| ObjectivesAs the global population ages and the average life expectancy of the population increases,the prevalence of atrial fibrillation(AF)is increasing every year,with statistics showing that the prevalence of AF doubled globally in 2019 from 1989 to 59.7 million.Risk factors associated with the increased prevalence of AF include advancing age,cardiovascular disease,overweight and obesity,alcohol consumption,obstructive sleep apnoea,hypertension,diabetes,coronary artery disease,heart failure and chronic kidney disease.The dangers of atrial fibrillation are enormous,with serious consequences such as strokes,heart failure and even sudden death due to the hemodynamic changes and hypercoagulable state of the blood caused by irregular fibrillation of the atria,resulting in a very heavy financial burden on families and society.Current therapeutic management of AF focuses on symptom reduction and prevention of complications.Heart rate control and rhythm control are used to improve symptoms,and anticoagulation is used to reduce the risk of stroke.However,these treatments are often inadequate and associated with potential adverse effects,and do not meet the needs of both patients and physicians.The mechanisms involved in the development of atrial fibrillation include trigger and maintenance mechanisms.The trigger mechanism is primarily abnormal electrical activity within the muscular cuff lesion that drives the development of AF.The maintenance mechanism is complex,with left atrial electrophysiology and structural remodeling playing an important role in the maintenance factors.It is because of the relative complexity of the maintenance mechanism that the success rate of ablation of persistent AF is low and the recurrence rate is high.The main reason for these treatment limitations may be the inadequate understanding of the pathogenesis of atrial fibrillation.The main manifestation of structural remodeling is myocardial fibrosis,which is one of the most critical factors in the development and maintenance of atrial fibrillation.The cellular and molecular mechanisms regulating atrial fibrosis are highly complex and the most important challenge is to exploit the great potential of these mechanisms for clinical translation with new biomarkers to elucidate the underlying pathophysiological mechanisms and detect treatments in individual AF patients,which is of great significance in exploring new approaches to combat atrial fibrosis.Non-coding RNAs(ncRNAs)were previously regarded as ’junk RNAs’ because they did not encode proteins,but they have recently received much attention for their important role in development,cell proliferation,differentiation,apoptosis and tumorigenesis.In recent years,non-coding RNAs have become a hot topic in cardiovascular diseases,been involved in pathophysiological processes such as myocardial infarction,atherosclerosis,thrombosis,fibrosis and inflammation,and a new risk marker for cardiovascular disease.Long strand non coding RNA(lncRNA),a group of nc RNAs >200 bases in length,has been found to regulate gene expression at multiple levels,including epigenetic,transcriptional and post-transcriptional regulation,and plays an important role in cancer,Alzheimer’s disease and cardiovascular disease.However,compared to other cardiovascular diseases such as heart failure and atherosclerosis,lncRNAs in AF have been less studied.This study was based on the construction of a rabbit model of atrial fibrillation induced by rapid electrical stimulation of the left atrium.lncRNAs associated with atrial fibrosis in atrial fibrillation were selected by bioinformatics,conservativeness and experimental validation based on the detection of differentially expressed transcripts in the left atrium of patients with and without atrial fibrillation by high-throughput sequencing in our group.The role of target lncRNAs in the development of fibrosis in a rabbit model of atrial fibrillation was investigated,including by regulating the function of lncRNAs,and the effect of target lncRNAs on fibrosis in atrial fibrillation was illustrated at the level of molecular mechanisms.To explore the lncRNAs involved in the pathogenesis of AF,with a view to translating preclinical discoveries of lncRNAs into clinical practice in the management and treatment of AF,and to identify biomarkers and therapeutic targets for the diagnosis and prognosis of AF.MethodsIn the first part of the study,rabbit atrial fibrillation was induced by rapid electrical stimulation of the left atrium.Two different electrical stimulation frequencies were used to assess the success rate of surgical modelling,the tolerance of the model rabbit to the experiment,the rate of induction of atrial fibrillation and the time to induction of atrial fibrillation.Color Doppler ultrasound was used to measure the anteroposterior diameter,left atrial volume and left atrial ejection fraction to assess the left atrial function of the model rabbits.Masson staining was applied to assess the extent of left atrial fibrosis under different stimulation groups,and the Image J software was used to do semi-quantitative analysis of Masson staining.The data of each group were expressed as x±s using SPSS25.0 statistical software,and t-test was used for comparison between pre-and postmodelling.In the second part of the study,RNA was extracted by TRIzol method and highthroughput sequencing was performed to detect the expression of gene transcripts and lncRNAs in patients with degenerative valve disease with/without persistent atrial fibrillation in the cardiac surgery department of the Northern War Zone General Hospital.The differential lncRNAs were verified by q RT-PCR,compared with the sequencing results,and Gene Ontology(GO)enrichment analysis was performed at the same time;suitable lncRNAs were selected according to the results of gene sequencing and GO analysis.The electrodes in the electrical stimulation group were sutured in the same way as in the intermittent stimulation group in Part I.In the sham operation group,the stimulation electrodes were sutured in the same position but without pacing.The stimulation electrodes were sutured in the same position but without pacing.All groups were terminated at 4 weeks after surgery,and the atrial tissue was stored separately at-80°C and fixed in a portion of 4% paraformaldehyde.The selected rabbit lncRNA was validated in the left atrial tissue of the RAP rabbit atrial fibrillation model by applying q RTPCR.The final lncRNAs were screened for further studies based on the above results.In vitro lncRNA control adeno-associated virus,lncRNA XR＿001793654.1 overexpression adeno-associated virus,was constructed at a viral titer of 2.1*10^12vg/m L.12 male adult New Zealand large-eared rabbits were randomly divided into XR＿001793654.1overexpression group and control group.A rabbit atrial fibrillation model with rapid electrical stimulation of the left atrium was constructed,and the corresponding adenoassociated virus was injected obliquely into the left atrial muscle at multiple points,and the left atrial tissues were left after 4 weeks of modeling,and the efficiency of virus infection was measured by freezing sections to observe the fluorescence intensity and q RTPCR to detect the expression of XR＿001793654.1 in both groups.The effect of XR＿001793654.1 on left atrial fibrillation was verified by using colour Doppler ultrasound to assess left atrial function and Masson staining to assess the degree of atrial fibrosis.In the third part of the study,based on XR＿001793654.1 screened in the second part,bioinformatics and database analysis revealed that ocu-miR-107 might interact with it and the target gene of miR-107-3p might be KLF13.The DNA fragment of XR＿001793654.1gene and the 3’UTR DNA fragment of KLF13,the target gene of miR-107-3p,were constructed as wild type,and its mutant DNA fragment was also synthesized and then the luciferase vector was constructed.The assay was used to verify whether KLF13 was the target gene of ocu-miR-107 and whether XR＿001793654.1 could interact with ocu-miR-107.Overexpression and silencing of ocu-miR-107 adeno-associated virus and negative control adeno-associated virus were also constructed.Eighteen adult New Zealand largeeared rabbits were randomly divided into XR＿001793654.1 overexpression negative control group,XR＿001793654.1 overexpression group,ocu-miR-107 overexpression group,ocu-miR-107 overexpression control group,ocu-miR-107 silencing group,ocumiR-107 silencing control group,XR＿001793654.1 001793654.1 overexpression and ocumiR-107 overexpression adeno-associated virus co-infection groups,and the construction model and viral infection and detection indexes were as in Part II.The expression of hasmiR-107 was measured by q RT-PCR assay in the left atrial tissue of AF/non-AF patients,respectively;the expression of ocu-miR-107 was measured in the electrical stimulation group/sham-operated group of RAP rabbit AF model;the effect of ocu-miR-107 on atrial fibrosis and left atrial function in rabbits with AF model was evaluated by Color Doppler ultrasound and Masson staining.Immunohistochemistry and Western Blot were performed to validate the predicted target protein KLF13 in human and rabbit tissues to determine the correlation between its expression level and AF fibrosis and whether XR＿001793654.1could interact with ocu-miR-107 to influence the expression of KLF13 and thus played a role in the development of AF fibrosis.Results Part11.During the working period of implantation in the model rabbit,the implant worked well,the P wave,QRS wave group and T wave were clear before stimulation,and the baseline was stable.After 72 hours of postoperative recovery,the programmed stimulation(continuous stimulation group stimulation voltage 2000 m V,stimulation pulse width 1ms,interval stimulation group stimulation voltage 2000 m V,stimulation pulse width 1ms,stimulation cycle 50 ms,stimulation 2s,pause 2s)was turned on.The heart rate of the rabbit model was significantly accelerated after stimulation and slowly decreased after stopping stimulation,indicating that the stimulation was effective.The rabbit model remained awake during the experimental cycle and could move freely in the cage,and there was no significant change in the living habits such as eating,drinking and sleeping compared to the preoperative period.2.In the continuous stimulation group,4 rabbits developed persistent atrial fibrillation after2 weeks,and the duration of atrial fibrillation was >48 h.In the interval stimulation group,no atrial fibrillation developed within 2 weeks,and 8 rabbits in the interval group developed persistent atrial fibrillation about 3 weeks after stimulation,and the duration of atrial fibrillation was >48 h.In the continuous group,4 rabbits developed atrial fibrillation between 3 and 4 weeks.This indicates that high-frequency programmed stimulation can produce atrial fibrillation within a short period of time(P < 0.05),but the mortality rate within 4 weeks during high-frequency stimulation was significantly higher in the continuous stimulation group compared with the interval stimulation group,and the interval stimulation group was significantly better tolerated than the continuous stimulation group(P < 0.05).3.Left atrial data measured according to Color Doppler ultrasound diagnostic instrument.There was no statistical difference in the comparison of baseline data between the two groups(P > 0.05).In both groups compared to the baseline data before stimulation,after stimulation,LAD,LAVmax and LAVmin were significantly greater and LAEF was significantly lower in both groups(P < 0.05),and significant left atrial enlargement and left atrial dysfunction could be observed,but there was no significant difference between the two groups(P > 0.05).This result indicates that the rabbit rapid pacing-induced atrial fibrillation model can result in a significant increase in left atrial diameter and volume,and a decrease in LAEF.4.Masson staining was performed on the left atrial samples from both groups to observe the extent of myocardial fibrosis in the left atrium.As shown,the collagen fibres were blue in color and the cardiomyocytes were red in colour.The results revealed that the atrial myocardial tissue in both groups was significantly fibrotic in disarray.The difference from normal atrial tissue with closely spaced atrial muscle without fibrotic changes was obvious.However,there was no significant difference in the degree of fibrosis between the two groups(P > 0.05).Part21,A total of 18,722 lncRNAs were measured by high-throughput sequencing,of which 55 transcripts were up-regulated and 11 transcripts were down-regulated,and the functions of the differentially expressed lncRNAs could be related to atrial fibrillation by GO enrichment analysis.2,The differentially expressed lncRNAs were compared with the rabbit gene library in a bidirectional blast to analyze the conservativeness,and the well-conserved lncRNAs were verified in human and rabbit atrial tissues,and finally XR＿001793654.1 was selected as the target lncRNA for further study according to the above conditions.3.lncRNA XR＿001793654.1 was functionally validated by in vivo experiments.In the AAV-XR＿001793654.1 group and the AAV-NC group,LAD,LAVmax and LAVmin were increased and LAEF was decreased(P < 0.05),and left atrial enlargement and left atrial dysfunction could be observed;compared with the Sham 4 weeks after surgery Compared with the Sham group at 4 weeks postoperatively,LAD,LAVmax and LAVmin in the AAVXR＿00179654.1 groups were significantly increased and LAEF was significantly decreased(P < 0.05);compared with the AAV-NC group 4 weeks after stimulation,LAD,LAVmax and LAVmin in the AAV＿00179654.1 group were significantly decreased and LAEF was significantly increased(P < 0.05).The results suggest that overexpression of XR＿001793654.1 can reduce the RAP-induced enlargement of left atrial diameter and volume,reduce the reduction of LAEF,and effectively regulate left atrial enlargement and dysfunction.Masson staining of left atrial samples from the AAV-XR＿001793654.1 and AAV-NC groups showed that the degree of atrial fibrosis was significantly reduced in the AAV-XR＿001793654.1 group compared with the AAV-NC group(P < 0.01).Part31.lncRNA XR＿001793654.1 has multiple binding sites to ocu-miR-107 according to bioinformatic analysis.Compared with non-atrial fibrillation patients with degenerative valve disease,the upregulation of has-miR-107 expression in the left ear tissue of atrial fibrillation patients with degenerative valve disease was significantly negatively correlated with lncRNA ENST00000623312,R2=0.8961,P<0.05.Also,the upregulation of lncRNA XR?＿001793654.1 and ocu-miR-107 in the left atrium of the RAP rabbit atrial fibrillation model were correlated,and XR＿001793654.1 was negatively correlated with miR-107-3p expression.After infection with XR＿001793654.1 overexpressing adeno-associated virus in the RAP rabbit AF model,q RT-PCR showed that the expression of miR-107-3p was down-regulated compared to the AAV-NC group,P < 0.05.This result confirmed that the expression of XR＿001793645.1 and miR-107-3p in the left atrial tissue of AF was negatively correlated relationship.Then,we identified potential binding sites for interaction between XR?＿001793654.1 and ocu-miR-107 by sequence comparison and bioinformatics analysis.The dual luciferase reporter gene assay showed that the fluorescence intensity of ocu-miR-107 sequence decreased after binding to the wild type of XR＿001793654.1 fragment compared to the negative control,P < 0.05,while no significant change in fluorescence intensity was observed after cotransfection of the ocumiR-107 sequence with the mutant of XR＿001793654.1 fragment.The dual luciferase reporter gene assay confirmed that XR＿001793654.1 could bind to the ocu-miR-107 sequence and that there was an interaction between the two.2.Left atrial infection experiments with ocu-miR-107 overexpression and silencing of adeno-associated virus were performed on the RAP rabbit model of atrial fibrillation.And the rabbits were electrically stimulated with the same stimulation frequency in the second part of the model for 4 weeks after sampling.q RT-PCR assay and fluorescence microscopy for infection efficiency showed that fluorescent signals could be found in the left atrium 4weeks after surgery,suggesting successful infection.The miR-107-3p expression was significantly down-regulated in the miR-107-3p inhibitor group compared to the inhibitor NC group,P < 0.01,and up-regulated in the miR-107-3p mimics group compared to the mimics NC group,P < 0.01.This indicates that overexpression of adeno-associated virus and silencing of adeno-associated virus were successful.Masson staining showed that fibrosis was reduced in the miR-107-3p inhibitor group and increased in the miR-107-3p mimics group.Colour Doppler ultrasound results showed that after 4 weeks of stimulation,LAD,LAVmax and LAVmin were significantly lower and LAEF was significantly higher in the miR-107-3p inhibitor group compared to the miR-107-3p mimics group(P < 0.05);this was similar to the Part II XR＿001793654.1 over The results were similar to those of the second XR＿001793654.1 over-expression group.The results suggest that the silencing of miR-107-3p can also reduce the degree of left atrial fibrosis and improve left atrial function in rabbits in the post-operative RAP model.3.Based on database information,biological information analysis and sequence comparison,it was found that ocu-miR-107 had a binding site to the 3’UTR of KLF13,suggesting that KLF13 may be a target gene for the action of ocu-miR-107.The results of the dual luciferase reporter gene assay showed that the fluorescence intensity of ocu-miR-107 sequence decreased after binding to the wild type of the 3’UTR fragment of KLF13 compared to the negative control,P < 0.05,while the mutant of ocu-miR-107 sequence and the 3’UTR fragment of KLF13 No significant change in fluorescence intensity was seen after cotransfection.The dual luciferase reporter gene assay confirmed that the 3’UTR of KLF13 could bind to the ocu-miR-107 sequence and there was an interaction between the two,and KLF13 should be the target gene of ocu-miR-107.Western Blotting of proteins extracted from atrial tissues of the RAP rabbit model after miR-107-3p adeno-associated virus infection showed that the expression of KLF13 was down-regulated in the group with miR-107-3p mimics and up-regulated in the group with miR-107-3p inhibitor,as detected by IHC The expression of KLF13 in both groups was detected by IHC,and the results were consistent with the results of Western blot.This suggests that miR-107-3p plays a role in AF fibrosis by regulating KLF13 expression.We measured the protein level in the central atrial tissue of AAV-XR＿001793654.1 group and showed that the expression level of KLF13 was upregulated in the AAV-XR＿001793654.1 group compared with that in the AAV-NC group.Similarly,we measured protein levels in the left atrial tissue of patients with degenerative valve disease and showed that KLF13 expression levels were downregulated in the left ear of patients with AF compared to the non-AF group.This result confirms that the low expression of XR?＿001793654.1 can affect the expression of the gene KLF13,which in turn may promote the development of atrial fibrosis in atrial fibrillation.4.Western Blotting results showed that upregulation of KLF13 expression level occurred after XR＿001793654.1 overexpression;however,miR-107-3pmimics after cotransfection could reverse the promotion of KLF13 after XR＿001793645.1 overexpression;compared with the AAV-NC+mimics-NC group The expression level of KLF13 was upregulated in the AAV-XR＿001793654.1+miR-107-3p mimics group,P < 0.05,but Masson staining showed no significant difference in the degree of fibrosis between the two,P > 0.05.This indicates that XR＿001793654.1 regulates the target gene through competitive binding of miR-107-3p KLF13 expression,which in turn affects the development of atrial fibrosis in atrial fibrillation.Conclusions1.The RAP rabbit atrial rapid pacing-induced atrial fibrillation procedure can induce atrial fibrillation lasting for more than 48 h under the conditions of suitable stimulation time and mode selection,with low mortality of selected interval stimulation and good tolerance in rabbits,and can successfully induce atrial fibrillation and left atrial fibrosis.2.lncRNA XR＿001793654.1 is closely related to atrial fibrillation and is well conserved.In vivo experiments have demonstrated that XR＿001793654.1 reduces the degree of left atrial fibrillation,has a certain protective effect on left atrial function and reduces the induction of atrial fibrillation in the RAP rabbit atrial fibrillation model.3.XR＿001793654.1 can play a role in atrial fibrosis in AF by competitively binding miR-107-3p through a sponge mechanism and thereby regulating the expression of the downstream target gene KLF13 and miR-107-3p can be able to reverse the function of XR＿001793654.1 in inhibiting atrial fibrosis in atrial fibrillation. |
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