Lipopolysaccharide inhibits myogenic differentiation of C2C12 myoblasts through the Toll-like receptor 4-nuclear factor-κB signaling pathway and myoblast-derived tumor necrosis factor-α

Background Circulating lipopolysaccharide (LPS) concentrations are often elevated in patients with sepsis or with various endogenous diseases that are associated with metabolic endotoxemia. Involuntary loss of skeletal muscle, termed muscle wasting, is commonly observed in these conditions, suggesting that circulating LPS might play an essential role in its development. Although impairment of muscle regeneration is an important determinant of skeletal muscle wasting, it is unclear whether LPS affects this process and, if so, by what mechanism. Here, we used the C2C12 myoblast cell line to investigate the effects of LPS on myogenesis. Methods C2C12 myoblasts were grown to 80% confluence and induced to differentiate in the absence or presence of LPS (0.1 or 1 μg/mL); TAK-242 (1 μM), a specific inhibitor of Toll-like receptor 4 (TLR4) signaling; and a tumor necrosis factor (TNF)-α neutralizing antibody (5 μg/mL). Expression of a skeletal muscle differentiation marker (myosin heavy chain II), two essential myogenic regulatory factors (myogenin and MyoD), and a muscle negative regulatory factor (myostatin) was analyzed by western blotting. Nuclear factor-κB (NF-κB) DNA-binding activity was measured using an enzyme-linked immunosorbent assay. Results LPS dose-dependently and significantly decreased the formation of multinucleated myotubes and the expression of myosin heavy chain II, myogenin, and MyoD, and increased NF-κB DNA-binding activity and myostatin expression. The inhibitory effect of LPS on myogenic differentiation was reversible, suggesting that it was not caused by nonspecific toxicity. Both TAK-242 and anti-TNF-α reduced the LPS-induced increase in NF-κB DNA-binding activity, downregulation of myogenic regulatory factors, and upregulation of myostatin, thereby partially rescuing the impairment of myogenesis. Conclusions Our data suggest that LPS inhibits myogenic differentiation via a TLR4–NF-κB-dependent pathway and an autocrine/paracrine TNF-α-induced pathway. These pathways may be involved in the development of muscle wasting caused by sepsis or metabolic endotoxemia.

Introduction Lipopolysaccharide (LPS), the major molecular component of the outer membrane of gramnegative bacteria, binds to Toll-like receptor 4 (TLR4) and induces formation of a TLR4-CD14 complex that increases nuclear factor-κB (NF-κB) activity [1,2]. LPS can cause a dysregulated inflammatory response leading to life-threatening organ dysfunction; a syndrome termed sepsis [3]. Increased levels of circulatory LPS are observed in patients with sepsis [4], elderly subjects [5,6] and individuals with diabetes mellitus [7], obesity [7], human immunodeficiency virus infection [8,9], cancer [10,11], liver cirrhosis [12], and end-stage kidney disease [13,14]. In the latter cases, increased LPS levels are caused by bacterial translocation from the intestinal tract to the circulation [15], a phenomenon known as metabolic endotoxemia [15]. Severe involuntary loss of skeletal muscle, termed muscle wasting, can be observed in all of these conditions [16], suggesting a potential role for circulating LPS in its development. Muscle wasting contributes to generalized weakness and debilitation, worsens quality of life, and increases mortality and economic burden [17]. Thus, there is an urgent need to advance our knowledge of its molecular pathogenesis.
One important cause of muscle wasting is breakdown of muscle protein through the ubiquitin-proteasome-dependent pathway [18]. Previous studies have shown that LPS activates the ubiquitin-proteasome pathway through TLR4 and induces catabolism both in cultured C2C12 muscle cells [19] and in rat muscle in vivo [20]. In agreement with these findings, increased ubiquitin-proteasome activity has been reported in elderly subjects [21] and in patients with metabolic endotoxemia due to diabetes mellitus [22], obesity [23], liver cirrhosis [24], and chronic kidney disease [25,26]. Damaged or degenerated myofibers are repaired or replaced through myogenesis, the process by which myoblasts fuse to form multinucleated myotubes. Although reduced myogenic capacity is another important determinant of skeletal muscle wasting [27-31], it is not known whether LPS affects this process.
LPS stimulates expression of proinflammatory cytokines, including TNF-α, not only in classical immune tissues but also in skeletal muscle [40,41,43]. Since TNF-α contributes to many pathogenic processes, including insulin resistance [44,45] and carcinogenesis [46], through both autocrine and paracrine mechanisms, it is possible that LPS-induced TNF-α secretion by myoblasts might also play a role in muscle wasting.
Here, we aimed to evaluate the effect of LPS on myogenesis, including the possible roles of TLR4-NF-κB signaling and autocrine/paracrine TNF-α on both positive and negative muscle regulatory factors. We found that selective inhibition of TLR4 signaling or neutralization of TNF-α activity had a beneficial effect on LPS-treated C2C12 myoblasts. Thus, TLR4-NF-κB signaling and myoblast-derived TNF-α play key roles in the impairment of muscle regeneration.
C2C12 myoblasts were treated with LPS from Escherichia coli 026:B6 (Sigma Aldrich, St. Louis, MO) dissolved in phosphate-buffered saline (PBS) at a concentration of 0.1 or 1 μg/mL for various times between 2 h and 144 h, as indicated. MyoD and myogenin expression and NF-κB (p65) DNA-binding activity were analyzed at 48 h, while expression of myosin heavy chain (MyHC) II, a myogenic marker, and myostatin was measured at 144 h. The TLR4 signaling inhibitor TAK-242 (Merck Millipore, Darmstadt, Germany) was added to cells at a final concentration of 1 μg/mL in dimethyl sulfoxide (DMSO, 0.1% vol/vol) immediately after the addition of LPS. The anti-TNF-α-neutralizing antibody (goat polyclonal, Cat. No. AB-410-NA; R&D Systems, Minneapolis, MN) was added at 5 μg/mL in PBS at 1 h prior to the addition of LPS. The anti-TLR2 neutralizing antibody T2.5 (mouse monoclonal, Cat. No. mab-mtlr2; InvivoGen, San Diego, CA) was added at 10 μg/mL in PBS at 1 h prior to the addition of LPS. The cell stimulators and inhibitors were present throughout the incubation. DM containing the appropriate concentrations of vehicle, LPS, TAK-242, and antibodies was exchanged every other day.
Quantitative analysis of TLR4 transcripts was performed in duplicate by real-time RT-PCR using SYBR Premix Ex Taq (Takara Bio), reaction capillaries (Roche Diagnostics, Mannheim, Germany), and a Light Cycler 1.5 (Roche Diagnostics). CK2 transcripts were quantified as an internal control. Real-time RT-PCR was performed as described previously [47,48], and included an initial denaturing step at 95˚C for 30 s; 40 cycles of denaturing at 95˚C for 5 s, annealing at 58˚C for 10 s, and extension at 72˚C for 15 s; and a final heating step for dissociation analysis. Crossing points were determined by the second derivative maximum method [49], and expression levels were calculated by a modified version of the standard curve method [50] using Light Cycler software version 3.5 (Roche Diagnostics).

Histological assessment
Cells were subjected to May-Grünwald and Giemsa staining to allow clear visualization of nuclei and myotube structures for quantitative measurements. After incubation in DM for 144 h, C2C12 cells were washed in cold PBS, fixed in 100% methanol, and stained as previously described [51] with minor modifications. Briefly, May-Grünwald staining solution (Wako) was diluted 1:3 in sodium phosphate buffer (1 mM NaH 2 PO 4 •H 2 O and 1 mM Na 2 HPO 4 , pH 6.0) and added to the cells for 5 min. Cells were then washed in distilled water and incubated in Giemsa staining solution (Wako) diluted 1:10 in distilled water for 10 min. Finally, cells were washed twice with distilled water and visualized with an inverted microscope (Olympus CK40, Tokyo, Japan) equipped with a camera (Olympus DP21). The myogenic index [52,53] was used as a morphological parameter of muscle differentiation. The number of nuclei in each myotube containing !3 nuclei and the total number of nuclei were counted in 5 randomly selected fields per well. The myogenic index (in %) was then calculated as: ([number of nuclei in myotubes in 5 fields] / [total number of nuclei in 5 fields] × 100) using ImageJ software version 1.39 (National Institutes of Health, Bethesda, MD). A total of 50-60 fields from 10-12 independent experiments was evaluated for each treatment group. Myotube widths were measured with ImageJ software using a modification of a published method [54][55][56]. In brief, cells were evaluated in 5 randomly selected fields per well. The width of each myotube containing !3 nuclei was measured at 3 different points on the cell and the average width per myotube was calculated. A total of 175-296 myotubes (50-60 fields, 10-12 independent experiments) was evaluated for each treatment group.

NF-κB assay
NF-κB (p65) DNA-binding activity was determined using a TransAM enzyme-linked immunosorbent assay (ELISA) kit (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. In brief, nuclear extracts of C2C12 cells were prepared using the Nuclear Extract Kit (Active Motif), added to the oligonucleotide-coated plate, and incubated with the NF-κB p65-specific primary antibody (1:1000 dilution) and horseradish peroxidase-conjugated secondary antibody (1:1000 dilution) contained in the ELISA kit. After washing, the colorimetric reaction reagents were added, and sample absorbance at 450 nm was measured in a spectrophotometer (Multiskan GO, Thermo Fisher Scientific). The assay was performed in duplicate.

Statistical analysis
The data are presented as the means ± standard error of the mean (SEM) unless otherwise indicated. Data were analyzed by one-way analysis of variance (ANOVA), and the treatment groups were compared with Tukey's post hoc test for honest significant difference. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 21.0 (IBM Corp., Armonk, NY). A p value <0.05 was considered statistically significant.

TLR4 mRNA is expressed constitutively in differentiating myoblasts
Although TLR4 is abundantly expressed in skeletal muscle of various vertebrates, including human and mouse [39][40][41], its expression pattern in differentiating myoblasts is unclear. We examined TLR4 mRNA expression at 2, 48, 92, and 144 h after induction of C2C12 differentiation and found that it is expressed constitutively (Fig 1A). Quantitation of TLR4 mRNA by real-time RT-PCR confirmed that similar levels were expressed throughout the differentiation period ( Fig 1B). Similarly, TLR4 mRNA levels did not change after culture of C2C12 cells in DM with LPS (0.1 or 1 μg/mL) for 144 h (Fig 1C), in agreement with previous observations by Frost et al. [43,57] and Lang et al. [41]. These results indicate that TLR4 would be expected to be available for LPS binding throughout C2C12 myogenesis and was unaffected by the presence of LPS.

LPS inhibits myogenesis in a dose-dependent manner
To examine the effect of LPS on myogenesis, C2C12 cells were cultured in DM for 144 h in the presence or absence of 0.1 or 1 μg/mL LPS. These concentrations were employed for consistency with a previous study [19] that showed that LPS induces protein breakdown via the ubiquitin-proteasome pathway in differentiated C2C12 cells. LPS was found to inhibit the formation of multinucleated myotubes (Fig 2A). To quantify the morphological changes, the proportion of total nuclei that were present in myotubes was calculated (the myogenic index; see Materials and Methods). As shown in Fig 2B, the myogenic index was significantly lower in LPS-treated than untreated myoblasts (control, 21 ± 3%; LPS 0.1 μg/mL, 10 ± 2%; LPS 1 μg/mL, 8 ± 2%). LPS also dose-dependently decreased the average myotube width (control, 20 ± 0.4 μm; LPS 0.1 μg/mL, 15 ± 0.5 μm; LPS 1 μg/mL, 11 ± 0.3 μm; Fig 2C and 2D), in agreement with earlier observations that LPS induces muscle proteolysis [19]. We also confirmed by western blotting that LPS dose-dependently suppressed expression of the differentiation marker MyHC II (LPS 0.1 μg/mL vs control, 44% decrease [p < 0.01]; LPS 1 μg/mL vs control, 85% decrease [p < 0.001]; Fig 2E and 2F). Thus, LPS directly inhibits C2C12 myogenesis in a dose-dependent manner in the absence of immune cells, which are the major sources of inflammatory cytokines.

Effect of LPS on muscle regulatory factors and NF-κB DNA-binding activity
The myogenic regulatory factors myogenin and MyoD are responsible for the induction and maintenance of early-phase muscle differentiation [32,33]. To understand the mechanism of LPS-induced myogenic inhibition, we examined the expression of these regulatory factors in LPS-treated C2C12 myoblasts. As shown in Fig 3A-3C, LPS treatment for 48 h dose-dependently We next examined whether LPS-induced inhibition of myogenesis is associated with increased expression of myostatin, which is a critical autocrine and paracrine inhibitor of skeletal muscle growth and differentiation [34][35][36]. Indeed, myostatin expression was increased dose dependently by incubation with LPS for 144 h (LPS 0.1 μg/mL vs control, 185% increase [p < 0.05]; LPS 1 μg/mL vs control, 283% increase [p < 0.001]; Fig 3D and 3E).
Previous studies have suggested that NF-κB mediates both the downregulation of MyoD and myogenin and the upregulation of myostatin [27-31, 37,38]. Thus, we next evaluated NF-κB (p65) DNA-binding activity in LPS-treated myoblasts. C2C12 nuclear extracts were prepared after 48 h of LPS treatment and analyzed by ELISA. This time point was chosen for convenience because the LPS-containing medium was refreshed every other day. As shown in Fig  3F, LPS treatment dose-dependently increased NF-κB DNA-binding activity (LPS 0.1 μg/mL vs control, 201% increase [p = 0.37]; LPS 1 μg/mL vs control, 417% increase [p < 0.01]). Collectively, these data suggest that LPS-induced MyoD and myogenin downregulation and myostatin upregulation is associated with increased NF-κB activity. The inhibitory effect of LPS on myogenesis is reversible Next, we investigated whether the inhibitory effect of LPS on myogenesis was reversible. To address this question, we adapted the methodology of Langen et al. [28], who showed that the toxic effect of TNF-α on C2C12 myogenesis was reversible. The cells were incubated with LPS for 48 h, washed, and cultured in fresh LPS-free DM. Myogenin expression was examined at 24 and 48 h after LPS washout. Notably, while myogenin expression was initially reduced by LPS, it was restored after washing and removal of LPS (Fig 4A and 4B). These results demonstrate that the inhibitory effect of LPS on myogenic differentiation is reversible, suggesting that it is not due to a nonspecific toxic effect such as induction of cell death.

LPS signals through both TLR2 and TLR4 to activate NF-κB
As demonstrated in Fig 5 and Fig 6, selective inhibition of TLR4 signaling did not fully suppress LPS-induced NF-κB activation and dysregulation of muscle regulatory factors. Since LPS is also a ligand for TLR2 [62][63][64], and TLR2 mRNA is expressed in C2C12 myoblasts [41,43], we considered that LPS might be signaling through both TLR2 and TLR4. To examine this, myoblasts were incubated with 1 μg/mL LPS in the presence or absence of a TLR2-neutralizing antibody (T2.5, 10 μg/mL), and NF-κB activity was measured after 48 h. This concentration of anti-TLR2 antibody was previously found to prevent serum amyloid A-induced TLR2-dependent signaling in C2C12 myotubes [56]. As shown in Fig 7A, addition of anti-TLR2 decreased LPS-induced NF-κB activation by about 30%, although the difference did not reach the level of statistical significance (p = 0.62). The selective TLR4 inhibitor had a greater effect on NF-κB activity (Fig 7B), suggesting that LPS signals predominantly through TLR4 but at least partially through TLR2 in these cells, This could be one possible explanation for the failure of TAK-242 to completely suppress the LPS-induced effects on NF-κB and myogenic regulatory factors.

Myoblast-derived autocrine/paracrine TNF-α is involved in LPS-induced inhibition of myogenesis
There is considerable evidence that LPS regulates the expression of proinflammatory cytokines, such as TNF-α and interleukin 6, in mouse myoblasts and skeletal muscle [40,41,43], and TNF-α has previously been shown to inhibit myogenic differentiation [27-31]. Therefore, we hypothesized that myoblast-derived TNF-α may be involved in LPS-induced inhibition of myogenesis. To test this hypothesis, we examined C2C12 cells after treatment with LPS (1 μg/ mL) in the absence or presence of a TNF-α-neutralizing antibody (5 μg/mL). This concentration of antibody was previously shown to inhibit TNF-α secretion and subsequent NF-κB activation induced by serum restriction of C2C12 myoblasts [65]. We found that the TNF-αneutralizing antibody partially but significantly reversed the LPS-induced downregulation of myogenin (LPS + anti-TNF-α vs LPS, 255% increase, p < 0.05; Fig 8A and 8B), MyoD (179% increase, p < 0.001; Fig 8A and 8C), and MyHC II (262% increase, p < 0.01; Fig 8D and 8E) expression, and additionally suppressed the LPS-induced upregulation of myostatin expression (40% decrease, p < 0.01; Fig 8D and 8F) and NF-κB activation (40% decrease, p < 0.05; Fig 8G). These results suggest that NF-κB activation and subsequent effects on myogenic regulatory factors induced by LPS are partially mediated by myoblast-derived TNF-α. Thus, it is possible that such an autocrine/paracrine TNF-α loop is involved in the impairment of muscle regeneration.
Based on these collective data, we propose a mechanistic model for the inhibition myogenesis by LPS in differentiating myoblasts (Fig 9).

Discussion
In the present study, we found that LPS inhibits C2C12 myogenesis through the TLR4-NF-κB and autocrine/paracrine TNF-α-mediated pathways. We found that LPS downregulated MyoD and myogenin expression and upregulated myostatin expression in a dose-dependent manner. Both pharmacological inhibition of TLR4 signaling and antibody-mediated neutralization of TNF-α reduced NF-κB activity and attenuated the LPS-induced dysregulation of muscle regulatory factors.
For our in vitro experiments, we employed two concentrations of LPS, 0.1 and 1 μg/mL, and observed a dose-dependent inhibitory effect on murine myoblast differentiation. A previous study suggested that humans were more than 10,000-fold more sensitive than mice to LPS [66], raising the possibility that human muscle regeneration could be much more vulnerable to the effects of LPS. Circulating LPS levels are commonly elevated in conditions such as sepsis [4] and endogenous diseases [5][6][7][8][9][10][11][12][13][14][15], and muscle atrophy can also be observed in these conditions [16]. Therefore, LPS-induced derangement of myogenesis might be a cause of muscle wasting in patients with sepsis or metabolic endotoxemia.
In this study, we demonstrated that the TLR4 signaling pathway mediated LPS-induced activation of NF-κB, downregulation of MyoD and myogenin expression, and upregulation of myostatin expression. Our findings are in agreement with several earlier observations that exogenous TNF-α-induced activation of NF-κB inhibited myogenesis in C2C12 cells by suppressing MyoD and myogenin expression [27-31]. NF-κB-mediated upregulation of myostatin has also been observed in other model systems, such as H 2 O 2 -treated cultured myoblasts [38] and mouse models of liver cirrhosis and hyperammonemia [37]. In contrast, several studies have suggested that myostatin expression in skeletal muscle is not increased in sepsis models. For example, Smith et al. observed that myostatin mRNA levels were reduced and myostatin protein levels were unchanged in rat skeletal muscle 16 h after cecal ligation and puncture [67]. Lang et al. reported that myostatin mRNA was not increased 24 h after LPS administration to rats [68]. One possible explanation for this discrepancy is the shorter endotoxin exposure times, since we observed increased myostatin expression in C2C12 cells after 144 h of LPS treatment. Martin et al. noted a time-dependent increase in myostatin mRNA expression after administration of LPS to mice; they found that the levels remained unchanged β-tubulin (internal standard) is shown. (B and C) Quantification of the data presented in (A). Data are the mean ± SEM of 7-15 independent experiments. (D) C2C12 myoblasts were cultured for 144 h as described in (A). A representative western blot probed with antibodies to MyHC II, myostatin, or β-tubulin (internal standard) is shown. (E and F) Quantification of the data presented in (D). Data are the mean ± SEM of 5-21 independent experiments. (G) Cells were treated as described in (A), and NF-κB activity was analyzed by ELISA. Data are the mean ± SEM of 7 independent experiments performed in duplicate. ***p < 0.001, **p < 0.01, *p < 0.05, ###p < 0.001, ##p < 0.01, #p < 0.05 by one-way ANOVA followed by Tukey's honest significant difference test.
https://doi.org/10.1371/journal.pone.0182040.g008 at 24 h after LPS injection but increased significantly after 76 h [69]. In the clinical setting, sepsis survivors often display systemic inflammation for protracted periods and also develop muscle wasting. Circulating myostatin is commonly elevated in patients with conditions such as chronic liver and kidney disease [25, 37,70], diabetes mellitus [71], and human immunodeficiency virus infection [72], and in the elderly [73,74]. All of these populations are likely to be chronically exposed to LPS due to bacterial translocation [15]. Taken together, these findings suggest that persistent exposure to LPS or inflammation may be required to induce myostatin.
We found that myogenin expression was rapidly restored after switching from LPS (1 μg/ mL)-containing medium to fresh medium, suggesting that the inhibition of myogenesis was reversible and not simply a toxic effect of LPS, such as induction of apoptosis. Shang et al. examined C2C12 cell viability after exposure to various concentrations of LPS [75]. They found that LPS at 1-10 μg/mL had no effect on C1C12 apoptosis, whereas higher concentrations (100-150 μg/mL) induced apoptosis through caspase-3 activation. Our findings are thus consistent with their data. Reversible inhibition of myogenesis has also been observed upon treatment of C2C12 myoblasts with TNF-α [28]. Taken together, these data suggest that persistent NF-κB activation may be required to block myogenesis and promote muscle wasting.
While the role of TLR4 in innate immunity is well characterized, its role in skeletal muscle development has been unclear. To address this knowledge gap, we examined the effect of a selective TLR4 signaling pathway inhibitor on the LPS-induced events. We observed that TAK-242 partially rescued the LPS-induced inhibition of myogenesis, activation of NF-κB, downregulation of myogenin, and upregulation of myostatin, suggesting that TLR4 is an upstream regulator of skeletal muscle myogenesis. Previous studies have suggested that TLR4 plays an important role in muscle protein breakdown. Doyle et al. [19] found that TLR4-mediated LPS signaling induced muscle catabolism via coordinate activation of the ubiquitinproteasome and autophagy pathways. According to Dehoux et al.
[20] and Martin et al. [69], ubiquitin ligase mRNA expression was induced in both rat and mouse skeletal muscle after LPS injection. In agreement with these findings, we observed that LPS dose-dependently decreased the myotube width, and that inhibition of TLR4 signaling prevented the atrophy. Collectively, these findings indicate that LPS may induce muscle wasting via synergistic effects on myogenesis and muscle proteolysis through TLR4. Skeletal muscle TLR4 is upregulated in diabetic and obese subjects [76] as well as in the elderly [5,77], suggesting that the TLR4-NF-κB pathway may be elevated in these populations. Thus, inhibition of the TLR4 signaling axis might be a useful method for preventing or reversing LPS-induced muscle wasting in patients with sepsis or metabolic endotoxemia. Future studies should address the effects of TLR4 antagonists on LPS-induced muscle breakdown.
We found that antibody-mediated neutralization of TNF-α reduced the LPS-induced increase in NF-κB DNA-binding activity, downregulation of myogenin and MyoD, and upregulation of myostatin in C2C12 cells, suggesting that LPS-induced autocrine/paracrine TNF-α might be involved in the impairment of muscle regeneration. Autocrine/paracrine regulation of TNF-α has also been observed in C2C12 myoblasts following serum restriction [65] and in various other cell lines and tissues, including cancer cells [46,78], immune cells [79,80], and microglia [81]. In skeletal muscle myoblasts, TNF-α is a strong activator of NF-κB [65] and of its own synthesis [82]. Therefore, as is the case in cancer cells [46,78], a positive TNF-α autocrine/paracrine loop in response to LPS may lead to persistent NF-κB activation in myoblasts, further inhibiting myogenesis and thus inducing muscle wasting.
Interestingly, we found that TNF-α neutralization, but not TLR4 inhibition by TAK-242, reversed the LPS-induced inhibition of MyoD expression. We speculate that MyoD expression might be more sensitive to regulation by TNF-α than by TLR4 signaling. In support of this possibility, the pattern of NF-κB activation by LPS and TNF-α has been shown to differ. In C2C12 myotubes, TNF-α was found to persistently activate NF-κB in a biphasic manner, while LPS did not [31]. Moreover, in human epithelial cells, LPS from Haemophilus influenzae (exogenous activation) and TNF-α (endogenous activation) synergistically induced NF-κB activation via two distinct signaling pathways [83]. In addition to the involvement of TLR2 mediated signaling, this could be another explanation for the failure of TAK-242 to fully ameliorate the harmful effect of LPS on differentiating myoblasts.
In vivo, LPS induces circulating immune cells to produce copious amounts of inflammatory cytokines, which have been implicated as potential mediators of muscle wasting via inhibition of myogenesis [27-31] and acceleration of muscle proteolysis [18,84,85]. In fact, inflammatory cytokine concentrations are elevated in the circulation of patients with sepsis [86] and metabolic endotoxemia [87][88][89]. Our study extends these observations by demonstrating that LPS itself can directly inhibit myogenesis through TLR4-NF-κB signaling and myoblast-derived TNF-α. Systemic and local inflammatory reactions may synergize to induce muscular wasting.
In this study, cells were co-incubated with TAK-242 dissolved in DMSO (final concentration 0.1% [vol/vol]) and LPS dissolved in PBS. We acknowledge that a DMSO vehicle control was not included in two experiments shown in Figs 5 and 6. High concentrations of DMSO (1-2% [vol/vol]) have been shown to augment the LPS effect on immune cells (i.e., increase inflammatory cytokine secretion) [90]; therefore, in our study, the relative effect of TAK-242 on the LPS response may have been weakened. Nevertheless, previous studies have shown that, even at concentrations as high as 1-2% (vol/vol), DMSO has no effect on NF-κB activity in various cell lines, reducing this concern [90][91][92].
We observed that TAK-242 significantly decreased myostatin expression when cells were stimulated with LPS at 1 μg/mL (30% decrease, p < 0.05) but not at 0.1 μg/mL (5% decrease, p = 0.7). At present, we do not have a plausible explanation for this discrepancy.
To date, no drugs have been approved for the treatment of skeletal muscle wasting. Our finding that blockade of the TLR4-NF-κB pathway or TNF-α can reverse impaired myogenesis suggests a new set of drug targets for clinical intervention in sepsis-or metabolic endotoxemia-induced muscle debilitation. Clinical trials with a TLR4 antagonist [93][94][95] and TNF-α inhibitor [96] have shown no improvement of the mortality rate of severe sepsis patients; however, those trials did not examine long-term muscle function [93][94][95][96]. The results presented here provide a rationale to test the effects of TLR4 and TNF-α antagonists on LPS-induced muscle wasting in sepsis or metabolic endotoxemia patients. Our data should also stimulate further studies to clarify the role of TLR4-NF-κB and TNF-α signaling in muscle wasting.

Conclusions
LPS inhibits C2C12 myogenesis by dose-dependently downregulating myogenin and MyoD expression and upregulating myostatin expression. TAK-242, a selective inhibitor of TLR4-mediated signaling, and a TNF-α-neutralizing antibody both reduced NF-κB activity and attenuated the downregulation of myogenic regulatory factors and upregulation of myostatin in LPS-treated C2C12 myoblasts. Consequently, myogenesis was partially restored. These data suggest that LPS inhibits myogenic differentiation of skeletal muscle myoblasts through TLR4-NF-κB signaling and myoblast-derived autocrine/paracrine TNF-α, raising the possibility that these pathways also contribute to the development of muscle wasting in patients with sepsis or metabolic endotoxemia.
Supporting information S1 Data. Excel spreadsheet providing the raw data for each figure.