Skeletal muscle fibers have the ability to increase their size in response to a mechanical overload. Finite element modeling data suggest that mechanically loaded muscles in vivo may experience not only tensile strain but also shear stress. However, whether shear stress affects biological pathways involved in muscle fiber size adaptation in response to mechanical loading is unknown. Therefore, our aim was twofold: (1) to determine whether shear stress affects growth factor expression and nitric oxide (NO) production by myotubes, and (2) to explore the mechanism by which shear stress may affect myotubes in vitro. C2C12 myotubes were subjected to a laminar pulsating fluid flow (PFF; mean shear stress 0.4, 0.7 or 1.4 Pa, 1 Hz) or subjected to uni-axial cyclic strain (CS; 15 % strain, 1 Hz) for 1 h. NO production during 1-h PFF or CS treatment was quantified using Griess reagent. The glycocalyx was degraded using hyaluronidase, and stretch-activated ion channels (SACs) were blocked using GdCl3. Gene expression was analyzed immediately after 1-h PFF (1.4 Pa, 1 Hz) and at 6 h post-PFF treatment. PFF increased IGF-I Ea, MGF, VEGF, IL-6, and COX-2 mRNA, but decreased myostatin mRNA expression. Shear stress enhanced NO production in a dose-dependent manner, while CS induced no quantifiable increase in NO production. Glycocalyx degradation and blocking of SACs ablated the shear stress-stimulated NO production. In conclusion, shear stress activates signaling pathways involved in muscle fiber size adaptation in myotubes, likely via membrane-bound mechanoreceptors. These results suggest that shear stress exerted on myofiber extracellular matrix plays an important role in mechanotransduction in muscle.
In response to mechanical loading skeletal muscle produces numerous growth factors and cytokines that enter the circulation. We hypothesized that myotubes produce soluble factors that affect osteoclast formation and aimed to identify which osteoclastogenesis-modulating factors are differentially produced by mechanically stimulated myotubes. C2C12 myotubes were subjected to mechanical loading by cyclic strain for 1 h, and postincubated with or without cyclic strain for 24 h. The effect of cyclic strain on gene expression in myotubes was determined by PCR. Conditioned medium (CM) was collected from cultures of unloaded and loaded myotubes and from MLO-Y4 osteocytes. CM was added to mouse bone marrow cells containing osteoclast precursors, and after 6 days osteoclasts were counted. Compared to unconditioned medium, CM from unloaded osteocytes increased osteoclast formation, while CM from unloaded myotubes decreased osteoclast formation. Cyclic strain strongly enhanced IL-6 expression in myotubes. CM from cyclically strained myotubes increased osteoclast formation compared to CM from unloaded myotubes, but this effect did not occur in the presence of an IL-6 antibody. In conclusion, mechanically loaded myotubes secrete soluble factors, among others IL-6, which affect osteoclast formation. These results suggest that muscle could potentially affect bone homeostasis in vivo via production of growth factors and/or cytokines.
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