The Function And Mechanism Of TCONS₀0075467 On Electrical Remodeling In Rabbits With Atrial Fibrillation

BackgroundAs the most common frequent arrhythmia in clinical,the incidence rate of Atrial fibrillation?AF?is high and increases with age.The total prevalence rate of AF in our country was 0.77%.AF affects the health and life quality of patients seriously.The thromboembolic complications such as stroke caused by the instability of heart hemodynamics result in not only high morbidity and mortality,but also heavy burden for the society.Although drug therapy and radiofrequency catheter ablation for atrial fibrillation have made great progress in recent years,the therapeutic effect of atrial fibrillation is still not satisfactory,which is closely related to its unclear mechanism.Atrial electrical remodeling has been proved to play an important role in the initiation and maintenance of AF.Atrial electrical remodeling occurs at the early stage of AF and leads to the shortening of atrial effective refractory period?AERP?and action potential duration?APD?,which can facilitate the initiation of AF and other remodeling.Explaining the molecular mechanism of electrical remodeling during AF will help to improve the prevention and treatment of AF.Long non-coding RNAs?lncRNAs?are generally defined as non-protein coding RNAs with more than 200 nucleotides?nt?in length.Compared with mRNAs,the expression profile of lncRNAs has distinct specificity in different tissues or different stages.LncRNAs may function through a variety of mechanisms,including chromatin remodeling,genomic imprinting,splicing regulation,and mRNA decay.Recently,mounting evidences reveal that differently expressed lncRNAs play indispensable rolesin many heart diseases,including cardiac hypertrophy,heart failure,myocardial infarction,interventricular septal defect and autonomic nervous remodeling during AF.But at present,there exist no systematic studies on the relationship between IncRNAs and atrial electrical remodeling,and what roles the IncRNAs play in the occurrence of atrial electrical remodeling.AimsIn order to identify the influence of right atrial electrical remodeling on AF and the roles of IncRNAs in electrical remodeling during AF,the IncRNA expression profiles of right atria?RA?in rabbit AF models were investigated by using high-throughput RNA sequencing?RNA-Seq?,and the electrical remodeling related IncRNA was identified.Then the electrophysiology and electrical remodeling were examined in RA and primary atrial cardiomyocytes after knockdown of the novel IncRNA.Furthermore the mechanisms of the IncRNA in the pathogenesis of electrical remodeling in AF were explored.It will help to illustrate the molecular pathogenesis of AF and find new therapeutic targets for AF.Methods1.Establishment of AF animal modelTwelve adult New Zealand white rabbits weighing about 2.5-3 kg of male or female sex were randomly distributed to AF group?n = 6?and control group?n = 6?.The atrial tachypacing?A-TP,10 Hz,600 beats/min?in RA was continued for 7 d in AF group.Through the right jugular veins,the endocardial electrode affiliated to pacemaker was inserted into the anterior wall of RA under X-ray guidance.The control group was underwent the similar surgery only without pacing.Cardiac electrophysiological tests were performed before and 7 days after the operation,including AERP and inducibility of AF.2.High-throughput sequencingThe total RNAs of right atria from 3 samples of control group and 3 samples of AF group were isolated using TRIzol reagent.The secondary generation high-throughput sequencing was performed to detect the expression of gene transcripts and IncRNAs.Some new IncRNAs were selected randomly,and the accuracy of the sequencing results was verified by the method of real-time quantitative PCR?qRT-PCR?.3.Identification of candidate IncRNAsGene Ontology?GO?enrichment analysis and Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway analysis were used to illustrated the transcripts with differential expression.Based on the expression changes,target genes prediction,co-expression genes analysis,literature retrieval,tissue specificity,conservatism analysis and so on,the new transcripts lncRNAs related to atrial electrical remodeling during AF were identified.4.Predicted mechanisms of IncRNAs?1?cis-mechanismBased on cis-mechanism,the lncRNAs transcripts could interfere with the expreesion of their adjacent genes.So the 10 kb upstream or downstream of the genomic location of IncRNAs in chromosome was detected to select gene transcrits.And the expression changes of these selected gene transcrits were verified.?2?trans-mechanism and co-expression analysisBased on the Basic Local Alignment Search Tool?BLAST?to predict the trans-mechanisms.There exist several binding sites on the IncRNAs sequence for binding with 3' untranslated regions?UTR?of mRNAs or miRNAs to regulate the expression of mRNAs.So we using the BLAST to search the possible binding sites on the 3,UTR of mRNAs and miRNAs.According to Pearson correlation coefficient?COR?and P value,we selected the transcripts co-expressed with IncRNAs based on the pipelines that |COR|>0.85,P<0.05.Then the GO analysis and KEGG analysis of these co-expressed transcripts were performed to predict the trans-mechanisms of lncRNAs.5.Knocking-down experiments?1?Infection of lentiviruses in vivoThe specific short hairpin RNA?shRNA?targeting TCONS₀0075467 and ocu-miR-328 were constructed and the lentiviruses were obtained from 293T cells.Meanwhile the negative control lentiviruses were acquired as the same way.The purpose of constructing these lentiviruses was to interference with the expression of TCONS₀0075467 and ocu-miR-328,and identify their functions.Twenty-five adult male or female rabbits were randomly distributed to five groups:negative control group?n=5?,infected with negative control lentiviruses;lenti-RNAi-TCONS₀0075467 group?n=5?,infected with lentiviruses for silencing TCONS₀0075467;lenti-miR-328 inhibitor group?n=5?,infected with lentiviruses for inhibiting ocu-miR-328;lenti-miR-328 mimics group?n=5?,infected with lentiviruses containing ocu-miR-328 precursor shRNA;and co-infected lentiviruses group,co-infected with the lentiviruses for silencing TCONS₀0075467 and the lentiviruses for inhibiting ocu-miR-328.After anaesthesia and thoracotomy,lentiviruses were directly injected into 10 separate sites through a microsyringe to distribute the transfer vectors uniformly over a large area.Samples were collected 7 days after infection.The atrial tissue sections were observed under fluorescence microscope to detective the efficiency of infection.AERP and AF inducibility were measured before infection,immediately after infection and 7 days after infection.?2?Infection of lentivirases in vitroThe primary atrial cardiomyocytes were extracted from rabbits atria.The cells were randomly divided into blank control group,negative control group,TCONS₀0075467 silencing lentiviruses group,ocu-miR-328 silencing lentiviruses group,ocu-miR-328 overexpressing lentiviruses group,co-infection group with TCONS₀0075467 silencing lentiviruses and ocu-miR-328 silencing lentiviruses.After 48 h,the fluorescence intensity of infected cells was observed under fluorescence microscope.After 96 h culture,RNA and protein were extracted after 96 h culture.6.Molecular biological assayTotal RNAs were isolated from tissue and cells after infection by using TRIzol reagent,and the expression levels of TCONS₀0075467 and ocu-miR-328 were detected by qRT-PCR.Western Blotting analysis were used to determine the protein expression of CACNA1C.7.Fluorescent in situ hybridization?FISH?On 4%paraformaldehyde fixed slides with monolayers of primary myocytes,using CY3-labelled probe for TCONS₀0075467 and Dylight 488-labelled probe for ocu-miR-328 at 5 ng/?l concentration.Hybridization was performed at 37 ? overnight,followed by visualization using fluorescence microscope?Nikon,Tochigi,Japan?.Slides were subsequently counterstained using DAPI.8.Luciferase activity assayThe ocu-miR-328 mimics and negative control mimics were synthesized.The DNA sequence of the 3'UTR of CACNA1C was acquired from Oryctolagus cuniculus cDNA library?NCBI GenBank?.The DNA fragments containing the wild type?WT?and mutation type?Mut?sequences of TCONS₀0075467 and 3'UTR of CACNA1C were synthesized.The oligonucleotides were ligated into NotI and XhoI sites by T4 Ligase in the psi-CHECK-2 luciferase reporter vectors?Promega,Madison,WI?.luciferase activities were measured with the Dual-Luciferase Reporter Assay System to verify the interaction between ocu-miR-328 and TCONS₀0075467.9.Whole-cell patch-clamp recordingPatch-clamp techniques were applied to primary myocytes after lentiviruses infected.The Tyrode and pipette solutions were configured.Briefly,the pipette of patch electrodes had the tip resistance of 3-5 M? when filled with pipette solution.The single cells were placed in a 1-ml chamber mounted on an inverted microscope?IX-70,Olympus,Japan?and perfused with Tyrode solution.Whole-cell recording were performed using an amplifier?Axopatch 700B?.Signals were filtered at 1 kHz and data were acquired by A/D conversion?Digidata 1550B,Axon Instrument?.L-type calcium current?ICaL?was recorded in the whole cell voltage-clamp mode and APD was recorded under the current-clamp mode.Results1.The rabbit AF models were successfully established through right atrial tachypacing.The AERP was significantly shortened and the inducibility of AF was increased in AF group.2.Through the next generation high-throughput sequencing of right atria from AF and non-AF rabbits,we acquired 99843 putative lncRNAs transcripts,in which 1220 transcripts displayed more than two-fold changes.Among them,237 transcripts were up-regulated and 983 transcripts were down-regulated.Subsequently,6 new IncRNAs were randomly selected to detective their expression levels by qRT-PCR.And the sequencing results were confirmed to be reliable by validating their expression trends.3.GO enrichment analysis and KEGG pathway analysis showed that the differentially expressed genes were mainly involved in cardiac development,ion transport and cell adhesion and so on biological processes.Through a series of bioinformatics and statistical filtering pipelines,such as fold-change screening,target genes prediction,co-expressed genes analysis,the function of target genes analysis and tissue specificity,we identified 3 new IncRNAs transcripts related to atrial electrical remodeling during AF,including TCONS₀0106987,XR₅16397,TCONS₀0075467.Among them,TCONS₀0106987 was up-regulated,XR₅16397 and TCONS₀0075467 were down-regulated.Then TCONS₀0075467 was selected as candidate IncRNA for further mechanism studies.4.The fluorescence intensity of frozen sections and cell slides were observed to detective the efficiency of infection.And qRT-PCR was used to verify the silencing efficiency.Intracardiac electrophysiological examination showed that the AERP did not reach statistical significance before and immediately after infection.After 7 days infection,inhibition of TCONS₀0075467 expression could significantly shorten AERP and elevated the inducibility of paroxysmal atrial tachycardia and AF.5.Sequence analysis using the NCBI BLAST revealed that TCONS₀0075467 was located on the forward strand of chromosome 13:141223079-141225530?OryCun 2.0?,and the transcript belongs to long intergenic ncRNAs?lincRNAs?,which do not overlap with any protein-coding loci.TCONS₀0075467 was found to be mostly localized in the cytoplasm of primary myocytes.GO enrichment analysis indicateed that TCONS₀0075467 was involved in biological processes such as ion transport and heart development.KEGG analysis indicated that it was closely related to calcium signaling pathway and various cardiomyopathy pathways.6.For the cis-mechanism,there exist no nearby genes reside within 10 kb of TCONS₀0075467 locus that associated with atrial electrical remodeling or ion channels.Based on the bioinformatics analysis,there are multiple binding sites between TCONS₀0075467 and ocu-miR-328.And the expression of TCONS₀0075467 was negatively correlated with ocu-miR-328 transcript level.The experiment of double luciferase report confirmed the interaction between TCONS₀0075467 and ocu-miR-328.After TCONS₀0075467 silencing,the expression level of ocu-miR-328 was up-regulated.7.In vivo,the cardiac electrophysiological examination of ocu-miR-328 silencing and overexpression models showed that the overexpression of ocu-miR-328 could significantly shorten the AERP,and increase the inducibility of paroxysmal atrial tachycardia and AF;conversely,the lower expression of ocu-miR-328 could extend the AERP,and decrease the inducibility of paroxysmal atrial tachycardia and AF.The double luciferase reporter experiment confirmed that CACNA1C was the target gene of ocu-miR-328.After overexpression of ocu-miR-328,the expression level of CACNA1C was down-regulated;and the lower expression of ocu-miR-328 increased the expression level of CACNA1C.8.After the lower expression of TCONS₀0075467,CACNA1C was down-regulated,the L-type calcium current was decreased and action potential duration was shortened.Compared with the TCONS₀0075467 silencing lentiviruses group,the AERP of co-infection group was prolonged,the inducibility of paroxysmal atrial tachycardia and AF were decreased,and the expression level of CACNA1C was up-regulated.But compared With the negative control group,these changes of co-infection group were not statistically significant.Conclusions1.There existed differentially expressed IncRNAs in right atria of AF and non-AF rabbit models.The abberantly expressed lncRNAs were closely involved in the development of atrial electrical remodeling during AF.2.We found that the new IncRNA transcript TCONS₀0075467 was closely related to the atrial electrical remodeling during AF.And the functional silence experiments confirmed the role of TCONS₀0075467 in AF inducibility.3.We further demonstrated that TCONS₀0075467 could sponge ocu-miR-328 in vitro and in vivo to regulate the downstream protein coding gene CACNA1C.In addition,ocu-miR-328 could partly reverse the effects of TCONS₀0075467 on electrical remodeling.Novelty1.We investigated the effects of the abberantly expressed lncRNAs on atrial electrical remodeling during AF from the perspective of IncRNAs.2.We explored the mechanisms of influence of lncRNAs on atrial electrical remodeling during AF through endogenous competitive binding mechanism to regulating target genes,which facilitate the mechanism studies of lncRNAs in AF pathogenesis and provide potential therapeutic targets for AF.3.We investigated the effects and mechanisms of lncRNAs on atrial electrical remodeling during AF through intracardiac electrophysiological examination and patch clamp technique,and by using the in vivo and in vitro infectious technology with lentiviruses mediated IncRNA silencing vector.