Administration of USP7 inhibitor P22077 inhibited cardiac hypertrophy and remodeling in Ang II-induced hypertensive mice

Figures

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  FIGURE 1. USP7 was overexpressed in patients with heart failure. (A,B) Immunohistochemical (IHC) staining of USP7 (upper) and BNP (lower) proteins in the heart tissues from normal control and HF patients (left). Scale bars, 50 μm. Quantification of the relative USP7- and BNP-positive areas (right; n = 3). (C) The concentration of USP7 protein level in serum from normal controls (n = 37) and patients with HF (n = 33) was detected by ELISA assay. The data are presented as the mean ± SD, and n represents the number of animals per group. *p < 0.05, and **p < 0.01.

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  FIGURE 2. The expression of USP7 was upregulated in Ang II-induced hypertrophic hearts and myocytes. (A) qPCR analysis of USP7 mRNA expression in Ang II-infused mouse hearts (n = 6). (B) Representative immunoblotting analysis of USP7 protein levels in Ang II (1,000 ng/kg/min) -induced hypertrophic hearts (left). Quantification of the relative USP7 protein level (right; n = 4). (C) Immunohistochemical staining of USP7 in Ang II (1,000 ng/kg/min)-infused mouse hearts (n = 6). Scale bar: 50 μm. (D) Representative immunoblotting analysis of USP7 protein level in NRCMs exposed to Ang II (100 nM) (left). Quantification of the relative USP7 protein level (right; n = 3). The data are presented as the mean ± SD, and n represents the number of animals per group. **p < 0.01.

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  FIGURE 3. Administration of USP7 inhibitor p22077 attenuates Ang II-induced maladaptive cardiac hypertrophy. (A) The basic structure of p22077. (B) Protocol for administration of p22077 in a mouse model of cardiac hypertrophy and remodeling. WT mice were treated with p22077 for 7 days and then infused with Ang II or Saline for additional 14 days. (C) Tail-cuff method measurement of systolic blood pressure (SBP) every 3 days after Ang II infusion (n = 6). (D–F) Representative M-mode echocardiography of LV chamber at day 7 and day 14. Measurement of EF% and FS% (n = 6 mice per group). (G–I) Representative heart sizes from each group. Scale bars, 0.5 cm. The ratios of HW/BW and HW/TL (n = 6 per group). *p < 0.05, and **p < 0.01.

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  FIGURE 4. Administration of p22077 attenuates Ang II-induced cardiac hypertrophy and cardiac fibrosis. (A,B) Histological examination of cardiac hypertrophy by TRITC-WGA staining (Left) and quantification of the relative myocyte cross-sectional area (200 cells counted per heart, Right; n = 6). Scale bars, 20 μm. (C,D) Histological examination of myocardial fibrosis detected by Masson’s trichrome staining (Left) and quantification of the relative fibrosis area (Right; n = 6). Scale bars, 50 μm. (E,F) Representative IHC and quantification of α-SMA-positive area (n = 6 mice per group). Scale bars, 50 μm. (G,H) Representative IHC and quantification of Collagen I-positive area (n = 6 mice per group). Scale bars, 50 μm. (I,J) qPCR analysis of ANP, BNP, collagen I and collagen III mRNA levels in the heart tissues. The data are normalized to the GAPDH expression (n = 6 per group). The data are presented as the mean ± SD, and n represents the number of animals per group. *p < 0.05, and **p < 0.01.

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  FIGURE 5. Administration of p22077 attenuates Ang II-induced inflammation and oxidase stress. (A,B) Immunofluorescent staining of heart sections for examination of F4/80-positive macrophages (green) and nuclei (DAPI, blue) (n = 6). Scale bar = 20 μm. Quantification of F4/80 positive area. (C,D) Representative IHC and quantification of CD68-positive area (n = 6 mice per group). Scale bars, 50 μm. (E,F) Dihydroethidium (DHE) staining of heart sections (left). Quantification of DHE intensity (n = 6). Scale bar = 20 μm. (G,H) qPCR analysis of IL-1β, IL-6, NOX2, and NOX4 in heart tissues (n = 6). The data are presented as the mean ± SD, and n represents the number of animals per group. *p < 0.05, and **p < 0.01.

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  FIGURE 6. Administration of p22077 regulates multiple signaling pathways in Ang II-induced cardiac remodeling. (A) Representative immunoblotting analysis of p-AKT, AKT, p-ERK, ERK, and Tubulin in heart tissues from each group (left). Quantification of the relative protein levels (right, n = 6). (B) Representative immunoblotting analysis of TGF-β1, p-Smad2, Smad2, Collagen I, Collagen III, and Tubulin (left). Quantification of the relative protein levels by densitometry (right, n = 6). (C) Representative immunoblotting analysis of NLRP3, p65, p-p65, and Tubulin (left). Quantification of the relative protein levels (right, n = 6). (D) Representative immunoblotting analysis of NOX2, NOX4, and Tubulin (left). Quantification of the relative protein levels (right, n = 6). The data are presented as the mean ± SD, and n represents the number of animals per group. *p < 0.05, and **p < 0.01.

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  FIGURE 7. A working model for the administration of USP7 inhibitor p22077 to suppress cardiac remodeling in Ang II-induced hypertensive mice. Hypertension induces the expression of USP7. Blocking USP7 activity by p22077 inhibits multiple signaling pathways, which involved in hypertrophy, fibrosis, inflammation, and oxidative stress, thereby attenuates cardiac remodeling in Ang II-induced hypertensive mice.