PVN-centered multi-omics and single-cell analyses suggest shared inflammatory and immune features in acute myocardial infarction-associated ventricular arrhythmia and atrial fibrillation

Figures

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  Figure 1. Myocardial infarction injury, arrhythmic phenotypes, and PVN inflammation/NF-κB activation jointly confirm successful establishment of the AMI model. (A) TTC staining of left ventricular transverse sections from Sham and AMI groups at 48 h and 7 days after surgery. Red indicates viable myocardium, whereas white/light areas indicate infarcted regions; scale bar = 2 mm. (B) Quantitative analysis of TTC staining showing the proportion of left ventricular infarct area (% LV). (C) Serum cTnI levels measured by ELISA. (D) Representative ECG telemetry waveforms. (E) PVC burden (PVC/hour). (F) Number of VT episodes. (G) VA burden quantified by ECG telemetry as total VT duration (seconds). (H) Representative immunofluorescence images of the PVN showing Iba1 (microglial marker), GFAP (astrocytic marker), and p-p65 (marker of NF-κB activation). (I–K) Quantitative analyses of PVN immunofluorescence: density of Iba1-positive cells (cells/mm2) (I), percentage of GFAP-positive area (%) (J), and proportion of p-p65-positive cells (%) (K). Data are presented as box plots; n = 8 per group. *p < 0.05; ****p < 0.0001.

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  Figure 2. Differential expression analysis of genes and metabolites. (A,B) Volcano plots showing differential gene expression between the AMI and Sham groups in the transcriptomic data at 48 h and 7 days, respectively. (C,D) Heatmaps of the top 20 DEGs in the transcriptomic data at 48 h and 7 days; yellow indicates the Sham group and pink indicates the AMI group. In the heatmaps, red represents high expression and blue represents low expression. (E,F) Volcano plots of DEMs between the AMI and Sham groups in the metabolomic data at 48 h and 7 days. (G,H) Heatmaps of the top 20 DEMs at 48 h and 7 days in the AMI dataset; yellow indicates the Sham group and blue indicates the AMI group. In the heatmaps, red denotes high expression and blue denotes low expression. (I) Interaction network of DEGs and DEMs, in which yellow nodes represent DEGs and green nodes represent DEMs. AMI, Acute Myocardial Infarction.

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  Figure 3. Scatter plot of causal effect estimates. (A–M) Scatter plots of MR effect estimates for ABCA3 (A), AKIP1 (B), ANKK1 (C), BLM (D), C1QA (E), CAPG (F), CD5 (G), DGKQ (H), DLG2 (I), IL12RB1 (J), PSMC3IP (K), SPCS2 (L), and ZNF318 (M) on VA. (N) Scatter plot of MR effect estimates for DEMs on VA. The slope of the fitted line represents the predicted strength of the causal relationship.

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  Figure 4. In vivo bilirubin intervention increased Serum direct bilirubin levels and reduced post-AMI arrhythmia burden while suppressing PVN inflammation and NF-κB activation. (A) Schematic of the experimental workflow: C57BL/6 mice underwent AMI or Sham surgery, followed by administration of bilirubin or vehicle starting 0-1 h after surgery (once every 24 h until day 7). ECG telemetry was recorded continuously during the 0-48 h postoperative period and for an additional 24 h on day 7. Blood samples were collected at 48 h and 7 days to measure direct bilirubin levels, and PVN tissues were harvested for immunofluorescence analysis. (B) Serum direct bilirubin levels at 48 h and 7 days. (C) PVC burden (PVC/hour). (D) Number of VT episodes. (E) Total VT duration (seconds). (F) Representative immunofluorescence images of the PVN showing Iba1 (microglia, green), GFAP (astrocytes, red), p-p65 (NF-κB activation, red), and DAPI (nuclei, blue). (G) Density of Iba1⁺ cells in the PVN (cells/mm2). (H) Percentage of GFAP⁺ area coverage in the PVN. (I) Proportion of p-p65⁺ cells in the PVN (%).

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  Figure 5. Co-Localization regions of genes and VA. (A–E) Co-localization regions of genes and VA: IL12RB1 (A), DGKQ (B), ABCA3 (C), ANKK1 (D), BLM (E) The left panel of each figure illustrates the distribution of SNPs' -log10(p) values from GWAS and eQTL studies, where smaller p-values correspond to higher positions on the Y-axis. The two right panels respectively show the distribution of SNP loci in eQTL and GWAS data, with the horizontal axis representing the SNP location and the vertical axis indicating the -log10(p) value of the SNP in GWAS or eQTL data.

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  Figure 6. Diagnostic efficacy of genes and molecular docking analysis. (A) Box plots depicting the expression levels of key genes across different groups in GSE41177; (B–E) ROC curves for genes ABCA3 (B), BLM (C), DGKQ (D), and IL12RB1 (E) in GSE41177; (F–I) Visualization of molecular docking results: ABCA3 with risperidone (F), BLM with streptonigrin (G), DGKQ with 2-methylcholine (H), and IL12RB1 with dinoprostone (I). Each panel presents an overall docking view followed by an interaction force diagram. The color gradient on the protein surface transitions from green to orange to red, indicating a progression from hydrophilic to hydrophobic amino acids. Blue dashed lines represent hydrogen bonds, light blue dashed lines denote weak hydrogen bonds, gray dashed lines indicate hydrophobic interactions, yellow dashed lines signify ionic bonds, green dashed lines represent π-π stacking interactions, and orange dashed lines denote cation-π interactions. AF, Atrial Fibrillation; ROC, Receiver Operating Characteristic; AUC, Area Under the Curve; TPR, True Positive Rate; FPR, False Positive Rate. Yellow indicates the control group, while pink represents the AF group. * indicates statistical…

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  Figure 7. Correlation and functional annotation analyses of Key genes. (A) Correlation analysis of the four key genes in the GSE41177 dataset, visualized using a chord diagram; (B) Chromosomal localization of the key genes; (C) Bar plots showing GO and KEGG functional annotation results for the key genes, including BP, CC, MF, and biological pathways (KEGG); the x-axis represents GO and KEGG terms; (D–G) Network diagrams displaying GO and KEGG enrichment analysis results for key genes: BP (D), CC (E), MF (F), KEGG (G) GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological Process; CC: Cellular Component; MF: Molecular Function. The selection criteria for GO and KEGG enrichment analysis were p-value < 0.05 and FDR (q-value) < 0.25. In the network diagrams, orange nodes represent terms, green nodes represent molecules, and lines indicate relationships between terms and molecules.

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  Figure 8. Differential gene expression and GSEA. (A,B) Volcano plot (A) and heatmap (B) of DEGs between the AF and control groups in GSE41177; (C) GSEA bubble plot for GSE41177; (D–G) GSEA results indicate upregulated pathways in the AF group, including Wilensky Dallapadula response, Thum contractile heart failure, Jiang normal hypoxia response, and Martin NF-κB target genes. AF: atrial fibrillation. Pink represents the AF group, while yellow indicates the control group. In the bubble plot, the size of the bubbles corresponds to the size of the gene sets, with color denoting the normalized enrichment score (NES); redder colors indicate higher NES values, while bluer colors indicate lower NES values. In the heatmap, red indicates high expression, while blue denotes low expression. GSEA selection criteria were adj.p < 0.05 and FDR value (q-value) < 0.25, with p-values adjusted using the BH method.

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  Figure 9. Immune infiltration analysis based on ssGSEA algorithm. (A) Box plot illustrating differences in immune cell infiltration abundance between the control and AF groups; (B) Heatmap depicting the correlation of immune cell infiltration abundance; (C) Bubble chart showing the correlation between key genes and immune cell infiltration abundance. ssGSEA: single-sample gene set enrichment analysis; AF: atrial fibrillation. ns indicates p-value ≥ 0.05, not statistically significant; * indicates p-value < 0.05, statistically significant; ** indicates p-value < 0.01, highly statistically significant; *** indicates p-value < 0.001, extremely statistically significant. Yellow represents the control group, and pink represents the AF group. Red denotes a positive correlation, blue denotes a negative correlation, with the color intensity indicating the strength of the correlation.

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  Figure 10. Single-Cell analysis. (A) Quality control violin plots for the GSE261170 dataset. (B) PCA visualization of cellular expression profiles across different samples. (C) UMAP clustering of 105,157 cells into 30 cell clusters. (D) Cell type annotation using the R package SingleR, classifying cells into nine cell types: smooth muscle cells, T cells, monocytes, NK cells, B cells, neutrophils, endothelial cells, hematopoietic progenitor cells (annotated as HSC-G-CSF by SingleR), and DC. (E) Bar plot showing the proportions of different cell types across samples. (F) Bar plot comparing the proportions of each cell type between the AF group and the control group. (G) Bubble plot illustrating the expression levels of four key genes across cell types, with darker colors indicating higher expression levels and larger bubble sizes indicating a higher proportion of cells expressing the gene within each cell type. (H) differential expression analysis across cell types based on scRNA-Seq data, showing the average log2FC of genes in the disease group relative to the control group; red dots indicate significantly upregulated genes, blue dots indicate significantly downregulated genes, and each dot…

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  Figure 11. IL12RB1 knockdown suppressed NF-κB activation and reduced Inflammatory Cytokine Expression and Secretion in the BV2 Cell H/R Model. (A) Schematic of the experimental workflow: BV2 cells were transfected with siNC or siIL12RB1, followed by H/R treatment, after which cells and culture supernatants were collected for qPCR, Western blot, and ELISA analyses. (B) Relative Il12rb1 mRNA expression measured by qPCR to verify knockdown efficiency. (C) Relative mRNA expression levels of inflammation-related genes Il-6 and Tnf measured by qPCR. (D) Protein levels of IL-6 and TNF-α in cell culture supernatants measured by ELISA (pg/mL). (E) Representative Western blot images. (F) Densitometric quantification of p-p65 bands. Data are presented as mean ± standard deviation, with n = 3 independent experiments; between-group comparisons were performed using two-tailed t-tests. Significance levels: ***p < 0.001, ****p < 0.0001.