Role of N6-methyladenosine methylation in transverse aortic constriction-induced cardiac fibrosis: insights from MeRIP-seq analysis

Abstract

This study aimed to investigate the role and potential mechanisms of N6-methyladenosine (m6A) methylation in a mouse model of transverse aortic constriction (TAC)-induced cardiac fibrosis using MeRIP-seq. A TAC-induced cardiac fibrosis mouse model was established, and cardiac function and structural parameters were assessed by echocardiography four weeks post-surgery. The global m6A methylation levels in myocardial tissues were evaluated using Dot blot analysis, and the expression levels of m6A-modifying enzymes (METTL3, METTL14, ALKBH5, FTO) were detected by qPCR and Western blot. Additionally, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was performed to identify differentially methylated sites, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to determine the biological functions and signaling pathways of m6A-methylated genes. The results showed that TAC surgery successfully induced a cardiac fibrosis model in mice, as indicated by significantly reduced EF and LVEF and increased LVIDd and LVIDs. Moreover, m6A methylation levels were elevated in myocardial tissues of TAC mice, accompanied by upregulation of METTL3 and METTL14 expressions and downregulation of ALKBH5 and FTO expression. MeRIP-seq revealed that m6A peaks were primarily enriched in the 3' UTR regions, with 1,466 differentially methylated sites identified between TAC and sham groups, including 717 hypermethylated and 749 hypomethylated sites. Functional enrichment analyses showed that these differentially methylated genes were involved in various biological processes, including signal transduction, transcriptional regulation, and ion channel activity, and were associated with pathways such as type 2 diabetes mellitus, signaling pathways regulating pluripotency of stem cells, and insulin signaling. Thus, our findings suggested that m6A methylation played a significant role in TAC-induced cardiac fibrosis by regulating key genes involved in myocardial remodeling and functional impairment.

Keywords: Cardiac fibrosis; Methylated RNA immunoprecipitation sequencing; N6-methyladenosine; Transverse aortic constriction.

Fig. 1

TAC induced impaired cardiac function and left ventricular remodeling in mice. A, B Echocardiographic assessment of aortic valve blood flow velocity in sham and TAC groups. C–I Cardiac function in mice was assessed using echocardiography. Key measurements included: Ejection fraction (EF), Left ventricular (LV) ejection fraction (LVEF), LV end-diastolic diameter (LVIDd), LV end-systolic diameter (LVIDs), LV posterior wall thickness, LV posterior wall thickness (LVPWd) (end-diastolic), LV posterior wall thickness (LVPWs) (end-systolic). J Masson staining was used to observe collagen deposition in the cardiac tissue of TAC-induced mice at a magnification of 400x. K HE staining showing pathological changes in the cardiac tissues. L Sirius red staining indicating fibrosis in the cardiac tissue of TAC-induced mice. ^**P < 0.01 vs. sham. Data is presented as mean ± SD

Fig. 2

Shows enhanced m6A methylation and dysregulation of m6A modifying enzymes in myocardial tissues of TAC-induced cardiac fibrosis model. A Dot blot analysis demonstrates increased global m6A methylation levels in myocardial tis-sues of the TAC group compared to the sham group (P > 0.05). (B–E) PCR analysis was used to test mRNA levels of key m6A methyltransferases (METTL3, METTL14) and demethylases (ALKBH5, FTO) in myocardial tis-sues from the TAC and sham groups. F–J Western blot analysis was performed on ALKBH5, METTL3, METTL14, and FTO in myocardial tissues. β-actin was used as internal control. ^*P < 0.05, ^**P < 0.01 vs. sham groups. Data is presented as mean ± SD

Fig. 3

General characteristics and topological characteristics of m6A methylation modification in cardiac tissue in TAC-induced cardiac fibrosis model. (A and B) Statistics on peak number and length statistics. (C) Distribution of peaks in chromosomes. (D) Distribution of peaks in the 5’ untranslated region (5’UTR), coding sequence (CDS), 3’UTR of genes. (E) Statistical distribution of peaks in each functional region of the gene

Fig. 4

Effect of TAC induction on m6A modification in mouse myocardial tissue. A Homer known motif enrichment results. B Difference peak volcano map. C The Gene Ontology (GO) pathway enrichment map of differentially peak related genes. D Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enriched in genes related to peaks