The Expression Of BRD4 In Patients With Nonvalvular Atrial Fibrillation And Its Role In The Occurrence Of Postoperative Recurrent And The Development Of Atrial Fibrosis

Objective:Atrial fibrosis is closely related to the occurrence and maintenance of atrial fibrillation,and the two are mutually causal.Treatment of atrial fibrosis can effectively reduce the incidence of atrial fibrillation and reduce the burden of atrial fibrillation,but the efficacy of current methods is limited.Bromine domain protein4（BRD4）is considered to be a potential target for the treatment of fibrosis diseases,and its correlation with atrial fibrosis and atrial fibrillation has only been reported by one study.There are no relevant studies on the role of BRD4 in patients with nonvalvular atrial fibrillation.Therefore,this study was clarified from five aspects through clinical research and animal experiments.1.To observe whether the level of BRD4 was significantly increased in peripheral venous blood of patients with non-valvular atrial fibrillation.2.To study the correlation between BRD4 and postoperative recurrence of left atrial fibrosis,LAVs,LAAPD,LVEF and atrial fibrillation.3.To prove whether inhibition the expression of BRD4 can significantly improve the degree of left atrial fibrosis in atrial fibrillation,and reduce the induced rate and duration of atrial fibrillation.4.To investigate whether BRD4 is involved in atrial fibrosis and atrial remodeling through TGF-β1/Smad signaling pathway,and thus in the occurrence and maintenance of atrial fibrillation.Methods:A total of 74 patients who were diagnosed with atrial fibrillation and undergoing radiofrequency ablation for the first time in Department of Cardiology of affiliated Hospital of Guizhou Medical University from February 2019 to August 2020 were selected as atrial fibrillation group（AF group）.Seventy-three hospitalized patients who were diagnosed as parodic supraventricular tachycardia and were diagnosed as atrioventricular node reentrant tachycardia or atrioventricular reentrant tachycardia by radiofrequency ablation for the first time were selected as the control group（PSVT group）.All the candidates were asked medical history in detail,underwent physical examination,electrocardiogram,and recorded basic clinical data in detail.All the included patients underwent complete transthoracic echocardiography before operation.After abstaining from food and drink for at least 8 hours,2ml of elbow venous blood was extracted from the supine position in the morning on an empty stomach and placed in the EDTA anticoagulant tube.The blood was examined within4 hours for q RT-PCR to detect the expression of BRD4 in m RNA level.Compare with the differences of BRD4 expression and LAAPD and LVEF between AF group and PSVT group.Patients in AF group were received transesophageal echocardiography in24 hours before surgery to determine whether there was thrombosis in the left atrium or left atrial appendage,and LAA-PEV was measured at the same time.All the enrolled patients signed the informed consent before surgery.Patients with paroxysmal supraventricular tachycardia were diagnosed as atrioventricular node reentrant tachycardia or atrioventricular reentrant tachycardia by intracardiac electrophysiological examination,and all patients in the AF group underwent intracardiac electrophysiological examination to rule out atrioventricular reentrant tachycardia.Before ablation,patients in the AF group,LAVS of left atrium with high density matrix mapping was observed using Abbott Ensite Velocity^TM 5.0 intelligent three-dimensional heart mapping system assisted by Ensite Velocity^TM body surface electrode under sinus rhythm.Work Mate^TM Claris^TM electrophysiological recording system was used to perform statistics and analysis on the local bipolar voltages at the measured points of the selected atria,and the overall mean value（m V）of the potential bipolar voltages at each point of the selected atria was calculated.The filtering of the bipolar voltages was set to 30-300 Hz.At the same time,the left atrium was artificially divided into anatomic areas of anterior wall,top wall and posterior wall.Left atrial low voltage regions（LAVS）was defined as bipolar voltage less than 0.5m V,Bipolar voltage more than 0.5m V is defined as normal voltage.Patients in AF group were divided into low-voltage region group（LAVS）and non-low-voltage region group（NLAVS）according to the voltage of the left atrium.Compare with the differences of BRD4 expression and LAAPD and LVEF and LAA-PEV between LAVS group and NLAVS group.Standard 12-lead ECG and/or 72-hour dynamic ECG were followed up at 1,3,6,12 and 18 months postoperatively.Early atrial fibrillation recurrence refers to the occurrence of atrial fibrillation or atrial flutter or atrial tachycardia lasting more than 30 s within 3 months after surgery.Since about 60% of early recurrence will self-correct,early recurrence is not included in the total recurrence rate,so 3 months after surgery is defined as the "blank period".Atrial fibrillation or atrial flutter or atrial tachycardia occurring 3 months after ablation,if the duration is more than 30 s,is considered as recurrence of atrial fibrillation,which can be divided into advanced recurrence（3-12 months after surgery）and long-term recurrence（recurrence more than12 months after surgery）according to the duration of recurrence.Based on this,patients in AF group were divided into postoperative sinus rhythm group（Nre AF）and recurrent atrial fibrillation group（re AF）.In this study,the time node for recurrence assessment was set at 12 months after surgery.If less than 12 months was followed up according to the longest follow-up month,all patients were followed up for more than6 months.According to the recurrence,patients in the AF group were divided into postoperative sinus rhythm group（Nre AF）and recurrent atrial fibrillation group（re AF）.Compare with the differences of BRD4 expression and LAAPD and LVEF and LAA-PEV and LAVS between Nre AF group and re AF group.Thirty-Six SD rats were purchased and randomly divided into blank control group（CTL）,atrial fibrillation model group（AF+CTL）,and atrial fibrillation model+JQ-1 group（AF+JQ1）,with 12 SD rats in each group.All rats in the AF+CTL group and AF+JQ1 group were required to undergo transesophageal electrophysiological examination and atrial rapid pacing to construct atrial fibrillation model.In the CTL group,multipole electrocardiogram catheters were only placed in the esophagus of rats,and atrial rapid pacing stimulation was not used to induce atrial fibrillation.The atrial fibrillation induction rate and duration of atrial fibrillation in each group were recorded.After the successful modeling of atrial fibrillation,rats were injected with（+）JQ1（Triphenylmethane triisocyanate,molecular formula:C23H25Cl N4O2 s,is a BRD4 inhibitor.8mg/Kg,once a week,consecutive 3 weeks,AF+JQ1 group）or equivalent volume of normal saline（CTL group,AF+CTL group）through tail vein.After the intervention,atrial rapid pacing stimulation was repeated to induce atrial fibrillation.The incidence of atrial fibrillation and the duration of atrial fibrillation in each group were recorded and the differences between the groups were compared.After the completion of the corresponding electrophysiological examination,the rats were anesthetized and killed,and the left atrium samples of the rats were collected for subsequent experiments.The left atrium samples of 6 rats in each group were collected and Masson’s trichrome staining was performed to observe the fibrosis of left atrium.The collagen volume fraction was calculated by software and the differences among the groups were compared.The left atrial tissues of the remaining 6 rats in the CTL,AF+CTL and AF+JQ1 groups were collected.Western blot was used to detect the expression of BRD4,TGF-β1,P-Smad2/3,Smad2/3,Smad7,Cx43 and type III collagen in each group at the protein level,and the differences of the above indexes in each group were compared.Results:1.The expression of BRD4 and LAAPD of patients in atrial fibrillation group were significantly higher than those of patients in paroxysmal supraventricular tachycardium group（P<0.05）.The LVEF of patients in the atrial fibrillation group was significantly lower than that of patients in the paroxysmal supraventricular tachycardia group（P<0.05）.All of the differences had statistically significant.2.The expression of BRD4 was significantly positively correlated with LAAPD（P<0.05）,and was negatively correlated with LVEF（P<0.05）,and the differences had statistically significant.3.The postoperative recurrence,left atrial low voltage,expression of BRD4 and LAAPD of patients with persistent atrial fibrillation were significantly higher than those of patients with paroxysmal atrial fibrillation（P<0.05）.The LVEF of patients with persistent atrial fibrillation was significantly lower than that of patients with paroxysmal atrial fibrillation（P<0.05）.All of the differences had statistically significant.The LAA-PEV of patients with persistent atrial fibrillation was lower than that of patients with paroxysmal atrial fibrillation（P > 0.05）,the difference had no statistically significant.4.The left atrial low voltage,expression of BRD4 and LAAPD of patients with postoperative recurrence of atrial fibrillation were significantly higher than those of patients with postoperative nonrecurrence of atrial fibrillation（P<0.05）.The LVEF and LAA-PEV of patients with postoperative recurrence of atrial fibrillation were significantly lower than those of patients with postoperative non-recurrence of atrial fibrillation（P<0.05）.All of the differences had statistically significant.5.The expression of BRD4 and LAAPD of patients with atrial fibrillation of left atrial low voltage were significantly higher than those of patients with atrial fibrillation of normal left atrial voltage（P<0.05）.The LVEF of patients with atrial fibrillation of left atrial low voltage was significantly lower than that of patients with atrial fibrillation of normal left atrial voltage（P<0.05）.All of the differences had statistically significant.The LAA-PEV of patients with atrial fibrillation of left atrial low voltage was lower than that of patients with atrial fibrillation of normal left atrial voltage（P>0.05）,the difference had no statistically significant.6.The much number of low voltage areas in the left atrium of patients with atrial fibrillation,the higher expression of BRD4（P<0.05）.The differences had statistically significant.The much number of low voltage areas in the left atrium of patients with atrial fibrillation,the larger of LAAPD,the lower of LVEF and LAA-PEV,all of the differences had no statistically significant（P>0.05）.7.The average induced rate of atrial fibrillation and the average duration of atrial fibrillation of AF+JQ1 group were significantly lower than those of AF+CTL group（P < 0.05）.All of the differences had statistically significant.8.Compared with the control group（CTL）,atrial fibrillation model group（AF+CTL）showed obvious fibrosis of the left atrium,and the collagen volume fraction was significantly increased（P<0.05）.Compared with the atrial fibrillation model group（A-F+CTL）,AF+JQ1 group showed significantly attenuating of fibrosis of the left atrium,and the collagen volume fraction was significantly decreased（P<0.05）.All of the differences had statistically significant.9.Compared with the control group（CTL）,the expression of protein levels of BRD4,TGF-β1,P-Smad2/3/Smad2/3 and type III collagen in AF model group（AF+CTL）were significantly increased（P<0.05）,while the expression of protein levels of Smad7 and Cx43 were signif-icantly decreased in AF+CTL group（P<0.05）.Compared with the AF model group（AF+CTL）,the expression of protein levels of BRD4,TGF-β1,P-Smad2/3/Smad2/3 and type III collagen in AF+JQ1group were significantly decreased（P<0.05）,while the expression of protein levels of Smad7 and Cx43 were significantly increased in AF+JQ1 group（P<0.05）.All of the differences had statistically significant.Conclusions:1.Elevated levels of BRD4 in peripheral venous blood may be used to predict left atrial enlargement,left atrial fibrosis,postoperative recurrence of atrial fibrillation,and reduced left ventricular ejection fraction in patients with nonvalvular atrial fibrillation.2.Left atrium dilated and left ventricular ejection fraction decreased in patients with nonvalvular atrial fibrillation.In patients with persistent atrial fibrillation,the left atrial low-voltage area increased significantly,the postoperative recurrence rate was higher,the left atrial enlargement was more obvious,and the left ventricular ejection fraction decreased more significantly.3.The postoperative recurrence rate of atrial fibrillation was significantly positively correlated with LAAPD and the low voltage region of the left atrium,and negatively correlated with LVEF and LAA-PEV.4.Patients with nonvalvular atrial fibrillation with increased left atrial low voltage area have enlarged left atrium and reduced left ventricular ejection function.5.BRD4 is associated with non-valvular atrial fibrillation.Inhibition of BRD4 expression can significantly improve the degree of left atrial fibrosis in rats with atrial fibrillation,and reduce the incidence of atrial fibrillation and the duration of atrial fibrillation.6.BRD4 may be involved in atrial fibrosis and atrial remodeling through the TGF-β1/Smad signaling pathway,thus participating in the occurrence and maintenance of atrial fibrillation.