Effect Of FHL3 Gene On The Formation Of Different Muscle Fiber Types Using Transgenic Mice Model

Muscles are composed of different muscle fibers. According to the different myosin heavy chain, muscle fiber types can be divided into I, IIa, IIb and IIx. The proportion of four different muscle fibers affects the quality of muscles, so the study of the conversion between different muscle fiber types is of great significance to improve the meat quality. FHL3 is a member of LIM protein family, and our previous studies have shown that over-expression of FHL3 gene inhibited the expression of MyHC I gene and promoted the expression of MyHC IIa and MyHC IIb gene in C2C12 myoblasts, but the effect of FHL3 gene on different muscle fiber types in vivo is still unclear. In this study, we verified that the different muscle fiber types were affected by FHL3 gene in transgenic mice. The results are as follows:1.The muscle tissue-specific expression transgenic vector pEGFP-N1L-MCK-FHL3 was constructed. FHL3 positive transgenic mice were selected by PCR technology. The copy number of foreign gene FHL3 in F0, F1 positive transgenic mice and insertion site of FHL3 gene were identified by Q-PCR and Tail PCR. The results revealed that the foreign gene FHL3 was inserted in mouse genome by the form of continuous multiple copies. Besides, the FHL3 gene of 51 th individual was inserted in mouse chromosome 4.2. The litter size in F0 generation, and the positive percentage, the growth curve in F1 generation were investigated. There was a significant increase in the litter size of F0 generation in FHL3 transgenic mice compared with wild-type mice. In addition, the positive percentage in mice born by F0 males was obviously lower than that born by F0 females. However, there was no significant difference of body weight at 2-8 weeks between transgenic and wild-type mice.3. The expression of FHL3 gene in tissues such as heart, liver, spleen, lung, kidney, brain, intestine, stomach, fat, muscle and testis were detected in two-month-old transgenic mice and wild-type mice by Q-PCR. The levels of FHL3 expression in cardiac muscle, skeletal muscle and testis of transgenic mice were significantly higher than that of wild-type mice.4. The quadriceps of mouse were paraffin-embedded, sliced and HE stained. The mean muscle fiber number and average cross-sectional areas of muscle fiber were examined quantitatively by Image J software, and no significant changes were observed between the wild-type mice and transgenic mice.5. The Q-PCR and Western blotting were used to determine the levels of FHL3, MyHC I, MyHC IIa and MyHC IIb gene in gastrocnemius of wild-type mice and transgenic mice. The results suggested that in transgenic mice the expression of FHL3, MyHC IIa, and MyHC IIb gene at mRNA and protein levels were significantly higher than that of wild-type mice, but the MyHC I gene expression was significantly lower.6. ATPase and SDH staining were performed to distinguish different muscle fiber types of 2-month-old mice soleus and quadriceps. The results showed that the type I fibers of transgenic mice were all significantly reduced in the soleus and quadriceps, while type II fibers were significantly increased compared with wild-type mice.In summary, using transgenic mice model we concluded that FHL3 may negatively regulate the expression of genes MyHC I, and positively regulate expression MyHC IIa and MyHC IIb gene, resulting in reduced type I muscle fibers and increased type II muscle fibers. This study illustrates the effect of FHL3 on different types of muscle fiber in vivo and provides a theoretical basis for improving the animal meat quality.