Comprehensive Analysis of Differences in N6-Methyladenosine RNA Methylation Groups in CVB3-Induced Viral Myocarditis and Identification of the Anti-Apoptotic Role of RBM15B

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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. N 6 -methyladenosine (m 6 A), 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 m 6 A methylation within the myocardium remain unexplored. In this study, we investigated alterations in global RNA m 6 A methylation levels during CVB3 infection using both in vitro and in vivo models, and further examined the regulatory role of the m 6 A 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 m 6 A dot blot analysis. Subsequently, m 6 A 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 m 6 A 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 m 6 A analysis revealed elevated m 6 A modification levels in CVB3 infection group. MeRIP-seq identified 327 significantly altered m 6 A 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 m 6 A 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 m 6 A methylation in CVB3-associated VMC and may provide novel therapeutic insights for managing CVB3-induced viral myocarditis. Keywords: viral myocarditis, coxsackievirus B3, m 6 A, RBM15B, HL-1 cell, apoptosis

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