Rapamycin preserves cardiac function in autoimmune myocarditis by reprogramming Cxcl9+ macrophages via the mTORC1–C/EBPβ–OSM axis

Figures

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  Fig. 1. Rapamycin preserves cardiac function and mitigates myocardial inflammation and fibrosis in EAM. A, Schematic of the experimental design. Male BALB/c mice received subcutaneous (s.c.) injections of α-myosin heavy chain (α-MyHC) emulsified in complete Freund's adjuvant (CFA) on days 0 and 7 to induce experimental autoimmune myocarditis (EAM). Rapamycin (1 mg kg−1, intraperitoneally [i.p.], every 2 days) or vehicle was administered from day 8 to day 21. Four groups were studied: Control + Vehicle, Control + Rapamycin, EAM + Vehicle, and EAM + Rapamycin. B, Representative M-mode echocardiographic images of each group. C, Quantification of EF, FS, SV, and CO (n = 5, 5, 10, and 10 for Control + Vehicle, Control + Rapamycin, EAM + Vehicle, and EAM + Rapamycin, respectively). D, Hemodynamic assessment of left-ventricular contractility showing peak rates of pressure increase (dP/dtmax) and decline (dP/dtmin) measured by Millar catheterization (n = 5, 5, 10, and 10 per group as in C). E, Representative H&E staining and quantitative analysis of heart sections illustrating inflammatory cell infiltration (n = 5, 5, 10, and 10 per group); Scale bars: 1 mm in low-magnification overview images…

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  Fig. 2. Rapamycin reprograms cardiac monocyte–macrophage states in EAM. A, Workflow for scRNA-seq of cardiac CD45+ immune cells from Control (Con), Rapamycin (Rap), EAM, and EAM + Rap groups, including tissue digestion, single-cell isolation, magnetic-activated cell sorting, library preparation, and data analysis. B, UMAP visualization of major cardiac immune compartments identified by unsupervised clustering and annotation. C, UMAP plots showing distribution of cells from each experimental group. D, Stacked bar charts showing the proportional composition of immune/stromal subsets among groups. E, Heatmap of odds ratios depicting enrichment of each subset across groups. F, GO enrichment of differentially expressed genes in the monocyte–macrophages (Mono_Macro) compartment comparing EAM and EAM + Rap. G, Gene set scoring of glycolysis, oxidative phosphorylation, inflammatory response, and senescence signatures in the myeloid compartment across groups. H–I, immunoblotting analysis of isolated cardiac macrophages showing phosphorylation of mTOR and S6 (H), and levels of HK2, NF-κB1, CDKN1A/p21, and mitochondrial complex I subunit mt-ND4 (I); β-actin serves as loading control. J, Seahorse…

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  Fig. 3. Rapamycin targets a pathogenic Cxcl9+ macrophage subset and reshapes inflammatory–metabolic states in EAM. A, UMAP visualization of reclustered monocyte–macrophage populations from cardiac single-cell RNA-seq, identifying Lyve1+, Plac8+, Cxcl9+, Spp1+, Top2a+, and Cd209a+ subsets. B, UMAP plots showing distribution of monocyte–macrophage subsets across experimental groups [Control (Con), Rapamycin (Rap), EAM, and EAM + Rap]. C, Proportional changes of monocyte–macrophage subsets among groups. D, GO enrichment analysis of biological processes associated with each macrophage subset. E, Heatmap showing odds ratios of enrichment of macrophage subsets across experimental groups. F, AUCell-based pathway scoring illustrating glycolysis, inflammatory, OXPHOS, and senescence signatures across macrophage subsets. G, UMAP feature plots displaying the spatial distribution and activity scores (AUC) of glycolysis, inflammatory, OXPHOS, and senescence pathways across macrophage subsets. H, Quantitative comparison of pathway activity scores in Cxcl9+ macrophages among Con, Rap, EAM, and EAM + Rap groups. I, Representative immunofluorescence staining of F4/80 and CXCL9 in cardiac tissue sections…

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  Fig. 4. Rapamycin reshapes the differentiation landscape and transcriptional control of pathogenic Cxcl9+ macrophages via the mTORC1–C/EBPβ axis. A, UMAP visualization of reclustered cardiac monocyte–macrophages with subset annotation, including Plac8+ monocytes, Spp1+ macrophages, and Cxcl9+ macrophages; black line indicates the Monocle3-inferred trajectory and white circle denotes the root. B, UMAP plot of the same embedding colored by pseudotime. C, Heatmaps showing representative gene expression dynamics along pseudotime for the Plac8+ monocyte and Cxcl9+ macrophage branches. D, GO enrichment analysis of pseudotime-associated genes along the Plac8+ to Cxcl9+ branch. E, Heatmap depicting regulon activity scores of selected transcriptional regulators identified along macrophage differentiation trajectories. F, Binary heatmap showing SCENIC-inferred regulon activation across macrophage subsets, with cell type and experimental group annotations. G, Venn diagram illustrating the overlap between pseudotime-dependent DEGs and key regulons identified in Cxcl9+ macrophages. H, Heatmap displaying regulon activity of selected transcriptional regulators across experimental groups. I, Specificity…

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  Fig. 5. Macrophage–cardiomyocyte crosstalk via OSM and its modulation by rapamycin. A, Volcano plot of DEGs in Langendorff-isolated cardiomyocytes comparing EAM + rapamycin versus EAM. B, GO enrichment of up- and down-regulated DEGs in cardiomyocytes. C, GSEA running-score plots for oxidative phosphorylation, striated muscle/cardiac muscle development, and regulation of immune response in cardiomyocytes. D, NicheNet-based macrophage to cardiomyocyte communication. Left, ranked ligand activity for macrophage-derived ligands; right, heatmap of predicted regulatory potential of ligand-specific target genes in cardiomyocytes. E, Chord diagram visualizing predicted interactions between macrophage subsets and cardiomyocyte targets. F, Violin plots showing Osm expression in Cxcl9+ macrophages across groups. G, Validation of OSM in BMDMs. Left, Osm mRNA expression measured by RT-qPCR in control and Cebpb-overexpressing (Cebpb-OE) BMDMs treated with vehicle or rapamycin. Right, OSM protein levels in BMDM culture supernatants quantified by ELISA under the same conditions (n = 6 per group). H, Schematic illustration of the conditioned medium (CM) co-culture system showing experimental design: BMDMs…

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  Fig. 6. Therapeutic neutralization of OSM in vivo improves cardiac structure and function in EAM. A, Schematic of the experimental design. Male BALB/c mice (6–8 weeks) were immunized with α-MyHC/CFA on days 0 and 7 to induce EAM. From day 9 to day 21, mice received intraperitoneal OSM-neutralizing antibody (OSM nAb, 2 mg kg−1, every other day) or isotype control; hearts and plasma were collected on day 21. B, Representative M-mode echocardiograms in control and EAM mice treated with isotype control or OSM nAb. C, Echocardiographic quantification of systolic function: EF, SV, CO, and FS (n = 8/group). D, Hemodynamic assessment by Millar catheterization showing peak instantaneous rates of LV pressure rise (dP/dtmax) and decline (dP/dtmin) (n = 8/group). E, Representative H&E staining of short-axis sections and higher-magnification insets (left) and quantitative inflammatory score (right) (n = 8/group). Scale bars, 1 mm (whole section) and 100 μm (insets). F, Representative Sirius Red staining (left) and quantification of collagen deposition (right) in the indicated groups (n = 8/group). Scale bars, 1 mm (whole section) and 100 μm (insets). G, Heatmap of cardiac mRNA expression (detected by…