Rel+ macrophages disturbing cardiac niche contributes to cardiac dysfunction following sepsis

Figures

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  Fig. 1. Non-contractile cardiomyocytes were identified as the main myocytes subpopulations in heart after sepsis. A, Uniform Manifold Approximation and Projection (UMAP) embedding of five cardiomyocyte subpopulations based on snRNA-seq data from ventricular tissue of normal and septic mice, colored by identified celltype (n = 3 mice per group). B, Abundance analyses of all cardiomyocyte subpopulations in the normal and septic mice. C, Heatmap showing the molecular signature and top 5 enriched GO pathways of each cardiomyocyte subpopulation. D, Violin plot showing the enrichment scores of contraction-related gene in each cardiomyocyte subpopulation. Scores were evaluated by the AddModuleScore algorithm. The analysis utilized a gene set of 230 contraction-related genes, which was curated from published literature and MSigDB database. E, Proportion of contractile cardiomyocytes (contractile CM, red) and non-contractile cardiomyocytes (non-contractile CM, Ankrd1+, green) in the normal and sepsis group. F, Staining of TNNT2 (contractile CM marker) in heart sections from normal and septic mice. Scale bar, 50 μm(n = 6 mice per group). G, Representative sarcomere shortening images of single…

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  Fig. 2. Transcription factor Egr1 plays an important role in the transition of cardiomyocytes from a contractile to a non-contractile phenotype after sepsis. A, Hierarchical clustering of the subpopulations. B, Cellular trajectory reconstructed for the transition towards non-contractile cardiomyocytes using monolce2. The arrow shows the direction of cellular state changes. C, Density curves showing the distributions of the four subpopulations along the trajectory. D, Density curves showing the distributions of the cardiomyocytes from two different conditions along the trajectory. E, Heatmaps showing the expression dynamics of the genes with significantly different patterns along the trajectory. F, GeneSwithes algorithm identifies key genes and transcription factors (TFs) along the trajectory. G, The expressions of representative candidate genes along the trajectory. H, The top enriched pathways along the trajectory. Green, down-regulated pathways. Orange, up-regulated pathways. I, Regulatory networks of key genes and TFs. J, The expression of Egr1 in different cell fate and conditions. K, Western blot analysis was performed to assess the interference efficiency of si-Egr1 and Egr1…

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  Fig. 3. Rel+ resident macrophage (Rel+ Mac) played a key role in the cardiomyocyte contractile phenotype switching after sepsis by metabolic reprogramming. A, UMAP embedding of seven monocyte/macrophage subpopulations. B, Dot plot illustrating the expression patterns of selected marker genes of each subpopulation. According to the expression of Ccr2, cardiac resident macrophage and monocyte-derived macrophage were identified. C, Heatmap showing the molecular signature and top 5 enriched GO pathways of each subpopulation. D, Relative proportion of each subpopulation in the monocyte/macrophage from each condition. E, Immunofluorescence staining confirmed the presence of the Mac2 (FPR1+) in cardiac tissues from sepsis. F, Subpopulation-specific transcription factors in each cell type. Scar bar = 50 μm. G, Differential metabolic pathways between Mac2 and other Macs. H, The expressions of glycolysis-related genes in Mac2 and other Macs. I, The ligand-receptor pairs for Rel+ Mac/non-contractile CM interactions. J, The pathways of respective target genes regulated by predicted ligands. K, Workflow for generating a cardiac resident macrophage-specific PKM2 knockout (PKM2−/−) mouse model. L,…

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  Fig. 4. Venous ECs and pericytes were associated with the occurrence of vascular leakage following sepsis. A, UMAP embedding of six endothelial cells subpopulations. B, Relative proportion of each subpopulation in endothelial cells from each condition. C, The expressions of the vascular permeability markers Plvap and Aqp1 in arterial EC, venous EC and capillary EC in the different conditions. D, The expressions of selected genes associated with vascular permeability in venous ECs in the different conditions. E, The cellular interactions in EC, SMC and PC. F, The differential number of interactions among EC, SMC and PC between normal and sepsis group. Red line, increased. Blue line, decreased. G, Cell-cell junction scores between vascular cell cluster. H, The down-regulated ligand-receptor pairs between PC and EC cluster in sepsis compared to normal group. I, The down-regulated ligand-receptor pairs between SMC, PC and EC cluster in sepsis compared to normal group

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  Fig. 5. Rel+ Mac interaction with EC and SMC contributed to the disturbance of microcirculation following sepsis (correlative observations requiring further validation). A, The incoming and outgoing interactions strength between vascular cells and macrophages in the different conditions. B, Dot plots of the cell-cell communication between vascular cells and macrophages. C, The selected ligand-receptor pairs between Venous EC or SMC2 and macrophages. D, Heatmap showing the COLLAGEN signaling network between the macrophages and vascular cells. G, The ligand-receptor pairs of COLLAGEN signaling between macrophages and vascular cells

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  Fig. 6. Rel+ Mac triggering pro-inflammatory transformation of fibroblasts FB3 played an important role in matrix remodeling and cardiac dysfunction in sepsis (correlative observations requiring further validation). A, UMAP embedding of four fibroblast subpopulations. B, Dotplot showing the molecular signature of each subpopulation. C, Relative proportion of each subpopulation in fibroblast of each condition. D, The top enriched pathways in FB3 from septic heart. E, Module scores of inflammatory in each fibroblast clusters at the different conditions. F, The expression of Lcn2 in each fibroblast clusters at the different conditions. G, The predicted genes interacted with Lcn2. H, The expression of Lcn2-targeted genes in FB3 at the different conditions. I, Immunofluorescence staining confirmed the co-location between Lcn2 and Mmp2 in FB3 from septic heart. J, Dot plots of the cell-cell communication between fibroblasts and macrophages. K, Heatmap showing the TNF and VCAM signaling network between macrophages and fibroblasts. L, The ligand-receptor pairs of VCAM1 and TNF signaling between macrophages and fibroblasts. M, The selected ligand-receptor pairs between macrophages and…

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  Fig. 7. Nanoparticle targeting Rel+ Mac metabolic reprogramming antagonized sepsis-induced cardiac dysfunction. A, Schematic diagram of lipid nanoparticle. B, Size distribution of lipid nanoparticle measured by phase analysis light scatting (PALS). C, Representative TEM images of PKMsi@NPs. D, Representative confocal images of PKMsi@NPs co-incubated with macrophages at different times. E, Effect of PKMsi@NPs on cell viability. n = 3 independent experiments. Scale bar, 50 μm. F, Immunofluorescence shows colocalization of macrophages (Cx3cr1) with PKMsi@NPs in cardiac tissues from Cx3cr1Cre;R26tdTomato mice. White stars indicate resident macrophages contains PKMsi@NPs. Scale bar, 50 μm. G, Representative echocardiography images of mice. Cardiac EF of sepsis mice after being treated with PKMsi@NPs (n = 8 mice each group). H, The levels of cTnI in the serum of sepsis mice after being treated with PKMsi@NPs (n = 8 mice each group). I, HE staining of heart tissues after injection of PKMsi@NPs (Bar = 50 μm) J, Microscopic injury score of heart tissue for all experimental groups (n = 6 mice each group). K, Survival rate of sepsis mice after treatment with PKMsi@NPs (n = 16 each group). Data were…

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  Fig. 8. Schematic illustration of Rel+ Mac disturbing cardiac niche contributes to cardiac dysfunction following sepsis. The noncontractile phenotype of cardiomyocytes was major type following sepsis. Rel+ Mac metabolic reprogramming with high glycolysis signatures and established a pro-inflammatory niche in septic heart. Furthermore, Rel+ Mac contributed to cardiomyocyte contractile phenotype switching after sepsis by ITGB1, ITGA9, LAMA2, ITGA4, IL6, TNF, VCAM1 and MMP13 signal axis. At same time, Rel+ Mac contributed to vascular leakage-associated venous endothelial cells and pericytes, vascular hypo-responsiveness associated smooth muscle cells, and cardiac matrix remodeling fibroblasts subpopulation FB3, which synergistic led to cardiac dysfunction after sepsis. Created with https://biorender.com