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

Abstract

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: HL-1 cell; RBM15B; apoptosis; coxsackievirus B3; m6A; viral myocarditis.

Figure 1

Establishment of CVB3 infection model and determination of total m⁶A. (A) Body weights of Sham and CVB3 groups; n = 6. (B) General morphology of the hearts of Sham and CVB3 groups. (C) Comparison of serum myocarditis inflammatory markers CK-MB and cTnT between Sham and CVB3 groups; n = 6. (D) Representative images of HE staining of mouse myocardium from Sham and CVB3 groups (scale bar = 50 μm); n = 6. (E) IF staining of CVB3 (red) in mouse myocardial tissue in the Sham and CVB3 groups, blue staining indicates DAPI nuclear staining (scale bar = 50 μm); n = 6. (F) m⁶A dot blot analysis of myocardial tissues from Sham and CVB3 groups of mice; n=3. (G) m⁶A dot blot analysis of Sham and CVB3 groups of HL-1 cell; n=3. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2

Significant dysregulation of m⁶A peaks during CVB3 infection of HL-1 cells. (A) Enrichment of peaks near gene transcription start sites. (B, C and D) Distribution of differentially methylated m⁶A peaks in Sham and CVB3 groups. (E) Volcano plot of genes with differential m⁶A peaks (|log2(FC)|>1 and p-value < 0.05). (F) Motif map of m⁶A modifications in the Sham and CVB3 groups.

Figure 3

Differential m⁶A modifications are involved in important biological pathways. (A) GO enrichment analysis of the hypermethylated peaks. (B) GO enrichment analysis of the hypomethylated peaks. (C) KEGG pathway analysis of the hypermethylated peaks. (D) KEGG pathway analysis of the hypomethylated peaks.

Figure 4

Differential expression gene analysis by RNA-Seq. (A) Heatmap showing mRNAs differentially expressed in three CVB3 group input samples and three Sham group input samples. (B) Volcano plot showing mRNAs differentially expressed between CVB3 and Sham groups with statistical significance (|log2(FC)| ≥1.0 and p < 0.5). (C) GO enrichment analysis of differential genes. (D) KEGG pathway analysis of differential genes.

Figure 5

Combined analysis between m⁶A-Seq and RNA-Seq. (A) Four-quadrant diagram of hyper-up, hyper-down, hypo-up, and hypo-down DMEG. (B) KEGG pathway analysis of hyper-up DMEG (C) KEGG pathway analysis of hyper-down DMEG. (D) KEGG pathway analysis of hypo-up DMEG (E) KEGG pathway analysis of hypo-down DMEG.

Figure 6

Validation of differentially expressed genes. (A) Wayne plots of in vivo and in vitro RNA-seq sequencing results. (B) Volcano plot showing mRNAs that were differentially expressed simultaneously after comparison of in vivo and in vitro RNA-seq sequencing results with statistical significance (|log2(FC)| ≥ 1.0 and p-value < 0.5). (C and D) In vivo and in vitro validation of differentially expressed genes. (E) IGV visualization showing three key m⁶A-modified genes. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7

Validation of m⁶A-associated differentially expressed genes (A) Heatmap of m⁶A regulators in sequenced mouse samples. (B and C) qRT- PCR results of 10 common m⁶A regulators in vivo and in vitro. (D and E) Analysis of RBM15B protein levels in mouse samples from Sham and CVB3 groups by Western blotting. (F and G) Analysis of RBM15B protein levels in HL-1 cell samples from Sham and CVB3 groups by Western blotting. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 8

Knockdown of RBM15B ameliorates HL-1 cell apoptosis caused by CVB3 infection. (A–C) After transfection of HL-1 cells with si-RBM15B or si-NC, the cells were harvested under the same infection conditions and analyzed by Western blotting to determine the expression of RBM15B, cleaved-PARP, Bax/Bcl2, and cleaved-caspase3; n = 3. (D) Cultures from the si-NC group and si- RBM15B group were collected 48 h after infection with CVB3, respectively, and viral plaque assay was performed using Hela cells; n = 3. (E) Calcein AM/PI double staining assay was used to assess the cytotoxicity of HL-1 cells after transfection with si-NC and si-RBM15B at CVB3 infection (48 h) (scale bar = 50 μm). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.