Selective Inhibition of the Immunoproteasome β5i Prevents PTEN Degradation and Attenuates Cardiac Hypertrophy

Figures

• 

  Figure 1. Effect of PR-957 on Ang II–upregulated proteasome subunit expressions and their corresponding proteasome activity. (A) The structure of PR-957. (B) Changes of proteasome activities in the LV tissues of Ang II–infused mice in the presence or absence of PR-957 for 2 weeks (n = 5). (C) Representative images of proteasome subunit expressions and the quantification of each protein band (n = 5). Data are expressed as mean ± SEM, and n represents the number of animals per group. *P < 0.05, **P < 0.01 vs. saline; #P < 0.05 vs. Ang II.

• 

  Figure 2. Administration of PR-957 reduces Ang II–induced cardiac hypertrophy in vivo. (A) Representative echocardiography images of Ang II–infused mice in the presence or absence of PR-957 for 2 weeks (top). Quantitative analysis of left ventricular (LV) ejection fraction (EF%, bottom, n = 5). (B) Representative images of heart size photographed with a stereomicroscope (top). Scale bar, 200 mm. Ratios of heart weight/body weight (HW/BW) and heart weight/tibial length (HW/TL, bottom, n = 5). (C) LV sections were stained with wheat germ agglutinin (WGA) to demarcate the cell boundaries (left). Scale bar, 100 μm. Quantification of the relative myocyte cross-sectional area (200 cells counted per heart) (right, n = 5). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of ANF and β-MHC (right, n = 5). Data are expressed as mean ± SEM, and n represents the number of animals per group. *P < 0.05, **P < 0.01 vs. saline; #P < 0.05 vs. Ang II.

• 

  Figure 3. Administration of PR-957 attenuates Ang II–induced fibrosis and inflammation in mice. (A) Left ventricular sections were stained with Masson’s trichrome to detect fibrosis (left). Scale bar, 50 μm. Quantification of the relative fibrotic area (right, n = 5). (B) Immunohistochemical staining of cardiac macrophages with anti-F4/80 antibody (left). Scale bar, 50 μm. Quantification of the F4/80 positive cells (right, n = 5). (C, D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of collagen I, collagen III, interleukin (IL)-1β, and IL-6 (n = 5). (D) Data are expressed as mean ± SEM, and n represents the number of animals per group. *P < 0.05, **P < 0.01 vs. saline; #P < 0.05 vs. Ang II ##P < 0.01 vs. Ang II.

• 

  Figure 4. PR-957 inhibits activation of hypertrophic, fibrotic, and inflammatory signaling pathways in mice. (A) Western blot analysis of the protein levels of PTEN, p-AKT, AKT, p-ERK, ERK, TGF-β1, p-p65, p65, p-IkBα, IkBα, and GAPDH in Ang II–infused mice in the presence or absence of PR-957 for 2 weeks. (B) Quantification of each protein band (n = 5). Data are expressed as mean ± SEM, and n represents the number of animals per group. *P < 0.05 vs. saline; #P < 0.05 vs. Ang II.

• 

  Figure 5. Blocking of phosphatase and tensin homolog on chromosome ten (PTEN) activity suppresses PR-957–mediated inhibitory effect on cardiac hypertrophy in mice. (A) The structure of VO-Ohpic. (B) Western blot analysis of PTEN, phospho-AKT, AKT, phospho-mTOR, mTOR, and GAPDH in LV tissues from mice after Ang II infusion in the presence or absence of PR-957 (12 mg/kg BW) together with VO-OHpic (10 mg/kg BW) (left) and quantification of each protein band (left, n = 5). (C) Representative echocardiography images of left ventricular chamber (top), and measurement of left ventricular ejection fraction (LV EF%, bottom). (D) Representative images of heart size (top) and ratios of heart weight/body weight (HW/BW) and heart weight/tibial length (HW/TL) (bottom, n = 5). (E) LV sections were stained with WGA to demarcate cell boundaries (left). Quantification of the relative myocyte cross-sectional area (200 cells counted per heart) (right, n = 5). (F) Quantitative real-time polymerase chain reaction analysis of the mRNA expression of ANF (right, n = 5). Data are expressed as mean ± SEM, and n represents the number of animals per group. *P < 0.05, **P < 0.01 vs. Ang II; #P < 0.05, ##P < 0.01 vs.…

• 

  Figure 6. Working model of PR-957–mediated cardioprotection against Ang II–induced cardiac hypertrophy. Ang II upregulates β5i protein level and its corresponding chymotrypsin-like activity, which in turn promotes degradation of phosphatase and tensin homolog on chromosome ten (PTEN), leading to the activation of AKT/mTOR, TGF-β/Smad2/3, and p-IkBα/NF-KB, thereby resulting in cardiac hypertrophy, fibrosis, and inflammation. Conversely, the inhibition of β5i by PR-957 markedly reverses these effects. Casein Kinase II (CK2); Phosphoinositide 3-kinase (PI3K); protein kinase A (PKA); AMP-responsive element binding protein (CREB); myocyte enhancer factor 2 (MEF2).