Involvement of plasminogen activator inhibitor-1 in p300/p53-mediated age-related atrial fibrosis

Figures

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  Figure 1. The connections among SERPINE1, EP300, CDKN1A, and TP53 in senescent atrial fibroblasts and patients with AF. (A) Heatmap of standardized and normalized initial RNA-seq data. (B) Heatmap displaying the expression profiles of SERPINE1, CDKN1A, TP53, and EP300 in the P4 (young) and P14 (senescent) human atrial fibroblasts. (C) Violin plots and box plots illustrating the differences in gene expression between the P4 (young) and P14 (senescent) human atrial fibroblasts. (D) GSEA identifying the potential biological functions of SERPINE1. (E–G) Heatmaps showing significantly enriched genes in the regulation of adherens junction, p53 signaling pathway, and TGF-β signaling pathway terms. (H) Heatmap presenting the expression profiles of SERPINE1, CDKN1A, TP53, and EP300 in the SR and AF groups. (I) Violin plots and box plots displaying the differences in gene expression between the SR and AF groups. (J) ROC analysis of individual SERPINE1, EP300, CDKN1A, and TP53 in merged datasets, with the x-axis representing sensitivity and the y-axis representing 1-specificity. The classification performance is represented by the AUC of the ROC. (K) Correlation analysis of the relevant genes…

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  Figure 2. Increased p53/p21 and PAI-1 were regulated by p300 in senescent human atrial fibroblasts. (A) Representative SA-β-gal staining and positive cells analysis of young (P3) and senescent (P11) human atrial fibroblasts. Scale bars, 100 µm. (B) Representative immunofluorescence staining and fluorescence intensity of Col1A1 in young and senescent human atrial fibroblasts. Scale bars, 50 µm. (C–D) Representative immunoblots and densitometric analysis of p300, p53, p21 and PAI-1 in young (P3) and senescent human atrial fibroblasts (P11) treated with curcumin (6, 9, or 12 µM) or C646 (5, 10, 15, or 25 µM). ∗P < 0.05, ∗∗P < 0.01; Data are mean ± SEM.

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  Figure 3. P300/p53 mediated the regulation of PAI-1 expression in senescent human atrial fibroblasts. (A) Representative SA-β-gal staining and positive cells analysis of senescent human atrial fibroblasts (P11) treated with or without p300shRNA. Scale bars, 100 µm. (B). Representative immunoblots and densitometric analysis of p300/p53/p21 and PAI-1 in senescent human atrial fibroblasts (P11) treated with p300 shRNA. (C) Representative immunoblots and densitometric analysis of p53/p21 and PAI-1 in senescent human atrial fibroblasts (P11) treated with p53 siRNA. ∗P < 0.05, ∗∗P < 0.01; data are mean ± SEM.

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  Figure 4. P300/p53 also regulated PAI expression in senescent mouse atrial fibroblasts. (A) Representative SA-β-gal staining and positive cells analysis of young (P3) and senescent (P11) mouse atrial fibroblasts. Scale bars, 100 µm. (B). Representative immunoblots and densitometric analysis of p300, p53 and PAI-1 in young (P3) and senescent mouse atrial fibroblasts (P11) treated with curcumin (6, 9, or 12 µM). (C) Representative SA- β-gal staining and positive cells analysis of senescent mouse atrial fibroblasts (P11) treated with or without p300siRNA. Scale bars, 100 µm. (D–E) Representative immunoblots and densitometric analysis of p300/p53/p21 and PAI-1 in senescent mouse atrial fibroblasts (P11) treated with p300 siRNA or p53 siRNA. ∗P < 0.05, ∗∗P < 0.01; data are mean ± SEM.

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  Figure 5. The activation of p300/p53/PAI-1 pathway contributed to the increased AF susceptibility. (A) Typical surface electrocardiogram (ECG) showing that AF was successfully induced by atrial burst stimulation in mice. And the inducible rate of AF in young and aged mice treated with different dosage of curcumin. n = 7–8. ∗P < 0.05 vs. 5 m mice, #P < 0.05 vs. 18 m mice. (B) Representative Masson staining of atrial specimens obtained from young (5 m) and aged (18 m) mice treated with curcumin (100 mg/kg/d). Scale bars, 50 µm. (C) Representative SA-β-gal staining of atrial tissue of young (5 m) and aged (18 m) mice treated with or without 100 mg/kg/d curcumin. Scale bars, 100 µm. (D) Representative immunoblots and densitometric analysis of p300, p53/ p21 and PAI-1 in in atrial tissues from young (5 m) and aged (18 m) mice (n = 6). (E–F) Representative immunoblots and densitometric analysis of p300, p53/p21 and PAI-1 in in atrial tissues from aged mice treated with different doses of curcumin (50 or 100 mg/kg/d) (n = 6) and from young (7 m) and aged (18 m) p300 (+/+) (wt) and p300 (−/+) (het) mice (n = 6). Data are mean ± SEM. ∗P < 0.05, ∗∗P < 0.01.