The Expression Profile Analysis Of Atrial MRNA Transcriptome And The Role Of IGF1 In Atrial Fibrosis In Experimental Atrial Fibrillation

Part One The expression profile analysis of atrial mRNA transcriptome in experimental atrial fibrillationBackgroundAtrial fibrillation(AF)is the most common persistent and rapid arrhythmia in the clinic.The incidence of AF is high and increases with age(up to 7.1%when>85 years old),and it is expected that patients with AF will increase 2 times in the next 25 years.Due to its high morbidity and mortality caused by thromboembolic complications,AF has serious harm on patient’s health and quality of life.But the therapeutic effect of AF is still unsatisfactory,which is closely related to its unclear mechanisms.The mechanisms of the occurrence and maintenance of AF mainly include electrical reconstruction,structural remodeling,autonomic nerve reconstruction and energy metabolism reconfiguration.We focus on exploring the molecular mechanism of atrial remodeling and searching new molecular targets for the treatment and prevention of AF.The protein molecules that carry out all kinds of life activities are derived from the precise regulation of genetic code.Genetic regulation from nucleic acid molecules is much more complicated than human imagination.Genome research and gene expression regulation research have opened a new chapter for the pathogenesis and prognosis of diseases.AimsThe aim of our study is to explore the expression profile of mRNAs transcriptome after atrial remodeling during AF;to determine the key molecular that play significant roles in atrial remodeling during AF by bioinformatics analysis;to illustrate the new molecular mechanism for atrial remodeling during AF and provide potential targets for optimizing the treatment of AF.Methods1.Establishment of AF animal modelTwenty healthy male Wistar rats were randomly divided into two groups,control group(n = 10)and AF group(n = 10).The electrode leads were inserted into the right atria through right jugular vein in the two groups underwent surgery.The AF group underwent rapid pacing(15 Hz,900 beats/min)for 10 days to establish rats AF models,while the control group was the same as AF group but without pacing.Cardiac electrophysiological tests were performed before and 10 days after the surgery,including AERP and inducibility of AF.2.Intracardiac electrophysiological examinationAfter pacing,the measuring electrode was inserted into the right atria through right jugular vein.The AERP was defined as the longest S1-S2 interval that failed to produce a response.Rapid irregular atrial rhythms lasting>10 s were regarded as successful inductions of AF.The atrial samples were stored at-80℃ until use.3.Sequencing analysis and verificationTotal RNAs were isolated from left atria by using TRIzol reagent.The differentially expressed profiles of mRNAs were examined through sequencing analysis.Some transcripts were selected randomly,and their expression levels were analyzed by real-time quantitative PCR(qRT-PCR)to verify the sequencing results.4.Bioinformatics analysisGene Ontology(GO)functional analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were performed to reveal and annotate the differentially expressed transcripts.Based on the KEGG analysis of aberrantly expressed genes,the molecules in the pathways were constructed to build a network and identify the key intersections between these pathways.Results1.The rats AF models were successfully built through right atrial tachypacing.Compared with control group,the AERP was significantly shortened and the inducibility of AF was elevated in AF group.2.Through the high-throughput sequencing of left atria from AF and non-AF rats,956 transcripts meet the pipelines that Fold Change is more than 2 times and p<0.05.And among them,395 transcripts were down-regulated and 561 transcripts were up-regulated.Subsequently,6 mRNAs transcripts were randomly selected and verified by qRT-PCR method,which proved that the sequencing results were true and reliable.3.GO enrichment analysis showed that the differentially expressed genes were mainly involved in the biological processes,such as cell proliferation,cell adhesion,transmembrane transport,protein hydrolysis and so on.And the KEGG pathway analysis indicated that the aberrantly expressed genes were associated with the biological pathways related to cardiomyopathy.Based on the constructed pathway molecular cascade network,we screened IGF1,CACNB4,ADCY5,ITGA4 and other crossing points.These molecules may play a variety of biological key roles through multi-channel.We selected IGF1 as a key element in our study to explore the mechanism of atrial remodeling during AF.ConclusionsThere existed abberantly expressed mRNAs transcripts in left atria of AF and non-AF rats models.These differentially expressed transcripts were closely related to the development of atrial remodeling during AF.Part Two The role of IGF1 in atrial fibrosis in rats with atrial fibrillationBackgroundAtrial fibrillation(AF)is a disorder of atrial electrical activity,which is one of the most frequent rapid arrhythmias,and has seriously harm on the patients’ physical and mental health and quality of life.Although many progress has been made in the treatment of AF,the therapeutic effect of AF is still not satisfactory,which is closely related to its unclear mechanism.Structural remodeling,electrical reconfiguration,autonomic nerve remodeling and energy metabolism reconfiguration play an important role in the occurrence and maintenance of AF.Among them,structural remodeling is a factor for the maintenance and recurrence of AF.The atrial structural remodeling is mainly manifested as significant interstitial fibrosis,and the occurrence of AF increases with the degree of atrial fibrosis.Atrial fibrosis can interfere with the excitation conduction of atrium,causing a one-way conduction slow or block,and increase the inducibility of AF.Thorough treatment of AF is the main battlefield of the 21 Century cardiovascular field:are there more in-depth mechanisms to explain the occurrence and maintenance of AF?Are there any other intervention targets to prevent and treat AF?IGF1 is a class of the metabolic effects of insulin like factor,which can promote cell growth and inhibit apoptosis.IGF1 is mainly synthesized in the liver,but muscle specific IGF1(mIGF1)can be synthesized by skeletal muscle and cardiac muscle,which can play an important role in the process of cell proliferation,differentiation,apoptosis,metabolism,embryonic development,tumor occurrence and development through endocrine and paracrine form.Studies have shown that after IGF-]is combined with IGF-IR,there are mainly two pathways activated:mitogen activated protein kinase(MAPK)pathway and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)pathway.And the study showed that IGF-1 was a kind of strong fibrotic factor,which could increase the expression of extracellular matrix protein.AimsThe purpose of this study is to explore the effect of mIGF1 factor on atrial fibrosis in AF rats;to reveal the mechanisms and pathways of IGF1 induced fibrosis;to illustrate new explanation for the molecular mechanism of atrial remodeling and provide potential targets for the optimal treatment of atrial fibrillation.Methods1.IGF1 interference experiment in vitro and cell viability detectionThe fibroblasts were extracted from atria of rats and cultured,and the plasmid transfection experiments were performed using 3-4 generation fibroblasts.The cells were randomly divided into negative control group,mIGF1 overexpression group and mIGF1 silencing group.After 48 h culture,total RNAs was isolated and the protein was extracted after 72 h.CCK8 kit was used to detect the cell viability after plasmids transfection.2.Infection of AAV9 adeno-associated viruses in vivoThe AAV9 type adeno-associated viruses including mIGF1 silencing shRNAs were constructed in vitro.Then 30 healthy male Wistar rats,weighing 240-260 g,were randomly divided into negative control group(n = 10),implantation of pacing control device,intravenous injection with negative control AAV;pacing group(n = 10),right atrial tachypacing(15 Hz),intravenous injection with negative control AAV;pacing +mIGF1 silencing viruses group(n = 10),right atrial tachypacing(15 Hz),intravenous injection with mIGF1 silencing AAV.The implantation of small animal pacing electrode is the same as Part One.A total of 1×10E11 TU adeno-associated viruses were injected through caudal vein after operation.The pacing was stopped after 10 days.The animals were killed 14 days after the viruses injected.3.Detection of AERP and AF inducibilityBefore operation and before the execution of animals,the electrophysiology was detected by multi-channel program stimulator in negative control group,pacing group,pacing + mIGFl silencing viruses group,in order to detect the AERP and AF inducibility.4.Detection of biological moleculesTotal RNAs were isolated from the infected cells and tissue by TRIzol reagent.The expression levels of IGF1、CTGF、AT1R、COL1A1 and COL3A1 were detected by qRT-PCR.The protein levels of IGF1、CTGF、AT1R、p/t-PI3K、p/t-Akt、Bcl2、Bax were detected by Western Blotting.5.Immunohistochemical and Masson stainingThe expression levels of CTGF and TGFβ1 were detected by immunohistochemical staining,and the expression of positive index was measured.The degree of fibrosis was identified by Masson staining on tissue sections.Results1.The mIGF1gene interference experiments were performed in fibroblasts.Compared with negative control group,after overexpression of mIGF1,the expression levels of CTGF and AT1R were up-regulated,phosphorylation levels of PI3K and Akt were up-regulated,the ratio of Bcl2/Bax and cell viability were increased,the expression levels of COL1A1 and COL3A1 were were up-regulated.Conversely,after the lower expression of mIGF1,the expression levels of CTGF and AT1R were down-regulated,phosphorylation levels of PI3K and Akt were down-regulated,the ratio of Bcl2/Bax and cell viability were decreased,the expression levels of COL1A1 and COL3A1 were were down-regulated.2.Compared with the negative control group,the AERP of pacing group shortened significantly,and the AF inducibility increased.Compared with the pacing group,the AERP of the pacing+ mIGFl silencing viruses group prolonged,but it was still lower than that of the negative control group,and the AF inducibility was improved.3.Immunohistochemistry and Masson staining showed that,compared with negative control group,the expression levels of mIGF1,CTGF,TGF-β1 in atrial tissue of pacing group were up-regulated,and the degree of fibrosis was aggravated.Conversely,compared with the pacing group,the expression level of mIGF1 and CTGF in atrial tissue of pacing + mIGF1 silencing viruses group were down-regulated,and the degree of fibrosis was attenuated.Conclusions1.We found that mIGF 1 was closely related to the structural remodeling during AF,and the role of mIGF1 in atrial fibrosis during AF was determined by functional silence experiments.2.The mIGF1 plays a fibrotic role by activating the PI3K-Akt pathway to increase the expression of CTGF and AT1R.Novelty1.Through sequencing analysis,the expression profiles of transcriptome related to atrial remodeling during AF were identified.2.To explore the molecular mechanism of mIGF1 regulating atrial fibrosis during AF,which could help to illustrate the mechanisms of the occurrence and maintenance of AF,and provide potential interventional targets for the prevention and treatment of AF.3.The effects and mechanisms of mIGFl on atrial structural remodeling during AF were detected by the regulation of gene expression mediated by adeno-associated viruses in vivo.