| Objective: Atrial fibrillation is the most common arrhythmia in clinical practice,affecting the survival and health of patients.Many risk factors may lead to structural remodeling of the atrial myocardium,thereby leading to the development of atrial fibrillation,including aging.Autophagy is essential for cellular homeostasis and is involved in the renewal of organelles and proteins.The purpose of this study is to investigate whether abnormal activation of autophagy in atrial fibrillation is related to fibrosis formation in heart tissues.This research also aims to establish the significance of autophagy function and its related genes as markers in atrial fibrillation.Methods: Part I:Utilizing publicly available gene expression data from atrial fibrillation and sinus rhythm(control group)heart tissues,we explored the role of autophagy in atrial fibrillation using computational bioinformatics methods.We first compared gene expression between atrial fibrillation and the control,identifying differentially expressed genes.We then identified a module with the highest correlation with atrial fibrillation and autophagy through weighted gene co-expression network analysis(WGCNA),and conducted functional identification.We further recognized autophagy-related genes among the differentially expressed genes.Using LASSO and Random Forests,we screened out the most influential key genes and plotted the receiver operating characteristic curves.In addition,we assessed the abundance of immune cells in each sample using single-sample gene set enrichment analysis and explored the correlation between key gene expression and immune cells.Part II: Peripheral blood samples from atrial fibrillation and control populations were collected,and the m RNA and protein expression levels of autophagy-related proteins(LC3A / B and p62)and key genes were detected using q RT-PCR and Western Blot.Part III: Establish a canine atrial fibrillation model.Inflammation,fibrosis,and tissue morphology changes in dog heart tissue were detected by HE staining and Masson staining.The m RNA and protein expression levels of autophagy-related proteins(LC3A / B and p62)and key genes were determined by q RT-PCR and Western-blot analysis.The expression of p62 was determined by immunohistochemistry.Results: Part I:(1)A substantial number of differentially expressed genes were identified between the atrial fibrillation group and the control group,among these,in GSE41177,8409 were identified,9314 were identified in GSE79768,and 3553 were identified in GSE115574,with 1184 differentially expressed genes in common.With the use of WGCNA,these common differentially expressed genes were used to construct a weighted gene co-expression network.Among them,the turquoise module had the greatest positive correlation with atrial fibrillation and autophagy.The genes in the turquoise module were significantly enriched in several signaling pathways,including endocytosis,protein processing in the endoplasmic reticulum,ubiquitin-mediated protein degradation,the insulin signaling pathway,and the m TOR signaling pathway.(2)The activity of the autophagy signaling pathway in the atrial fibrillation group was significantly higher than in the control group,and 29 differentially expressed autophagy-related genes were identified.By intersecting the results of LASSO regression analysis and random forest analysis,we obtained the final four key genes(GSK3B,QSOX1,RAB3GAP2,SYNPO2).Compared with the sinus rhythm group,GSK3 B and RAB3GAP2 were highly expressed in the atrial fibrillation group,while QSOX1 and SYNPO2 were lowly expressed.The AUC value of the key genes was all above 0.7.Moreover,type 2 T helper cells had the greatest positive correlation with autophagy,while activated B cells and CD56 dim natural killer cells had the greatest negative correlation with autophagy.Part II:(1)Through the detection of autophagy-related proteins,it was found that both the m RNA and protein levels of LC3 A /B and p62 in patients with atrial fibrillation were significantly higher than those in the control group(P? < 0.05).The detection results of the key genes GSK3 B,QSOX1,RAB3GAP2,and SYNPO2 showed that compared with the control group,the expressions of GSK3 B and RAB3GAP2 in the atrial fibrillation group were significantly increased,while the expressions of QSOX1 and SYNPO2 were significantly reduced(P??<0.05).Part III:(1)The levels of cardiac inflammation and fibrosis in dogs with atrial fibrillation model were significantly higher than those in the control group,especially in the elderly atrial fibrillation group.In the autophagy inhibitor treatment group,the inflammation and fibrosis levels of myocardial tissues in dogs with atrial fibrillation were improved.The results of q RT-PCR and Western blot tests found that compared with the young control group,the expression levels of LC3 A / B and p62 were significantly increased in the elderly control,young atrial fibrillation,and elderly atrial fibrillation groups(P??<0.05),and in dogs with atrial fibrillation treated with autophagy inhibitors,the protein expression levels of LC3 and p62 significantly decreased.Compared with the young control group,the expressions of GSK3 B and RAB3GAP2 were significantly increased in the elderly control,young atrial fibrillation,and elderly atrial fibrillation groups(P? <0.05),and the expressions of QSOX1 and SYNPO2 were significantly reduced(P? < 0.05).Then,in dogs with atrial fibrillation treated with autophagy inhibitors,the abnormal expression of the key genes was improved.Conclusions:(1)This study found that autophagy is significantly activated in atrial fibrillation,and the identified co-expression module is significantly correlated with atrial fibrillation and autophagy.Enrichment analysis found that the module genes are mainly related to the m TOR signaling pathway and protein processing signals.The key genes have a high ability to distinguish between atrial fibrillation and sinus rhythm,indicating that they may serve as potential biomarkers for atrial fibrillation.These findings provide new insights into atrial fibrillation and may help in the prediction and treatment of atrial fibrillation in the future.(2)The experimental results validate the results of bioinformatics analysis,showing the expression of key genes in AF and the possible activation of autophagy in AF.These results provide us with important experimental evidence to further explore the role of autophagy and immune response in AF and how these mechanisms can be exploited to prevent and treat AF.(3)Autophagy inhibitors reduce the autophagy activities in the elder AF canines in contrast to the AF models,which are accompanied with the decreased expression of autophagy-related genes.These findings may reveal an potential role of autophagy in the pathogenesis of AF and also suggest autophagy inhibitors as potential strategies for the treatment of AF. |
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