Department of Health, Exercise and Sports Sciences, University of New Mexico, Albuquerque, New Mexico, United States.
Molecular Medicine Program, University of Utah, Salt Lake City, Utah, United States.
Am J Physiol Cell Physiol. 2023 Jul 1;325(1):C300-C313. doi: 10.1152/ajpcell.00007.2023. Epub 2023 Jun 19.
Toll-like receptor 4 (TLR4) activation by lipopolysaccharides (LPS) increases proinflammatory cytokine production and upregulation of muscle atrophy signaling pathways. Muscle contractions can suppress LPS/TLR4 axis activation by reducing the protein expression of TLR4 on immune cells. However, the mechanism by which muscle contractions decrease TLR4 remains undefined. Moreover, it is not clear whether muscle contractions affect TLR4 expressed on skeletal muscle cells. The purpose of this study was to uncover the nature and mechanisms by which stimulated myotube contractions using electrical pulse stimulation (EPS) as an in vitro model of skeletal muscle contractions affect TLR4 expression and intracellular signaling to combat LPS-induced muscle atrophy. C2C12 myotubes were stimulated to contract via EPS with and without subsequent LPS exposure. We then examined the isolated effects of conditioned media (CM) collected following EPS and soluble TLR4 (sTLR4) alone on LPS-induced myotube atrophy. Exposure to LPS decreased membrane-bound and sTLR4, increased TLR4 signaling (decreased inhibitor of κBα), and induced myotube atrophy. However, EPS decreased membrane-bound TLR4, increased sTLR4, and prevented LPS-induced signaling and myotube atrophy. CM, which contained elevated levels of sTLR4, prevented LPS-induced upregulation of atrophy-related gene transcripts muscle ring finger 1 (MuRF1) and atrogin-1 and reduced myotube atrophy. Recombinant sTLR4 added to media prevented LPS-induced myotube atrophy. In summary, our study provides the first evidence that sTLR4 has anticatabolic effects by reducing TLR4-mediated signaling and atrophy. In addition, the study reveals a novel finding, by demonstrating that stimulated myotube contractions decrease membrane-bound TLR4 and increase the secretion of sTLR4 by myotubes. Excessive Toll-like receptor 4 (TLR4) activation causes muscle atrophy. Muscle contractions can limit TLR4 activation on immune cells, but its impact on TLR4 expressed on skeletal muscle cells remains unclear. Here, we demonstrate in C2C12 myotubes for the first time that stimulated myotube contractions reduce membrane-bound TLR4 and increase soluble TLR4, preventing TLR4-mediated signaling and myotube atrophy. Further analyses revealed soluble TLR4 independently prevents myotube atrophy, supporting a potential therapeutic role in combating TLR4-mediated atrophy.
Toll 样受体 4（TLR4）通过脂多糖（LPS）的激活增加促炎细胞因子的产生和肌肉萎缩信号通路的上调。肌肉收缩可以通过减少免疫细胞上 TLR4 的蛋白表达来抑制 LPS/TLR4 轴的激活。然而，肌肉收缩降低 TLR4 的机制尚不清楚。此外，肌肉收缩是否影响骨骼肌细胞上的 TLR4 尚不清楚。本研究的目的是揭示使用电脉冲刺激（EPS）作为骨骼肌收缩的体外模型刺激肌管收缩对 TLR4 表达和细胞内信号的影响，以对抗 LPS 诱导的肌肉萎缩。用 EPS 刺激 C2C12 肌管收缩，并在有或没有随后 LPS 暴露的情况下进行。然后，我们研究了 EPS 后收集的条件培养基（CM）和单独的可溶性 TLR4（sTLR4）对 LPS 诱导的肌管萎缩的单独影响。LPS 的暴露降低了膜结合和 sTLR4，增加了 TLR4 信号（减少 IκBα），并诱导了肌管萎缩。然而，EPS 降低了膜结合的 TLR4，增加了 sTLR4，并阻止了 LPS 诱导的信号和肌管萎缩。含有升高水平 sTLR4 的 CM 可防止 LPS 诱导的萎缩相关基因转录物肌环指 1（MuRF1）和 Atrogin-1 的上调，并减少肌管萎缩。添加到培养基中的重组 sTLR4 可防止 LPS 诱导的肌管萎缩。总之，我们的研究首次提供了证据表明 sTLR4 通过降低 TLR4 介导的信号和萎缩具有抗分解代谢作用。此外，该研究还揭示了一个新发现，即刺激肌管收缩可降低肌管上的膜结合 TLR4 并增加肌管的 sTLR4 分泌。过度的 Toll 样受体 4（TLR4）激活会导致肌肉萎缩。肌肉收缩可以限制免疫细胞上的 TLR4 激活，但对骨骼肌细胞上表达的 TLR4 的影响尚不清楚。在这里，我们首次在 C2C12 肌管中证明，刺激肌管收缩可降低膜结合的 TLR4 并增加可溶性 TLR4，从而防止 TLR4 介导的信号和肌管萎缩。进一步的分析表明，可溶性 TLR4 可独立地防止肌管萎缩，这为对抗 TLR4 介导的萎缩提供了一种潜在的治疗作用。
