Small‐molecule inhibition of MuRF1 attenuates skeletal muscle atrophy and dysfunction in cardiac cachexia

Figures

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  Figure 1. An overview of the screening process that finally prioritized the compound EMBL ID#704946 in this study (A). An AlphaScreen of 130 000 compounds was first performed, which resulted in 79 hits based on their ability to inhibit the interaction between titin and MuRF1's ‘central domain’ (B–C). These interacting fragments were expressed as GST and His‐tagged fusion proteins so that complex formation could be monitored in an AlphaScreen assay with glutathione donor and Ni‐NTA acceptor beads, respectively (C). Of these 79 compounds, a total of 9 were further synthesized and tested for inhibition towards MuRF1 autoubiquitination (D), with 3 compounds (#704946, #704946‐5, #704946‐8) showing the greatest inhibition (E). These final three compounds were then tested for cytotoxicity in myoblasts (F) and myocytes (G), with increasing concentrations of ID#704946 clearly showing a lower activity in lactate dehydrogenase. Refer to text for further details. Data are presented as mean ± SEM.

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  Figure 2. Expression of MuRF1 at the mRNA level (A) and atrophy (B) was elevated in myotubes following 24 h incubation with dexamethasone (DEX; 10 μmol/L). In contrast, pre‐treatment for 2 h with ID#704946 at 10 μmol/L (but not 0.1 μmol/L) reduced MuRF1 mRNA levels and prevented fibre atrophy. Data are presented as mean ± SEM. *P < 0.05 vs. CON. § P < 0.05 vs. DEX.

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  Figure 3. Physical characteristics of sham and mice treated with monocrotaline fed either normal chow (MCT) or EMBL ID#704946 compound (MCT + 704946). Data confirmed that MCT treatment induced cardiac cachexia independent of the chow administrated, as demonstrated by an impaired weight gain (A), increased pulmonary congestion (B), increased heart weight (C), and right ventricular (RV) hypertrophy (D), the latter visualized by representative haematoxylin and eosin‐stained sections (E). Data are presented as mean ± SEM, with n = 20 (sham), n = 27 (MCT), and n = 27 (MCT + 704946) per group. *P < 0.01 vs. sham.

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  Figure 4. Skeletal muscle wet weights (normalized to tibia length) for the EDL (A), soleus (B), and tibialis anterior (TA) (C). In addition, the fibre cross‐sectional area for the TA muscle is also presented (D) and representative histological sections (E). Data are presented as mean ± SEM, with n = 20 (sham), 27 (MCT), and 27 (MCT + 704946) per group. *P < 0.05 vs. sham; § P < 0.01 vs. sham and MCT + ID#704946.

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  Figure 5. In vitro skeletal muscle contractile function, as assessed during isometric contractions (A) and also isotonic contractions, whereby shortening velocity (B) and power (C) were determined. MCT‐treated mice demonstrated impairments in shortening velocity and power by around 20% compared with shams, but this was essentially prevented in MCT mice fed the ID#704946 compound. Data are presented as mean ± SEM, with n = 16 per group. *P < 0.05 vs. sham; § P < 0.01 vs. sham and MCT + ID#704946.

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  Figure 6. The proteomes expressed in TA muscle samples were analysed using mass spectrometry to identify differentially expressed protein in the different groups (n = 3 per group). (A, B) Differentially expressed proteins found in MCT/sham mice (A) and in MCT + ID#704946/MCT mice (B) are shown by volcano plots. (C) The set of 51 differentially expressed proteins from group A and of 70 proteins from group B share a group of 5 proteins that are differentially expressed in both sets with high significance. We therefore consider them as candidates to be modulated by the ID#704946 compound during MCT stress: These five proteins are detailed in the table (D). Of these, two proteins were further investigated by western blot (n = 9 per group) including eIF2B subunit‐delta (E) and BAX (F), with representative blots also presented (G). Data are presented as mean ± SEM. *P < 0.05 vs. sham; § P < 0.01 vs. MCT.

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  Figure 7. Protein expression and representative blots for MuRF1 (A), MAFBx (B), and CARP (C) for sham, MCT, and MCT + ID#704946 mice. Our data revealed that MCT treatment leads to an increase in MuRF1 and CARP expression, but this was attenuated in mice fed the #704946 compound. No changes were detected in the levels of MAFBx. Data are presented as mean ± SEM, with n = 12–16 per group. *P < 0.05 vs. sham and MCT + ID#704946.

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  Figure 8. Protein expression for the contractile proteins myosin heavy (A) and light chains (B), sarcomeric actin (C), as well as ubiquitinated actin (D), with the respective representative blots also presented (E) for sham, MCT, and MCT + ID#704946 mice. In addition, proteasome activity was also measured in each group (F). Data are presented as mean ± SEM, with n = 12–16 per group. *P < 0.05 vs. sham and MCT + ID#704946.