Transcriptional Expression And Chromatin Accessibility In Angiotensin Ⅱ-induced Myocardial Hypertrophy Mice

Objective To investigate gene expression profile and chromatin accessibility after cardiac hypertrophy by high throughput transcriptome sequencing(RNA-seq)and assay for targeting accessible-chromatin using high-throughput sequencing(ATAC-seq).Methods Dividing C57 mice into two groups,the experimental group and the control group are established by intraperitoneal injection of AngⅡ and normal saline respectively,and then we demonstrate the myocardial hypertrophy by immunohistochemical staining and echocardiography tests.Subsequently we take mouse cardiac tissue for two different high-throughput sequencing(namely RNA-seq and ATAC-seq)respectively.On the one hand,the genes differentially expressed after hypertrophy are screened by RNA-seq,and some of the characteristic marker genes are verified by qPCR.On the other hand,ATAC-seq is used to study chromatin openness,by analyzing the distribution of ATAC-seq peaks on different cis-regulatory element regions,which are visualized in several marker genes.Results(1)The results of echocardiography show that,compared with the control group,the left ventricular end-diastolic diameter(LVEDd),ejection fraction(EF)and fraction shortening(FS)are significantly decreased in the hypertrophic group;At the same time,diastolic thickness of left ventricular posterior wall(LVPWd)and left ventricular posterior wall thickness at end systole(LVPWs)are significantly increased.Besides,interventricular septum thickness at end-diastole(IVSd),interventricular septal thickness at end-systole(IVSs)and left ventricular end systolic diameter(LVIDs)are increased or decreased severally albeit with no significance.(2)The results of HE staining show that the myocardial cells in the control group are oval with well-ordered arrangement,and the size of their nuclei are uniform.In addition,the myocardial fibers are orderly arranged.However,in the hypertrophic group,the myocardial cells are hypertrophic and the myocardial fibers are disorderly arranged.(3)Masson staining results show that the myocardial cells are red and collagen is blue.There is no collagen hyperplasia in the myocardial space in the control group.In the hypertrophic group,the interstitial fibrosis and collagenous accumulation occurred.(4)The results of GO analysis for differentially expressed genes(DEGs)show that the biological processes of GO are mainly concentrated in inflammatory response,immune response,angiogenesis,myocardial contraction,calcium homeostasis and other biological functions.The result of KEGG pathway analysis indicate the DEGs are concentrated in PI3K-Akt pathway,cardiac hypertrophy,calcium signaling pathway,Rap1 pathway,cAMP pathway,Ras pathway,Foxo pathway,AMPK pathway,cGMP-PKG pathway and MAPK pathway.(5)After verification by qPCR,it is found that the expression levels of characteristic marker genes related to hypertrophy are significantly increased.(6)The number of ATAC-seq peaks in a whole they are mainly located in the intergene region and intron region of genes.The peaks around the exon,promoter and transcription start site of the gene in the hypertrophic group increased slightly compared with the control group.Importantly,ATAC-seq peaks is enhanced near the transcription start site of genes associated with hypertrophy,such as Nppa,Nppb and Acta1.Conclusion The myocardial hypertrophy model is successfully constructed by using Ang Ⅱ.By analyzing the results of RNA-seq,ATAC-seq,qPCR,immunohistochemical staining and echocardiography tests,the following conclusions are drawn:(1)After the occurrence of myocardial hypertrophy in mice,myocardial function is disrupted and fibrosis occurred.(2)The expressions of 16076 genes are changed dramatically in the hypertrophic group,compared with control group,especially for the genes related to myocardial function and disease.(3)The occurrence of cardiac hypertrophy lead to enhance chromatin openness of some genes association with myocardial function,and their expression level is upregulated.For the down-regulated genes,their chromatin openness are inhibited.The results of this study will provite provide data support and theoretical basis for future investigation on myocardial hypertrophy.