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. 2020 Jan 20;10(1):694.
doi: 10.1038/s41598-020-57714-3.

TAK-242, a specific inhibitor of Toll-like receptor 4 signalling, prevents endotoxemia-induced skeletal muscle wasting in mice

Yuko Ono  1   2 , Yuko Maejima  3 , Masafumi Saito  4 , Kazuho Sakamoto  5 , Shoichiro Horita  3 , Kenju Shimomura  3 , Shigeaki Inoue  4 , Joji Kotani  4
Affiliations

TAK-242, a specific inhibitor of Toll-like receptor 4 signalling, prevents endotoxemia-induced skeletal muscle wasting in mice

Yuko Ono et al. Sci Rep. .

Abstract

Circulating lipopolysaccharide (LPS) concentrations are often elevated in patients with sepsis or various endogenous diseases related to bacterial translocation from the gut. Systemic inflammatory responses induced by endotoxemia induce severe involuntary loss of skeletal muscle, termed muscle wasting, which adversely affects the survival and functional outcomes of these patients. Currently, no drugs are available for the treatment of endotoxemia-induced skeletal muscle wasting. Here, we tested the effects of TAK-242, a Toll-like receptor 4 (TLR4)-specific signalling inhibitor, on myotube atrophy in vitro and muscle wasting in vivo induced by endotoxin. LPS treatment of murine C2C12 myotubes induced an inflammatory response (increased nuclear factor-κB activity and interleukin-6 and tumour necrosis factor-α expression) and activated the ubiquitin-proteasome and autophagy proteolytic pathways (increased atrogin-1/MAFbx, MuRF1, and LC-II expression), resulting in myotube atrophy. In mice, LPS injection increased the same inflammatory and proteolytic pathways in skeletal muscle and induced atrophy, resulting in reduced grip strength. Notably, pretreatment of cells or mice with TAK-242 reduced or reversed all the detrimental effects of LPS in vitro and in vivo. Collectively, our results indicate that pharmacological inhibition of TLR4 signalling may be a novel therapeutic intervention for endotoxemia-induced muscle wasting.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
TAK-242 reduces LPS-induced atrophy of C2C12 myotubes. (A,B) Western blot analysis (A) and quantification (B) of MyHC expression in C2C12 myotubes treated for 48 h with vehicle, LPS (1 μg/mL), or LPS and TAK-242 (1 μM). Data in (B) were normalised to β-tubulin protein levels, and the ratio in vehicle-treated control cells was set to 1. N = 5/group. Full-length blots are presented in Supplementary Figure S4. (C) Representative immunofluorescence staining of MyHC in C2C12 myotubes treated as described for (A,B). Scale bar = 100 μm. (D–F) Distribution of myotube widths (D), mean myotube width (E), and fusion index (F) of C2C12 cells treated as described in (A,B). Myotube width was measured as described in the Methods. N = 97–114. The fusion index was calculated from five randomly selected fields as described in the Methods. For all panels, data are presented as the mean ± s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 vs vehicle control; ###p < 0.001, ##p < 0.01 vs LPS-treated group. P-values were derived from one-way ANOVA followed by Tukey’s honest significant difference test or Kruskal-Wallis test followed by Dunn’s post hoc tests with Bonferroni correction.
Figure 2
Figure 2
TAK-242 suppresses LPS-induced activation of inflammatory and proteolytic pathways in C2C12 myotubes. (A,B) C2C12 myotubes were treated with vehicle (0.1% vol/vol DMSO) or TAK-242 (1 μM) and then with PBS or LPS (1 μg/mL) 1 h later. After 4 h, cell culture supernatant was collected and TNF-α (A) and IL-6 (B) concentrations were measured by ELISA. N = 6/group. (C,D) qRT-PCR analysis of TNF-α (C) and IL-6 (D) mRNA levels in C2C12 myotubes treated for up to 8 h as described in (A,B). N = 4/group. (E,F) qRT-PCR analysis of atrogin1/MAFbx (E) and MuRF1 (F) mRNA in C2C12 myotubes treated for 3 h. N = 4–5/group. (C–F) Data were normalised to CK2 mRNA levels and are shown as fold increase over the vehicle-treated controls. (G,H) Western blot analysis (G) and quantification (H) of Atrogin-1/MAFbx in C2C12 myotubes treated for 4 h. Data were normalised to β-tubulin protein levels, and the ratio in vehicle-treated control cells was set at 1.0. N = 7/group. Full-length blots are presented in Supplementary Figure S5A. (I,J) NF-κB (p65) binding activity in C2C12 myotubes treated for 4 h with vehicle, LPS (1 μg/mL), or LPS plus TAK-242 (1 μM and 0.1 μM). NF-κB (p65) DNA-binding activity in myotubes was analysed using a TransAM ELISA kit. Data are shown as sample absorbance at 450 nm (I) or fold increase (J) over the vehicle-treated controls. N = 4/group. (K,L) Western blot analysis (K) and quantification (L) of LC3-II expression in C2C12 myotubes treated for 24 h. Data were normalised to β-tubulin protein levels, and the ratio in vehicle-treated control cells was set at 1.0. N = 14/group. Full-length blots are presented in Supplementary Figure S5B. All panels, data are presented as the mean ± s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 vs vehicle control; ###p < 0.001, ##p < 0.01, #p < 0.05 vs LPS-treated group. P-values were derived from one-way ANOVA followed by Tukey’s honest significant difference test or Kruskal-Wallis test followed by Dunn’s post hoc tests with Bonferroni correction.
Figure 3
Figure 3
TAK-242 reduces LPS-induced muscle atrophy and weakness in mice. (A–D) Wild-type C57BL/6 mice (8–12-week-old males) were injected with vehicle (PBS containing 0.9% DMSO) or TAK-242 (3 mg/kg) and then with PBS or LPS (1 mg/kg) 1 h later. After 2 days, mice were assessed for body weight (A), TA muscle weight (B), and grip strength (C). Food intake (D) was measured every 24 h up to 48 h. N = 4–5/group. (E,F) Western blot analysis (E) and quantification (F) of MyHC expression in TA muscles at 2 days after administration of LPS (1 mg/kg) and TAK-242 (3 mg/kg) as described for (A–D). Data were normalised to β-tubulin protein levels, and the ratio in vehicle control-treated mice was set at 1.0. N = 7–8/group. Full-length blots are presented in Supplementary Figure S6. (G–I) Representative images of H&E-stained TA muscle sections (G) and quantification of the distribution (H) and mean (I) cross-sectional areas of TA muscle fibres at 2 days after administration of LPS (1 mg/kg) and TAK-242 (3 mg/kg) as described for (A–D). The cross-sectional area of TA muscle fibres was measured as described in the Methods. Scale bar, 100 μM. N = 506–523/group. For all panels, data are presented as the mean ± s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 vs vehicle control, ###p < 0.001, ##p < 0.01, #p < 0.05 vs LPS-treated group by one-way ANOVA followed by Tukey’s honest significant difference test.
Figure 4
Figure 4
TAK-242 reduces LPS-induced inflammatory and muscle proteolytic pathways in mice (A,B) Wild-type C57BL/6 mice (8–12-week-old males) were injected with vehicle (PBS containing 0.9% DMSO) or TAK-242 (3 mg/kg) and then with PBS or LPS (1 mg/kg) 1 h later. After 4 h, plasma samples were prepared and TNF-α (A) and IL-6 (B) concentrations were measured by ELISA. N = 3–4/group. (C–F) qRT-PCR analysis of TNF-α (C), IL-6 (D), atrogin-1/MAFbx (E), and MuRF1 (F) mRNA in TA muscles at 4 h after administration of LPS (1 mg/kg) and TAK-242 (3 mg/kg). Data were normalised to CK2 mRNA levels and are shown as fold increase over the vehicle-treated controls. N = 5–6/group (C,D), or 4–9/group (E,F). (G,H) Western blot analysis (G) and quantification (H) of atrogin-1/MAFbx in TA muscles at 4 h after administration of LPS (1 mg/kg) and TAK-242 (3 mg/kg). Data were normalised to β-tubulin protein levels, and the ratio in vehicle control-treated mice was set at 1.0. N = 8/group. Full-length blots are presented in Supplementary Figure S7A. (I,J) NF-κB (p65) DNA-binding activity in TA muscles at 4 h after administration of LPS (1 mg/kg) and TAK-242 (3 mg/kg and 0.3 mg/kg) was analysed using a TransAM ELISA kit. Data are shown as sample absorbance at 450 nm (I) or fold increase (J) over the vehicle-treated controls. N = 7–9/group. (K,L) Western blot analysis (K) and quantification (L) of LC3-II expression in TA muscles at 24 h after administration of LPS (1 mg/kg) and TAK-242 (3 mg/kg). (L) Data were normalised to β-tubulin protein levels, and the ratio in vehicle control-treated mice was set at 1.0. N = 5–8/group. Full-length blots are presented in Supplementary Figure S7B. For all panels, data are presented as the mean ± s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 vs vehicle control; ###p < 0.001, ##p < 0.01, #p < 0.05 vs LPS-treated mice by one-way ANOVA followed by Tukey’s honest significant difference test.
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