SIRT1 mediates the inhibitory effect of Dapagliflozin on EndMT by inhibiting the acetylation of endothelium Notch1

Figures

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  Fig. 1. DAPA ameliorates ISO-induced myocardial perivascular fibrosis and cardiac dysfunction by targeting endothelial cells. A Images of transthoracic echocardiograms in M-mode (n = 6). B, H&E staining of the lengthwise section of the heart. Scale bar = 100 μm. C Representative images showing Masson staining of perivascular fibrosis in heart sections. Scale bar = 100 μm. D Representative images of perivascular fibrosis stained with PSR, and red represents collagen fiber deposition. Scale bars = 100 μm. E tSNE plots and UMAP plots. Each point depicts a single cell, colored according to cluster designation. F The proportion of the corresponding cell types. G Cell proportion of 2 sample sources in each cell type. H Rose diagrams showing the differentially expressed genes (DEGs) numbers after HF in each cell type. I Top 10 of GO terms and pathways enriched in DEGs of HF and Normal. J Circle plot showing the total intercellular signaling network in the HF group. Blue indicates weakening, while red indicates strengthening after HF. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. Vehicle group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 vs. the ISO group

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  Fig. 2. DAPA ameliorates ISO-induced myocardial perivascular fibrosis and cardiac dysfunction by impairing EndMT. A Results for the GSEA enrichment of DEGs. B Enrichment analysis of biological processes involved in the regulation of EMT or EndMT-related pathways. C Enrichment analysis of KEGG signaling pathways involved in regulating EMT or EndMT-related pathways. D Enrichment of EMT or EndMT marker genes. E The Western blotting analysis of EndMT-related proteins and quantitative analysis (n = 3). F The Western blotting analysis of SIRT1 and quantitative analysis (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. Vehicle group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 vs. the ISO group

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  Fig. 3. DAPA inhibits TGF-β1-induced EndMT. A Western blotting analysis of HUVECs and MAECs treated with TGF-β1 for 48h and the quantitative results. B A representative Western blotting and quantitative analysis of EndMT markers in HUVECs and MAECs treated with DAPA for 48h with or without TGF-β1. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. control group; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. TGF-β1 group

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  Fig. 4. SIRT1 is involved in DAPA-mediated protection of endothelial cells in EndMT. A Western blotting analysis of EndMT markers and SIRT1 in HUVECs and MAECs treated as indicated (The statistical results are presented in Additional file 1: Figure S3A). B Representative images of the EdU proliferation assay of HUVECs and MAECs treated as indicated. Red fluorescence indicates proliferating cells (the statistical results are presented in Additional file 1: Figure S3B). Scale bars = 100 μm. C Representative images of the Transwell assay of HUVECs and MAECs treated as indicated. The Figure indicates the number of migrated cells. Scale bars = 100 μm. D Immunofluorescence analysis of CD31 (green) and α-SMA (red) double-stained HUVECs and MAECs, and the nuclei were stained with DAPI (blue). Scale bars = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. control group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 vs. the TGF-β1 group

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  Fig. 5. DAPA inhibits EndMT in ISO-induced mice by activating SIRT1. A Transthoracic echocardiogram in M-mode (n = 6). B H&E staining of the lengthwise section of the heart. Scale bar = 50 μm. C Representative images of Masson staining showing perivascular fibrosis in heart sections. Scale bar = 50 μm. D Representative images of PSR staining showing perivascular fibrosis in heart sections. Red represents collagen fiber deposition. Scale bars = 50 μm. E Representative IHC analysis for CD31 and α-SMA in the vascular wall in ventricular transverse sections from different groups of mice. Scale bars = 50 μm. F Western blotting analysis of EndMT-related proteins and SIRT1 in the mouse myocardium (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. Vehicle group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 vs. the ISO group

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  Fig. 6. DAPA inhibits SIRT1 ubiquitination and degradation by inhibiting TGF-β1-induced SIRT1 phosphorylation. A The positions of phosphorylated residues on human and mouse SIRT1 were marked. B Shown is a sequence analysis of human and mouse SIRT1. The serine position is indicated by the number. C Representative Western blotting showing SIRT1 expression in vivo and in vitro treated as indicated and quantified. D SIRT1 immunoprecipitation reaction was performed in endothelial cells and subjected to Western blot analysis for detecting SIRT1 ubiquitination. E The effects of MG132 on SIRT1 protein expression upon TGF-β1. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05 vs. the TGF-β1 group

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  Fig. 7. DAPA promotes nucleus translocation of SIRT1. A The overall binding view and local binding view of the SIRT1-DAPA complex obtained based on docking; the yellow dotted line represents the hydrogen bond, the green line represents the amino acid forming a hydrogen bond with the small molecule DAPA in the protein binding pocket, the cartoon represents the SIRT1 protein, and the purple stick is DAPA. B Chemical structure of DAPA and biotin-labeled DAPA (Bio-DAPA). The eluted product was obtained using a biotinylated protein interaction pull-down kit and further analyzed by Western blotting experiments. Total lysates were used as an input control. C CETSA between DAPA and SIRT1. The curve was fitted using GraphPad Prism 9.5.1. D Nucleus-cytoplasm distribution of SIRT1 in cells treated with TGF-β1and DAPA. E Immunofluorescent staining of SIRT1 in HUVECs and MAECs treated as indicated. Scale bars = 50 μm. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control group; #p < 0.05, and ##p < 0.01vs. the TGF-β1 group

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  Fig. 8. DAPA inhibits EndMT by regulating the Notch1 signaling pathway. A SIRT1 is implicated in the regulation of KEGG signaling pathways (Show only the top ten). B Representative Western blotting and quantitative analysis of NICD in HUVECs and MAECs treated with TGF-β1 for 48 h. C Representative Western blotting and quantitative analysis of NICD in HUVECs and MAECs treated with different concentrations of DAPA for 48h in the presence of TGF-β1. D Representative Western blotting showing NICD expression in vivo treated as indicated and quantified. E The binding conformation of SIRT1 and activated Notch1. The left image is the cartoon, the right image is the surface, and the yellow area is the binding region. F Co-IP of NICD and SIRT1. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. control group; #p < 0.05, ##p < 0.01 vs. the TGF-β1 group

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  Fig. 9. DAPA targets SIRT1 to deacetylate NICD and promote its proteolysis. A Immunoblot analysis of total acetylation level. B PPI networks of SIRT1. C NICD acetylation level was measured by IP using an anti-acetylation antibody and Western blotting. D The effects of MG132 on NICD protein expression upon DAPA. E Western blotting analysis of NICD expression levels in the nucleus and cytoplasm of HUVECs and MAECs treated as indicated and quantified. F Immunofluorescent staining of SIRT1 in HUVECs and MAECs treated as indicated. Scale bars = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. control group; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 vs. the TGF-β1 group