Activation of β2-Adrenergic Receptor Alleviates Viral Myocarditis by Regulating Energy Metabolism in Monocyte-derived Macrophages via the AMPK Pathway

Figures

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  Fig. 1. Activating β2-AR attenuates inflammation and fibrosis in VMC. A-B Representative flow cytometry images (A) and statistical results (B) of β2-AR expression on CD45+CD64+F4/80+CCR2+ macrophages in control and myocarditis model mice at 1 week and 2 weeks post-CVB3 infection (n = 6/group). C-D Representative H&E-stained images of myocardial tissue from each group at 1 week (C) and 2 weeks (D) post-CVB3 infection (original magnification, 200 × ; scale bars = 50 µm). E–F Representative Masson's trichrome staining images from each group at 1 week (E) and 2 weeks (F) post-CVB3 infection (original magnification, 200 × ; scale bars = 50 µm). G-H Representative cardiac ultrasound images of the mice in each group at 1 week (G) and 2 weeks (H) post-CVB3 infection. (I) Statistical analysis of the myocardial pathological scores for each group at 1 week and 2 weeks post-CVB3 infection (n = 6/group). J Statistical analysis of cardiac collagen volume fractions in each group at 1 week and 2 weeks post-CVB3 infection (n = 6/group). K-L Statistical analysis of the LVFS (K) and LVEF (L) of each group at 1 week and 2 weeks post-CVB3 infection (n = 6/group). M–O Real-time qPCR analysis of CCL2 (M),…

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  Fig. 2. Effects of activating β2-AR on the frequencies and subsets of monocytes/macrophages. A Representative flow cytometry images of cardiac macrophage subsets among CD45+CD64+F4/80+ cells in the control, CVB3 + vehicle, and CVB3 + formoterol-treated groups at 1 week and 2 weeks post-infection. B-C Statistical analysis of CCR2+ macrophages (B) and CCR2− macrophages (C) among cardiac macrophages in the control, CVB3 + vehicle, and CVB3 + formoterol-treated groups at 1 week and 2 weeks post-infection (n = 6/group). D Representative flow cytometry images of Ly6Chigh and Ly6Clow monocytes among blood CD45+CD11b+Ly6G− cells in the control, CVB3 + vehicle, and CVB3 + formoterol-treated groups at 1 week and 2 weeks post-infection. E–F Statistical analysis of Ly6Chigh monocytes (E) and Ly6Clow monocytes (F) among blood CD45+CD11b+Ly6G− cells in the control, CVB3 + vehicle, and CVB3 + formoterol-treated groups at 1 week and 2 weeks post-infection (n = 6/group). G-H Representative flow cytometry images (G) and statistical analysis (H) of Ly6C+ monocytes among splenic CD45+CD11b+Ly6G− cells in the control, CVB3 + vehicle, and CVB3 + formoterol-treated groups at 1 week and 2 weeks post-infection…

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  Fig. 3. Effects of activating β2-AR on the polarization and function of cardiac CCR2+ macrophages and CCR2− macrophages. A-B Representative flow cytometry images (A) and statistical analysis (B) of CCR2+iNOS+ M1 macrophages among CD45+CD64+F4/80+CCR2+ macrophages in the control, CVB3 + vehicle, and CVB3 + formoterol groups at 1 week and 2 weeks post-infection (n = 6/group). C-F Representative flow cytometry images and statistical analysis of TNF-α (C-D) and CCL2 (E–F) secreted by CD45+CD64+F4/80+CCR2+ macrophages in the control, CVB3 + vehicle, and CVB3 + formoterol groups at 1 week and 2 weeks post-infection (n = 6/group). G-H Representative flow cytometry images (G) and statistical analysis (H) of CCR2+CD206+ M2 macrophages among CD45+CD64+F4/80+CCR2+ macrophages in the control, CVB3 + vehicle, and CVB3 + formoterol groups at 1 week and 2 weeks post-infection (n = 6/group). I-J Representative flow cytometry images (I) and statistical analysis (J) of IL-10 secretion by CD45+CD64+F4/80+CCR2+ macrophages in the control, CVB3 + vehicle, and CVB3 + formoterol groups at 1 week and 2 weeks post-infection (n = 6/group). K-L Representative flow cytometry images (K) and statistical analysis (L)…

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  Fig. 4. Activating β2-AR attenuates inflammation and fibrosis in VMC via CCR2+ macrophages. A Schematic illustrating macrophage depletion using clodronate liposomes (Lipo-Clod). The mice received intraperitoneal injections of 200 µl clodronate liposomes or PBS-containing liposomes (Lipo-PBS) 2 days before CVB3 infection, followed by injections on days 5 and 10 post-infection. B-C Representative flow cytometry images (B) and statistical analysis (C) of CD11b+Ly6G− monocytes among splenic CD45+ cells in the Vehicle and Lipo-Clod groups at 1 week and 2 weeks post-CVB3 infection (n = 6/group). D Schematic showing CCR2+ monocyte/macrophage inhibition via INCB3344 which was administered from day 1 to day 7 or day 14 post-CVB3 infection. E–F Representative flow cytometry images (E) and statistical analysis (F) of Ly6Chigh monocytes among blood CD45+CD11b+Ly6G− cells in the Vehicle and INCB3344 groups at 1 week and 2 weeks post-CVB3 infection (n = 6/group). G-H Representative H&E staining (G) and Masson’s trichrome staining (H) images of the myocardium from the Vehicle, Lipo-Clod, Formoterol, and Formoterol + Lipo-Clod groups at 1 week and 2 weeks post-CVB3 infection (original magnification, 200…

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  Fig. 5. Effects of activating β2-AR on the metabolic profiles of cardiac tissue from mice with VMC. A-B PCA score plots of cardiac metabolites in negative-ion (A) and positive-ion (B) modes showing separation between the control and VMC groups. C-D Permutation test diagram for metabolite analysis in the control vs. VMC groups (C) and in the VMC vs. formoterol-treated groups (D). E–F Volcano plots showing differential cardiac metabolites between the control and VMC groups (E) and between the VMC and formoterol groups (F). G-H Column charts displaying upregulated (red) and downregulated (green) differentially abundant metabolites in the control group vs. the VMC group (L) and the VMC group vs. the formoterol group (M), shown on a log2 scale. I-J MSEA showing the most altered functional metabolic pathways in the control group vs. the VMC group (R) and VMC vs formoterol group (S). The length of each bar represents fold enrichment, and color intensity (yellow to red) indicates statistical significance. Abbreviations: The data are presented as the means ± SEMs. MSEA, metabolomic set enrichment analysis; PCA, principal components analysis; SEM, standard error of the mean; VMC, viral…

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  Fig. 6. Effects of β2-AR on the metabolic profiles of LPS-induced BMDMs. A Western blot analysis and quantification of β2-AR expression (n = 3/group). B-C PCA score plot of metabolites in the control vs. LPS groups (B) and in the LPS- vs. formoterol-treated groups (C), illustrating metabolic differences between the groups. D-E Permutation test diagrams in the control vs. LPS group (D) and the LPS vs. formoterol group (E). F-G Heatmaps displaying clustering of differentially abundant metabolites between the control and LPS groups (F) and between the LPS and formoterol groups (G). H-I KEGG enrichment plots of differentially abundant metabolites in the control vs. LPS group (H) and the LPS vs. formoterol group (I). The rich factor represents the ratio of differentially abundant metabolites in a given pathway to the total number of metabolites annotated in that pathway, with larger rich factor values indicating greater enrichment. Smaller p-_values indicate more significant enrichment. Abbreviations: The data are presented as the means ± SEMs. BMDMs, bone marrow-derived macrophages; KEGG, Kyoto Encyclopedia of Genes and Genomes; LPS, lipopolysaccharide; PCA, principal components analysis;…

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  Fig. 7. β2-AR regulates energy metabolism, polarization and function of LPS-induced BMDMs through the AMPK pathway. A PCA clustering of the transcriptome data of the control, LPS-, and formoterol-treated groups. B-C Heatmap and volcano plot of differentially expressed genes (DEGs) in the control vs. LPS group (B) and the LPS vs. formoterol group (C). D Venn diagram depicting total genes and the number of DEGs across the groups. E Transcriptome-based expression levels of Adrb2, Cd86, and Mrc1 in the control and LPS groups (n = 4/group). F Correlation analysis of Adrb2 expression with Cd86 and Mrc1 expression. G Statistical plot of the GO secondary annotation classification of DEGs in the control vs. LPS group and the LPS vs formoterol group. H GSEA enrichment plot for the TCA cycle and respiratory electron transport in the control vs. LPS group and for oxidative phosphorylation in the LPS vs. formoterol group. I GSEA enrichment plot for the AMPK signaling pathway in the control vs. LPS group and the LPS vs. formoterol group. J-K Western blot analysis (J) and statistical quantification (K) of p-AMPKα, AMPKα, p-AMPKβ, and AMPKβ levels (n = 3/group). L Real-time qPCR analysis of CD86, CD206,…

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  Fig. 8. Schematic representation of the mechanism by which β2-AR protects against AVM. β2-AR enhances mitochondrial energy metabolism, mitigates mitochondrial injury, and decreases inflammation and fibrosis through the activation of the AMPK pathway. Abbreviations: AMPK, adenosine monophosphate (AMP)-activated protein kinase; AVM, acute viral myocarditis; β2-adrenergic receptor, β2-AR