Yanan Hu,1,2,* Jiahui Lin,3,* Lu Yi,4 Sha Cheng,5 Gao You,6 Huan Chang,7 Hanmin Liu,1,2 Zuocheng Yang,8 Shuyue Chen9 

1Department of Pediatric Pulmonology and Immunology, West China Second University Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu, 610041, People’s Republic of China; 3Department of Gastroenterology, The Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, People’s Republic of China; 4Department of Hengyang Medical School, The First Affiliated Hospital, University of South China, Hengyang, Hunan, 421001, People’s Republic of China; 5Department of Gastroenterology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, People’s Republic of China; 6Cardiovascular Surgeon Department, Intensive Care Unit, Xiangya Hospital, Central South University, Changsha, Hunan, 410013, People’s Republic of China; 7Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, People’s Republic of China; 8Department of Pediatrics, The Third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, People’s Republic of China; 9Department of Plastic Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Zuocheng Yang; Shuyue Chen, Email yang_zcr@126.com; duiyu07@163.com

Background: Viral myocarditis (VMC) is a leading cause of sudden cardiac death in children and young adults, with Coxsackievirus B3 (CVB3) identified as the primary viral pathogen responsible. N6-methyladenosine (m6A), the most abundant and reversible RNA methylation modification in mammals, plays a pivotal role in regulating numerous biological processes. However, the potential effects of CVB3 infection on m6A methylation within the myocardium remain unexplored. In this study, we investigated alterations in global RNA m6A methylation levels during CVB3 infection using both in vitro and in vivo models, and further examined the regulatory role of the m6A methyltransferase RBM15B in vitro.
Methods: First, the total quantity of m6A was quantified in Balb/c mice and HL-1 cells with CVB3 infection via m6A dot blot analysis. Subsequently, m6A methylation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were performed on cell model, while RNA-seq was conducted on animal tissues. We further analyzed the expression of m6A regulatory genes and their involvement in key pathways linked to VMC pathogenesis to elucidate underlying mechanisms. Given the pronounced expression of RBM15B in vitro, we knocked down RBM15B and assessed its regulatory effects on CVB3-infected HL-1 cells using Western blotting, viral plaque assays, and Calcein AM/PI double staining.
Results: Quantitative m6A analysis revealed elevated m6A modification levels in CVB3 infection group. MeRIP-seq identified 327 significantly altered m6A peaks (116 upregulated, 211 downregulated). RNA-seq detected 1,597 upregulated and 2,942 downregulated mRNAs. Integrated analysis of MeRIP-seq and RNA-seq identified 38 hypermethylated-upregulated, 23 hypermethylated-downregulated, 65 hypomethylated-downregulated, and 13 hypomethylated-upregulated genes. GO and KEGG pathway analyses of these differentially methylated genes highlighted their roles in broad biological functions. Furthermore, qRT-PCR validation of mice RNA-seq data confirmed significant differences in four key genes (Igtp, ApoI9b, Ddit3, and Irgm3), along with altered expression of m6A regulators (IGF2BP2, EIF3H, RBM15B, and YTHDC2), with RBM15B showing the most pronounced changes. RBM15B knockdown in HL-1 cells reduced CVB3 replication (viral plaque assay) and attenuated apoptosis induced by CVB3 infection (Calcein AM/PI staining and Western blotting).
Conclusion: These findings establish a foundation for exploring the role of m6A methylation in CVB3-associated VMC and may provide novel therapeutic insights for managing CVB3-induced viral myocarditis.

Keywords: viral myocarditis, coxsackievirus B3, m6A, RBM15B, HL-1 cell, apoptosis