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PO3-9-13

Lipopolysaccharide impairs myogenic differentiation of C2C12 myoblasts

[Speaker] Kazuho Sakamoto:1,2
[Co-author] Yuko Ono:2, Junko Kurokawa:1
1:Department of Bioinformational Pharmacology, University of Shizuoka, Japan, 2:Department of Pharmacology, Fukushima Medical University School of Medicine, Japan

Sepsis often causes severe skeletal muscle atrophy which forces long-term bedrest and artificial respiration to patients. Lipopolysaccharide (LPS), an endotoxin from Gram-negative bacteria, is often elevated in patients with sepsis and induce global inflammatory reactions. LPS might play an essential role in the development of sepsis-related muscle wasting. 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 adopted the C2C12 myoblast cell line to investigate the effects of LPS on myogenesis. In this study, 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. LPS concentration-dependently and significantly reduced the formation of multinucleated myotubes. Furthermore, the protein expression of myosin heavy chain II, myogenin, and MyoD were decreased. On the other hand, LPS increased the protein expression of myostatin and up-regulate NF- κB DNA-binding activity. 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. Our data suggest that LPS inhibits myogenic differentiation via a TLR4/NF- κB-dependent pathway and TNF- α-induced pathway. These pathways may be involved in the development of muscle wasting caused by sepsis.
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