| Objective:Atrial fibrillation(AF)is the most common persistent arrhythmia in clinical treatment,which can bring about a variety of complications and great burden to family and society.The treatment methods of AF mainly include medication and non-medication.Currently,radiofrequency ablation,an important mean of non-drug therapy,exerts an increasingly pivotal effect in AF treatment.However,the high recurrence rate after surgery significantly affects the long-term efficacy,in which the role of internal autonomic nerve reconstruction in the postoperative recurrence of AF is nonnegligible.Whereas,how autonomic nerve reconstruction occurs is still unclear.The purpose of this study is to excavate the mechanism of internal autonomic nerve reconstruction after denervation of AF,and to provide a new explanation for the mechanism in the postoperative recurrence of AF.Methods:1.Establishment of radiofrequency ablation dog modelsTwelve healthy adult mongrel dogs(males and females)were randomly divided into four groups(three dogs in each group):one-month control group,one-month radiofrequency ablation group,6-month control group and 6-month radiofrequency ablation group.Dogs in control groups underwent thoracotomy,and dogs in radiofrequency ablation groups were ablated at right top and bottom ganglia.Dogs were killed after feeding for 1 month and 6 months,and the atrial muscle tissue 1cm away from the ablation area was taken for follow-up assays.2.High-throughput sequencing and validationA total of twelve samples(three samples in each group)were subjected to high-throughput sequencing to find out differentially expressed long non coding RNAs(lncRNAs)and mRNAs,the results of which were determined via quantitative real-time PCR(qRT-PCR)and Western blotting(WB).3.Bioinformatics analysisDifferentially expressed lncRNAs were used for prediction of target genes,and differentially expressed mRNAs were applied for gene ontology(GO)enrichment analysis and kyoko encyclopedia of genes and genomes(KEGG)pathway analysis.In combination with co-expression analysis and our previous research findings that the expression level of GAP43 will increase after radiofrequency ablation in dogs and atrial fibrillation induction rate increases after 1 month and 6 months of ablation,lncRNA-LncRNA056298 was screened out to be related with internal autonomic nerve reconstruction and GAP43 was assumed to be the target gene of lncRNA-LncRNA056298.4.Prediction of the functional mechanism of LncRNA056298(1)cis-predictionTranscripts within 10kb upstream and downstream of LncRNA056298 were screened to determine whether LncRNA056298 had correlation with adjacent transcripts in expression.(2)trans-predictionBased on the condition that pearson correlation coefficient(PCC)>0.85 and P<0.05,mRNAs co-expressed with LncRNA056298 were screened,followed by GO enrichment analysis and KEGG pathway analysis for predicting the trans-mechanism of LncRNA056298.5.Cell experimentLncRNA056298 Smarter Silencer and negative control Smarter were constructed.The primary cardiomyocytes from dogs were extracted,cultured and transfected via applying Lipofectamine 2000.Cells were divided into negative control group(transfection with LncRNA056298 negative control Smarter)and silence group(transfection with LncRNA056298 Smarter Silencer).Total RNA was harvested at 48h and total protein was extracted at 72h after treatment.6.Animal grouping and lentivirus infectionLncRNA056298 negative control and overexpression lentivirus were established,with the titer of 1×109TU/ml.Twelve healthy adult mongrel dogs without gender restrictions were randomly and averagely divided into two groups:negative control lentivirus group(Injection of LncRNA056298 negative control lentivirus in right atrium muscle)and overexpression lentivirus group(Injection of LncRNA056298 overexpression lentivirus in right atrium muscle).Atrial effective refractory period(AERP)and AF induction rate were measured 10d before and after lentivirus injection.Experimental dogs were sacrificed after feeding for 10d to take out right atriums for subsequent experiments.7.Detection of molecular biological indicatorsThe expression levels of LncRNA056298 and GAP43 mRNA and protein were detected using qRT-PCR and Western blotting,respectively.And the changes of indicators related to internal autonomic nerve reconstruction,CHAT,TH,GAP43 and PGP9.5 contained,were analyzed by immunohistochemistry.Results:1.Comprehensive analysis of transcripts with different expressions was performed based on GO enrichment analysis,KEGG pathway analysis,co-expression analysis,literature search and the findings of previous studies.LncRNA056298,with its predicted target gene GAP43,was screened out for further study.2.In primary canine cardiomyocytes,GAP43 expression was downregulated after LncRNA056298 silencing,as revealed by qRT-PCR and Western blotting.After in vivo infection of LncRNA056298 overexpression lentivirus,qRT-PCR and Western blotting demonstrated that GAP43 expression was upregulated.10d later,AERP was significantly shortened and the induction rate of AF was elevated.The results of immunohistochemistry showed that internal autonomic nerve reconstruction-associated markers containing GAP43,TH,CHAT and PGP9.5 were markedly upregulated in overexpression lentivirus group,the above results suggest that cardiomyocytes can secrete GAP43 to cause neural remolding.3.The cis mechanism by which lncRNAs regulate target genes is a position-based regulation mechanism,the condition for exerting cis mechanism is that the distance between lncRNAs and their target genes is less than 10 kb and their expression is correlated,the basic local alignment search tool(BLAST)discovered that LncRNA056298 was localized in the noncoding sequence of GAP43 antisense strand.The results of qRT-PCR and Western blotting exhibited that LncRNA056298 and GAP43 were correlated with each other in expression level,which pushed us to speculate that LncRNA056298 might affect GAP43 expression through cis-mechanism.Conclusion:LncRNA056298 affect the occurrence of internal autonomic nerve reconstruction by influencing GAP43 expression.Internal autonomic nerve reconstruction may be an important factor for AF recurrence after radiofrequency ablation,and LncRNA056298 may affect GAP43 expression via cis-mechanism.Significance:This study elucidates the mechanism in internal autonomic nerve reconstruction after radiofrequency ablation from the perspective of lncRNAs,which provides a new explanation for the poor long-term prognosis of AF after radiofrequency ablation. |
