N6-methyladenosine RNA landscape in the aged mouse hearts

Xia Jing¹fu Zhao Liao¹yu Jing Wu¹yang Zi Xu¹peng Zhi Yang¹liang Hai Mo²Shun Xu¹Xinguang Liu¹Jun Tao^3*dong Xing Xiong^1,4*

• ¹Dongguan Key Laboratory of Aging and Anti-Aging, The First Dongguan Affiliated Hospital, Guangdong Medical University, dongguan, China
• ²Department of Cardiology, Dongguan First Hospital affiliated to Guangdong Medical University, Dongguan, China
• ³Department of Hypertension and Vascular Disease, The First Affiliated Hospital of Sun Yat-sen, guangzhou, China
• ⁴Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University, Dongguan, China

Objective: Cardiac aging is a major risk factor for the development of cardiovascular diseases. Although evidence suggests an association between N6-methyladenosine (m6A) modification and numerous cardiovascular diseases, its role in cardiac aging remains unclear.This study was conducted to elucidate the role of m6A modification in cardiac aging and the molecular mechanisms involved.Methods: Global methylation levels and the expression of major m6A regulators were compared between young and aged hearts. Transcriptome-wide m6A landscape analysis was conducted using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify aberrant m6A peaks.Furthermore, gene set enrichment analysis (GSEA) was performed to identify gene sets associated with cardiac aging. Functional validation of key molecules was carried out through in vitro experiments.The overall m6A level remained constant; however, the expression of the methyltransferase METTL14 and the demethyltransferase FTO were significantly upregulated in aged hearts. Knockdown of METTL14 alleviated H₂O₂-induced senescence phenotypes, as reflected by a reduction in the number of SA-β-gal positive cells and a decrease in p21 expression. Compared with young hearts, the dysregulated m6A peaks were significantly enriched in genes associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, and the PI3K-Akt signaling pathway.GSEA showed that these genes were enriched in the aging of heart and aorta cardiomyocytes. Additionally, 255 genes with siginificantly changed of both m6A peaks and RNA expression were identified by combining MeRIP-seq and RNA-seq data. Among these genes, EFEMP1 was significantly upregulated in aged hearts, accompanied by enhanced m6A modification. Treatment with the methyltransferase inhibitor cycloleucine significantly suppressed the expression level of EFEMP1. In AC16 cells, silencing EFEMP1 suppressed H2O2-induced cell senescence. Furthermore, we found a positive correlation between METTL14 and EFEMP1 in multiple datasets related to cardiac aging.Our findings indicate that m6A modification plays an essential role in the process of cardiac aging. EFEMP1 may serve as a potential new therapeutic target for age-related cardiac diseases.