Mechanical loading induces expression of osteo-anabolic and osteo-catabolic factors by myotubes: Role in myogenic adaptation

Muscle and bone share numerous signaling pathways in the regulation of bone and muscle mass. However, of some crucial bone mass regulatory factors, such as RANKL, it is not known whether they are expressed in muscle, or whether they have an effect on muscle physiology. In addition, for some factors that are expressed in muscle and bone alike, i.e. COX-2, BMPs, Wnt signaling proteins, it is known that they play an important role in adaptation of bone mass to mechanical loading, but it is yet unknown whether the expression of these factors is affected by mechanical loading in muscle. Therefore we aimed to investigate 1) whether mechanical loading by cyclic uni-axial strain (CS) affects the expression of COX-2, BMP2, BMP6, and 7, Wnt signaling proteins, and RANKL by C2C12 myotubes, and 2) whether C2C12 myotube metabolism is affected by RANKL. C2C12 myotubes were subjected to CS (15% 1 Hz, 1 h), and gene expression of COX-2, BMP2, BMP6, BMP7, Wnt signaling proteins, and RANKL was analyzed immediately after 1 h CS using PCR. Prostaglandin E2 (PGE2) concentrations in the medium were measured at 3 h and 6 h post-CS. C2C12 myotubes were treated with rmRANKL (0, 0.2, 2, and 20 ng/mL). After 2h and 20h RANKL treatment, samples were collected for mRNA, DNA, and total protein analysis. After 20h RANKL treatment myotube thickness was measured. CS increased COX-2 mRNA levels (2-fold), but CS did not affect PGE2 production or mRNA levels of BMPs in myotubes. CS increased mRNA levels of Wnt1 (70-fold), and Wnt7a (200-fold), and decreased mRNA levels of Wnt4 (8-fold), and RANKL (1.8-fold). Addition of RANKL to myotubes increased total protein content and decreased myotube diameter, possibly as a result of changes in expression of the NFκB target genes c-fos and NFATC1. Our data show that cyclic uni-axial strain affects the expression of COX-2 and Wnts signaling proteins, as well as a novel factor RANKL. These typical osteo-anabolic and osteo-catabolic factors are produced by muscle cells in response to mechanical loading, and may play a role in the regulation of muscle fiber size, but may also affect bone remodeling via paracrine and endocrine signaling.