Effects Of FHL3 On Expression Of Different Types Of MyHC Genes In C2C12 Myoblast

Muscle fibers type plays an important role in determining muscle growth and meat quality. Different fiber type myosin heavy chain (MyHC) genes, such as MyHC slow, MyHC 2a, MyHC 2x and MyHC 2b are considered as molecular markers to distinguish muscle fiber type. Therefore, it is of great significance to discover genes regulating the expression of MyHC and its molecular mechanism, which may contribute to improve the meat quality and lean production. FHL3, a member of LIM protein superfamily, regulates myoblast differentiation, the cell reconstruction and downstream gene expression through interacting with proteins such as MyoD and CREB. However, whether FHL3 regulates MyHC genes expression or not is currently not clear. In this study, the expression of different types of MyHC genes in the mRNA level was measured after knocking down the expression of FHL3 in C2C12 cells. The results are as follows:1. The expression change of FHL3 gene, MyHC slow,2a,2x and 2b genes were compared at Oh,48h,96h,144h of C2C12 cell differentiation by the real-time PCR and Western Blotting. The expression of FHL3 gradually decreased from the myoblast to myotube. The expression of MyHC slow gene increased at the early stage of myotube formation, and then declined after myotube formation. The expression of MyHC 2a gene gradually increased with the cell differentiation, and reached its expression peak at the time of myotube formation. During the differentiation of C2C12 cells, both MyHC2b and MyHC 2x did not significantly varied at the early stage of cells differentiation, but started to increase at the time of myotube formation.2. Interference experiment was conducted by three small fragments of chemical synthesis and siRNA#3 with the best interference effect was screened out. C2C12 myoblasts were transfected with these small fragments, then differentiated 24h,48h and 72h after transfection 24 hours. The expression of FHL3 at differentiated 24h and 48h decreased significantly, respectively.3. Based on the above results, C2C12 myoblasts were transfected with siRNA#3 and then differentiated 72h after transfection 24h. The relative expressions of the MyHC slow and MyHC 2a decreased significantly and no significant change for MyHC 2b, whereas MyHC 2x increased significantly. However C2C12 myoblasts were transfected with siRNA#3, grown for 48 hours and then differentiated 48 hours. The relative expression of f MyHC slow decreased significantly, while MyHC 2b and MyHC 2x increased significantly. Combining the above results, the expressions changes of MyHC slow and MyHC 2x was similar in two treatments. 4. C2C12 cells which were differentiated 24h,48h and 72h respectively were transfected with siRNA#3, and then grown for 48 hours. The relative expressions of the FHL3 gene at the differentiated 24h and 48h cells decreased significantly and highly significantly respectively. The expressions of the MyHC slow and MyHC 2a of the differentiated cells at 24h and 48h decreased significant, highly significant. The expressions of MyHC slow at transfection firstly or differentiation firstly decreased with the decrease of the FHL3 expression.In this study, it was proven that FHL3 had significant effects on MyHC genes in the C2C12 myoblast. FHL3 negatively regulated the expression of MyHC 2b and MyHC 2x, while positively regulated the expression of MyHC slow and MyHC 2a. This study gives us an important clue to further elucidate how the FHL3 regulated the expressions of MyHC genes. However more experiments will be required in order to study this mechanism between FHL3 and MyHC genes.