But whether miRNA-208a-3p contributes to atrial electrical remodeling by targeting Cx40 or not is unknown in AF.In this study,as to investigate the potential roles of mi RNA-208 a on Cx40 remolding in human AF,we measured mi RNA-208a-3p and Cx40 in patients with AF or sinus rhythm(SR)and Cx40 in AC16 cell line treated with miRNA-208a-3p mimic or inhibitor.The luciferase assays were performed to comfirm if Cx40 was directly targeted by miRNA-208a-3p.Method: The tissues of right atrial appendage(RAA)of patients with SR or AF were obtained.The expression of miRNA-208a-3p and the mRNA and protein expression of Cx40 were measured by in situ hybridization histochemistry(ISHH),real time quantity polymerase chain reaction(RT-qPCR),Western-blot and immune histochemical(IHC)respectively.The miRNA-208a-3p mimic or inhibitor was transient transfected into human cardiac myocytes(AC16 cell line)so as to construct the overexpression or low expression of miRNA-208a-3p AC16 cell model,then the mRNA and protein of Cx40 were determined in AC16 cells.Finally,whether Cx40 is the target gene of miRNA-208a-3p was verified by Luciferase reporter assay.Result:(1)The analysis of clinical data between patients with AF(n=19)and SR(n=18)showed there were no differences in age,gender,cardiac ejection,cardiac function but inner diameter of left atrium(65.16�9.81 vs 46.06�7.57)(p<0.05).The expression of miRNA-208a-3p in RAAs of patients with AF was higher than that of patients with SR [20.510(70.167)vs 1.169(5.991)](p<0.05).The expression of Cx40 mRNA in RAAs of patients with AF [0.354(0.527)] was no differences in patients with SR [0.372(0.303)](p>0.05).The Cx40 protein level significantly decreased(0.394�0.168 vs 0.300�0.104)(p<0.05)and the lateralization of Cx40 was observed in AF patients.The miRNA-208a-3p level in RAAs of patients was negative correlated with the Cx40 protein(r=-0.345,p<0.05).(2)There was no differences in Cx40 mRNA in AC16 cells among the groups transfected with miRNA-208a-3p mimic(0.455�0.110)or mimic negative control(0.587�0.127)or nothing 0.547(0.291)(p>0.05).But the level of Cx40 protein in AC16 cells transfected with miRNA-208a-3p mimic was lower than that in other two groups [0.061�0.020 vs 0.175(0.060)and 0.061�0.020 vs 0.211�0.023](p<0.05).There was no differences in Cx40 mRNA in AC16 cells among the groups transfected with miRNA-208a-3p inhibitor(3.889�3.718)or inhibitor negative control(4.720�3.761)or nothing(2.427�2.272)(p>0.05).But the level of Cx40 protein in AC16 cells transfected with miRNA-208a-3p inhibitor was higher than that in other two groups [0.243�0.079 vs 0.078�0.033 and 0.243�0.079 vs 0.071�0.050](p<0.05).There were no differences in luciferase activity between pmirGLO-hGJA5 3'-UTR transfected with miRNA-208a-3p mimic(30.084�0.790)and miRNA mimic NC(25.163�1.030)(p>0.05).There were no differences in luciferase activity between pmirGLO-mut-hGJA5 3'-UTR transfected with miRNA-208a-3p mimic(27.996�3.130)and mi RNA mimic NC(24.014�0.468)(p>0.05).Conclusion: miR-208 a is an important upstream negative regulatory factor of Cx40 and upregulation of miR-208 a is responsible for Cx40 remolding in human chronic AF.This may represent a potential therapeutic target for AF.