Expression Of Galectin-3 And Its Correlation With Atrial Fibrosis In Valvular Atrial Fibrillation

BackgroundAtrial fibrillation(AF)is most common atrial arrhythmia in clinics,with a higher prevalence in patients with structural heart diseases,while the valvular heart disease remains the most common concomitant pathology in AF patients.However,the underlying mechanism of AF in patients with heart valve disease is still elusive.The electronic remodeling and structural remodeling are considered as the major reasons of AF.With the deeper and wider researches on AF,it is found that the atrial interstitial fibrosis plays a key role in the onset and maintenances of AF.Galectin-3,as an inflammation cytokine,which can participate in atrial fibrosis and cardiac remodeling associated with heart failure.Some studies show that Galectin-3 mainly induces the stimulation of fibroblasts and infiltration of macrophages,increases the expression of various kinds of proteins,thus accelerate the process of myocardial fibrosis.Meanwhile the expression of Galectin-3 is related to CyclinD1.There are two markers of fibroblast activation.?-SMA is a marker of muscle fibroblasts,bFGF is a mitogenic agent for fibroblasts,both can reverse the process of fibrosis from one side.In recent years,the studies also found that serum Galectin-3 level correlated with atrial fibrillation.Nevertheless,the expression of Galectin-3 in myocardium and the role of Galectin-3 in the development of valvular AF is still unknown.Objectives1.To investigate the alternations of Galectin-3 in left and right atrial myocardium and serum of valvular AF patients.2.To explore the correlationship of Galectin-3 expression levels with atrial fibrosis and the development of AF.MethodsA total of 50 Patients with mitral valve disease,who were hospitalized in Changzheng hospital affiliated to Second Military Medical University from Jan,2016 to Dec,2016 to undergo cardiac surgery,were enrolled in this study.They were divided into the persistent AF group(AF group)and sinus rhythm group(SR group)according to whether they were complicated by AF.There were 29 cases of patients in AF group and 21 cases of patients in SR group.There was no statistical difference between two groups in baseline characteristics such as gender,age,weight,BMI,cardiac function(NYHA grade).General information such as gender,age,height,weight,BMI,smoking status,the history of arrhythmia,previous use of drug(?-blocker,digitalis)were collected.All included patients should agree to participate in the study and sign the consent.Blood samples for liver and kidney function,thyroid function,total cholesterol,triglyceride,blood glucose,serum potassium,serum sodium,B brain natriuretic peptide(BNP),C-reactive protein(CRP)were taken at the first morning after admission.Left and right atrium were resected in operation for further study.Echocardiography was used for measure left atrial diameter(LAF),left ventricular end diastolic diameter(LVDd),left ventricular end-diastolic diameter(LVDs),and the Simpson's method was used to measure left ventricular ejection fraction(LVEF).Preoperative level of serum Galectin-3 was measured by Enzyme-Linked Immunosorbent Assay(ELISA).Hematoxylin-eosin stain was used to observe the pathology of cardiac tissue;Masson stain was used to check the fibrotic changes of cardiac tissue,by which the Collagen volume fraction(CVF)% was calculated.Realtime quantitative PCR(Q-PCR),Western-Blot(WB),Immunohistochemistry(IHC)were adopted to determine the expression level of mRNA and protein of TGF-?,bFGF,?-SMA,cyclin D1,type ?,type ? collagen.Galectin-3 levels and fibrosis levels in different groupsAll data was analyzed by SPSS 21.0,and it is statistical significant when P<0.05.Results1.Baseline demographic data.There was no statistical difference between two groups in baseline characteristics including age,gender,BMI,valve lesion type,cardiac function(NYHA),LVEF,BNP.However,the LAD of AF group was significantly high than SR group(53.97±8.37 vs 41.81±8.18,P < 0.01).2.Galectin-3 expressionThere was a significant difference between two groups in serum Galectin-3,which of the AF group was higher than the SR group 2471.87±734.81 vs 1781.84±624.08 pg/ml,P=0.001);Both in left and right atrium,the protein(LAA :0.91±0.33 vs 0.72±0.31,P=0.043,RAA :0.97±0.31 vs 0.78±0.28,P=0.035)and mRNA(LAA :2.49±0.39 vs 2.23±0.34,P=0.016,RAA :2.43±0.37 vs 2.17±0.35,P=0.012)expression level of Galectin-3 were higher in AF group than SR group.3.Morphologic alternation of atrial fibrosisHE stain showed a decreased number an irregular distribution of cardiomyocytes in both left and right atrium of AF patients,without obvious difference between left and right atriumMasson's stain showed that the positive expression in both left and right atrium of AF group is much more intensive than SR group,which manifested with more blue-stained fiber and irregular distribution.In addition,the CVF in both atriums of AF group was higher than SR group(LAA:36.41± 5.29 % vs 32.11 ±8.23 %,P=0.044,RAA:36.23±5.64% vs 31.23±8.64%,P=0.027)?However,the CVF didn't differ between left and right atrium(P>0.05).4.Galectin-3 related Cyclin D1 alternations in atrial myocardium(1)DistributionImmunohistochemistry results showed that there were the expression of CyclinD1 in both atriums,while the positive expression is more intensive in AF group than SR group.(2)Protein expression levelCyclin D1 increased significantly in protein(LAA :0.92±0.15 vs 0.79±0.20,P=0.013,RAA :0.87±0.16 vs 0.76±0.19,P=0.037)expression level of AF group,but there was no disparity between left and right atrium;(3)mRNA expression levelThe Cyclin D1 expression levels of AF group in left and right atrial myocardium were significantly higher than those of SR group(LAA :1.90±0.38 vs 1.67±0.35,P=0.037,RAA : 1.89±0.30 vs 1.71±0.35,P=0.044),but there was no significant difference in the expression of Cyclin D1 between left and right atrial muscles in two groups.5.?-SMA,bFGF expression in atrial myocardium(1)DistributionImmunohistochemistry results showed that there were the expression of ?-SMA,bFGF in both atriums.bFGF expression of AF group was significantly higher than SR group,while the difference is not significant in terms of?-SMA.(2)Protein expression levelProtein expression level of bFGF increased significantly in AF group(LAA :0.27±0.09 vs 0.22±0.08,P=0.047,RAA :0.28±0.07 vs 0.23±0.08,P=0.038),while the ?-SMA also increased without statistical significance(LAA :0.84±0.23 vs 0.75±0.22,P=0.171,RAA :0.82±0.20 vs 0.75±0.20,P=0.186).There was no difference between left and right atrium in the expression of bFGF and ?-SMA.(3)mRNA expression levelThe mRNA expression level of bFGF in left and right atrial myocardium of AF group was higher than that of SR group(LAA :0.63±0.14 vs 0.55±0.13,P=0.044,RAA :0.60±0.13 vs 0.51±0.10,P=0.009).The expression of ?-SMA mRNA in the two groups was not statistically significant(LAA :3.07±0.35 vs 2.90±0.33,P=0.091,RAA :3.06±0.35 vs 2.93±0.30,P=0.173).There was no significant difference in ?-SMA and bFGF expression between left and right atrium.Through the expression of bFGF and ?-SMA,it can be inferred that the activity of fibroblasts is enhanced in AF patients.6.Col? and Col ? Expression in atrial myocardium.(1)DistributionImmunohistochemistry results showed that there were the expression of Col? and Col ?in both atriums.Col? expression of AF group was significantly higher than SR group,while the difference is not significant in terms Col ?.(2)Protein expressionThe expression of Col? protein in left and right atrial myocardium of AF group was higher than that of SR group(LAA :0.56±0.09 vs 0.49±0.09,P=0.013,RAA :0.52±0.07 vs 0.47±0.11,P=0.035).There was no significant difference between the two groups(LAA :1.10±0.27 vs 1.01±0.21,P=0.181,RAA :1.16±0.26 vs1.04±0.22,P=0.092).There was no significant difference in Col? and Col ? expression between left and right atrium.(3)mRNA expression levelThe mRNA expression of Col? in left and right atrial myocardium of AF group was significantly higher than that of SR group(LAA:1.07±0.11 vs 0.97±0.17,P=0.024;RAA:1.06±0.21 vs 0.92±0.18.P=0.011),but there was no significant difference between the two groups(LAA:1.08±0.27 vs 1.02±0.29,P=0.450,RAA:1.12±0.31 vs 1.11±0.20,P=0.820)in mRNA expression of Col ?.There was no significant difference in Col? and Col ? expression between left and right atrium.In the AF group,the expression of Col? was increased and the expression of Col ? was not significantly increased,but the ratio of Col? / Col ? was increased,suggesting that the atrial fibrosis deepened in AF patients,which resulted from the Galectin-3 related activation of fibroblast.7.Correlation analysis of Galectin-3 and atrial fibrosis.In both left and right atrium,the expression level of Galectin-3 was positively correlated with LAD,CVF,Col?,Cyclin D1.In the left atrium,the expression level of Galectin-3 was positively correlated with Col ?.However,there is no significant correlation between the expression of Galectin-3 and the expression of Col ? in right atrial atrium.It is also correlated with ?-SMA,bFGF without a statistical results in both left and right atrium(P >0.05).Conclusions1.The expression Galectin-3 in atrial myocardium were significantly increased in AF patients,which implies that Galectin-3 is associated with the development of AF.2.Galectin-3 can enhance the activity of fibroblast by affecting the expression of Cyclin D1,hence promoting atrial fibrosis by increasing the expression of ColI,which may play a role in the underlying mechanism of AF.3.No significant difference between left and right atrium in atrial myocardium was found.