The Effects Of FGF13 In Skelatal Muscle Proliferation And Differention In Mice

Skeletal muscle mainly performs the function of contraction and relaxation, and participates in various movements in the body. In the research of animal and poultry, the quantity and quality of skeletal muscle are regarded as the main indexes to measure meat quality. Skeletal muscle growth and development is a quite complex process, including muscle derived stem cells to differentiate into muscle cells and monocytes, cell fusion into multinucleated myotubes, mature muscle fibers and so on. And this process involves a number of myogenic regulatory factors(MRFs). Although the process of skeletal muscle growth and development in mouse has been well-explored, the factors involved in the regulation of myogenesis are needed to be further explored.FGF13 belongs to members of the FGF subfamily(FHF), which are not secreted because lack the signal peptide. FGF13 is widely distributed in the developing brain and nervous system. In addition, FGF13 has high expression level in brain and skeletal muscle of patients with Borjeson-Forssman-Lehmann syndrome(BFLS). Recently, one study reported that high expression level of FGF13 was observed in culture of single muscle fibers. However,functions of intracellular FGF13 in skeletal muscle remain largely unknown. Real-time RT-PCR was performed to detect expression of FGF13 during C2C12 muscle cell proliferation and differentiation. To further understand the role of FGF13, we examined its effects on proliferation and differentiation by Western blotting analyses of cells transfected with FGF13 siRNA or FGF13 over-expression plasmids, or treated with chemical MEK inhibitors. Effects of FGF13 on related signal pathways were determined in C2C12 cell proliferation and differentiation. Next, we constructed the mice model of skeletal muscle injury, and injected FGF13 siRNA in the muscle of local tissues. The recovery of muscle injury was detected using frozen section and immunofluorescence. Furthermore, proteins interacting with FGF13 were screened from mouse cDNA library by yeast two-hybrid system.This study will be contributed to explore FGF13 functions on molecular level and provides some novel clues to explain the mechanism of skeletal muscle growth and development.The specific results are as follows:1. The expression of FGF13 mRNA in C2C12 cells reached its highest level on the fourth day of myogenic differentiation. The levels of MyOG and MyHC increased significantly after FGF13 eliminated. In contrast, the levels of MyOG and MyHC decreased significantly when FGF13 overexpressed during C2C12 cell differentiation in vitro. Hence,we hypothesized that FGF13 inhabits myoblast differentiation.2. FGF13 inhibiting ERK1/2 activity by phosphorylation was similar to that of the inhibitor PD98059. Thus, we could conclude that FGF13 inhibiting myogenic differentiation was indeed dependent on the ERK1/2 signaling pathway.3. Spry1 promoted myotube formation by inhibition of ERK1/2 pathways. FGF13 and Spry1 play inverse roles during myogenic differentiation and that FGF13 inhibited myogenic differentiation by down-regulating Spry1 expression. We conclude that FGF13 promoted ERK1/2 activation and phosphorylation by reducing Spry1 expression during C2C12 cell differentiation.4. FGF13 down-regulation of CyclinE and up-regulation of p27 expression lead to slower cell proliferation by inhibiting Spry1. However, the ERK1/2 pathway did not seem to affect cell proliferation. We built muscle gastrocnemius muscle injury model with BaCl2 and intramuscular injected si-FGF13, and the number of inflammatory cells decreased and MyOG expression increased in injured tissue. The result suggested that FGF13 played a negative role in the repair of muscle injury.5. The bait plasmid pGBKT7-m FGF13 was constructed and FGF13 interacting proteins were screened by yeast two-hybrid system. Nine candidate proteins were ontained, in which Shcbp1 and ARHGAP5 may interact with FGF13 to affect ERK1/2 and P38 signaling pathways. But the specific binding forms and functions are needed to be further verified.In summary, this study illustrated that FGF13 down-regulated Spry1 expression, thereby activating the ERK1/2 pathway via phosphorylation and inhibiting C2C12 cell differentiation.Consequently, FGF13 may participate as a repressor during myoblast differentiation via the ERK1/2 pathway. In addition, FGF13 was a negative regulator for C2C12 cell proliferation.Although FGF13 inhibited Spry1 to retard cell proliferation or differentiation, ERK1/2pathway activation occurred only during C2C12 cell differentiation. Generally, FGF13 could inhibit C2C12 cell proliferation and differentiation by down regulating Spry1. Nine candidate proteins interacting with FGF13 were screened out by yeast two-hybrid system, which participate in energy metabolism, cell proliferation and differentiation, migration and movement, autophagy, autoimmunity and signal transduction.