(Pro)renin receptor promotes cardiomyocyte senescence via tripartite motif-containing 24-mediated stabilization of p53 in diabetic cardiomyopathy

Figures

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  Figure 1 PRR expression is associated with senescence markers in diabetic hearts (A) Gene set enrichment analysis (GSEA) of cardiac transcriptomic data (GSE26887) showing enrichment of the cellular senescence pathway in diabetic patients. (B) Bubble plot illustrating GSEA enrichment of cellular senescence-related and inflammatory pathways in diabetic hearts. (C,D) SA-β-Gal staining and quantification in myocardial tissues from normal diet (ND) controls and high-fat diet/streptozotocin (HFD/STZ)-induced DCM model mice, scale bar: 100 μm. (E,F) Immunofluorescence detection and quantification of γ-H2AX, scale bar: 10 μm. (G,H) Immunohistochemical staining and quantification of p16, scale bar: 20 μm. (I,J) Immunohistochemical staining and quantification of p21, scale bar: 20 μm. (K,L) Immunohistochemical staining and quantification of PRR expression, scale bar: 20 μm. (M‒P) Correlation analysis between PRR expression and the levels of SA-β-Gal, γ-H2AX, p16, and p21. Data are presented as the mean ± SD (n = 10). ** P < 0.01, *** P < 0.001, unpaired t-test.

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  Figure 2 PRR overexpression promotes cellular senescence in the diabetic myocardium (A,B) SA-β-Gal staining and quantification in myocardial tissues from HFD/STZ-induced DCM mice treated with adenovirus encoding PRR (Ad-PRR) or control EGFP (Ad-EGFP), scale bar: 50 μm. (C–E) Immunohistochemical staining and quantification of p16 and p21, scale bar: 40 μm. (F,G) Immunofluorescence detection and quantification of γ-H2AX in cardiac tissue, scale bar: 10 μm. Data are presented as the mean ± SD (n = 10). *P < 0.05, **P < 0.01, one-way ANOVA.

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  Figure 3 PRR overexpression promotes cardiomyocyte senescence and enhances fibroblast activation under HG/PA conditions (A,B) SA-β-Gal staining and quantification in primary cardiomyocytes treated with low glucose/high osmolarity (LG/HO), high glucose and palmitic acid (HG/PA), HG/PA+Ad-EGFP, or HG/PA+Ad-PRR, scale bar: 20 μm. (C–E) Western blotting and quantification of p16 and p21 expression across treatment groups. (F,G) Immunofluorescence staining and quantification of γ-H2AX, scale bar: 20 μm. (H,I) ELISA measurement of secreted SASP factors (TGF-β1 and TGF-β2) in the supernatants of cultured cardiomyocytes. (J,K) Western blotting and quantification of α-SMA expression in fibroblasts after exposure to conditioned medium (CM) collected from cardiomyocytes. (L,M) Cell proliferation of fibroblasts following CM treatment was assessed via CCK-8 assay, scale bar: 100 μm. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA.

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  Figure 4 PRR overexpression aggravates myocardial fibrosis and impairs cardiac function in DCM mice (A) Masson’s trichrome staining showing fibrotic changes in heart sections, scale bar: 50 μm. (B,C) Quantification of interstitial and perivascular collagen deposition. (D) Immunohistochemical staining of collagen type I (COL I) and collagen type III (COL III) in myocardial tissue, scale bar: 100 μm. (E,F) Quantification of the COL I-positive area and the COL I/III ratio. (G) Representative echocardiographic images acquired by M-mode, PW Doppler, and tissue Doppler imaging. (H,I) Quantification of the left ventricular ejection fraction (LVEF) and fractional shortening (LVFS). (J,K) Assessment of diastolic function on the basis of the E/A and E/e′ ratios. Data are presented as the mean ± SD (n = 10). * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA.

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  Figure 5 PRR promotes cardiomyocyte senescence through the stabilization and activation of p53 (A,B) Western blotting and quantification of p53 protein levels in HG/PA-treated cardiomyocytes overexpressing PRR. (C) CHX chase assay assessing p53 protein stability after PRR overexpression. (D) Quantification of p53 degradation rates. (E,F) Effects of the proteasome inhibitor MG132 and the lysosomal inhibitor bafilomycin A1 on p53 protein levels. (G,H) Western blotting and quantification of p21 expression following the inhibition of p53 transcriptional activity via pifithrin-α (PFT-α). (I,J) ELISA measurement of the concentrations of TGF-β1 and TGF-β2 in the culture supernatants of cardiomyocytes with PRR overexpression or PFT-α. (K,L) SA-β-Gal staining and quantification in treated cardiomyocytes, scale bar: 20 μm. Data are presented as the mean ± SD (n = 3). * P < 0.05, ** P < 0.01, one-way ANOVA.

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  Figure 6 PRR stabilizes p53 by inhibiting TRIM24-mediated ubiquitination and degradation in HG/PA-treated cardiomyocytes (A) Volcano plot of differentially expressed genes (DEGs) from transcriptomic analysis of HG/PA-treated cardiomyocytes with PRR overexpression. (B–D) Heatmaps of DEGs enriched in p53 binding (B) and ubiquitin ligase activity (C) and their overlap (D). (E,F) Western blotting of TRIM24 protein expression in HG/PA-treated cardiomyocytes overexpressing PRR. (G) Coimmunoprecipitation analysis of the interaction between TRIM24 and p53 in PRR-overexpressing cells. (H) Ubiquitination assay evaluating the effect of PRR on TRIM24-mediated p53 ubiquitination. (I,J) Western blotting and quantification of p53 protein levels. (K,L) Western blotting was used to assess the effect of MG132 treatment on TRIM24-mediated p53 degradation. (M,N) SA-β-Gal staining and quantification of positive cardiomyocytes. (O,P) ELISA was used to measure the concentrations of TGF-β1 and TGF-β2 in the culture supernatants. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, one-way ANOVA.