Ro5‐4864, a ligand of the mitochondrial translocator protein, protects against heart failure in mice via regulation of the p62‐Keap1‐Nrf2 axis

Figures

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  Figure 1. Echocardiographic parameters at baseline and after 8 weeks of TAC or Sham A, representative M‐mode echoes in Sham‐Saline (n = 12), Sham‐Ro5 (n = 5), TAC‐Saline (n = 9) and TAC‐Ro5 (n = 12) mice; fractional shortening (B), left ventricular (LV) mass, corrected for body weight (C) and LV volume (D) derived from echocardiography in the four groups. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

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  Figure 2. Ro‐5 decreases TSPO expression in TAC‐induced heart failure A, heatmaps of the proteome from four experimental groups. B, volcano plots of TAC‐Saline and TAC‐Ro5 animals demonstrating the downward shift in protein expression in TAC‐Ro5 animals, including TSPO. C, protein expression by LC/MS of TSPO in the four groups. Data are expressed as mean ± SD. * P ≤ 0.05, ** P ≤ 0.005, **** P ≤0.0001.

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  Figure 3. Ro‐5 decreases markers of heart failure and cardiac fibrosis (A) proteomic analysis (AU) of natriuretic peptide A expression in heart lysate from sham and TAC mice treated with saline or Ro5‐4864 (n = 5 per group). B, AU of collagen alpha‐1 (XII) expression in heart lysate from sham and TAC mice treated with saline or Ro5‐4864 (n = 5 per group). C, D, western blot quantification of periostin (POSTn), a marker of fibrosis, in heart lysate from sham and TAC mice treated with saline or Ro5‐4864 (n = 6 in each group). Data are expressed as mean ± SD. * P ≤0.05, ** P ≤0.005, *** P ≤0.001.

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  Figure 4. Effect of Ro5 on immune system and inflammation Graphic representation of proteomic pathways which were modified in TAC‐Ro5 versus TAC‐saline hearts using liquid chromatography/mass spectrometry (n = 5 per group). Pathways are listed on the left, the –logP value is on the X‐axis and the number of proteins is represented by the size of the circle. As noted, the most significant changes were in pathways involving inflammation and the immune system.

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  Figure 5. Glutathione system expression Quantified proteomic analysis (AU) of glutathione reductase (A), glutathione synthetase (B), glutathione peroxidase 1 (C), thioredoxin reductase 1 (D), immunoglobulin heavy constant 2b (E) and immunoglobulin κ (F) expression in heart lysate from sham and TAC mice treated with saline or Ro5‐4864 (n = 5 per group). Data are expressed as mean ± SD. * P ≤0.05, ** P ≤0.005, *** P ≤0.001, **** P≤ 0.0001.

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  Figure 6. Effects of Ro5 on Nrf2 signalling pathway in TAC‐induced heart failure A, representative western blots of Nrf2, Keap1, P62 and GAPDH in saline and Ro5‐treated hearts under sham and TAC conditions. B, representative western blots of NQO1 and α‐tubulin. C, changes in protein expression of Nrf2, Keap‐1, p62 normalized to GAPDH expression in heart tissue lysates from all groups (n = 4–5 per group). NQO1 signal was normalized to the intensity of α‐tubulin (used instead of GAPDH as the MW of NQO1 and GAPDH are close). Data are expressed as mean ± SD. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.001, **** P ≤ 0.0001.

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  Figure 7. Markers of oxidative stress A, MitoSOX Mitochondrial Superoxide Indicator intensity of in vitro murine cardiomyocytes treated with H2O2 ± Ro5. Untreated control n = 25, Ro5‐treated group n = 67, H2O2‐treated group n = 21, H2O2+Ro5 treated group n = 29. B, left mean fluorescent intensity of in vitro murine cardiomyocytes treated with H2O2 ± Ro5 using the indicator DCF (n = 16 H2O2, n = 33 H2O2 + Ro5). C, superoxide dismutase 2 (SOD2) expression in the hearts from saline and Ro5‐treated mice under sham and TAC conditions. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.005, **** P < 0.0001.