FHL3 Differentially Regulates The Expression Of MyHC Isoforms Through Interaction With Functional MyoD And PCREB

Different fiber types of My HC isoform genes are considered as molecular markers to distinguish muscle fiber types. Therefore, it is of great significance to discover genes regulating the expression of My HC and its molecular mechanism, which may contribute to improve the meat quality and pig production. FHL3 is one of members of LIM protein superfamily, which can not bind with DNA promoters and regulates target gene expression by interacting with other transcriptional factors. However, how FHL3 regulates My HC genes expression is currently not clear. In this study, we presented a molecular mechanism for the regulation of My HC expression by FHL3 in C2C12 myoblasts. The results are as follows:1. C2C12 myoblasts were transfected with pc DNA3.1-FHL3 vector and si RNA FHL3(si FHL3). Quantitative real-time PCR, Western blotting and immunofluorescence were used to detect the expression of FHL3 and My HC isoforms gene. The results showed that FHL3 down-regulated the expression of My HC 1/slow and up-regulated the expression of My HC 2a and My HC 2b, while there is no significant effect on the My HC 2x expression. 2. C2C12 myoblasts were co-transfected with pc DNA3.1-FHL3 and si Myo D. The results showed that My HC 2a and My HC 2b expression was also increased by Fhl3 overexpression after knockdown of Myo D, but there was no effect of Fhl3 overexpression on the My HC 1/slow expression after Myo D knockdown. C2C12 myoblasts were also co-transfected with pc DNA3.1-Myo D and si FHL3. The results showed that Myo D overexpression stimulated My HC 1/slow expression and knockdown of FHL3 strengthened the promoting function of Myo D. The above results indicated that FHL3 down-regulated My HC 1/slow though inhibiting the transcriptional Myo D, but regulates the expression of My HC 2a and My HC 2b through other transcription factors. 3. C2C12 myoblasts were co-transfected with My HC 2a promoter deletions fragments and pc DNA3.1-FHL3. The transcription activities of the region containing c AMP response elements(CRE) were improved by FHL3 overexpression. The results of CREB overexpression and knocdown exprement suggested that CREB promoted the expression of My HC 2a gene. 4. C2C12 myoblasts were co-transfected with pc DNA3.1-FHL3 and si CREB/pc DNA-3.1-CREB. The results comfired that FHL3 promoted the My HC 2a expression through the CREB, and p CREB played the more important roles in expression the regulation of My HC 2a by FHL3. Co-immunoprecipitation experiments showed that FHL3 could interact with CREB, p CREB, and Myo D coactivator p300, and when Myo D and CREB co-existed, FHL3 preferred to bind to p CREB than Myo D. 5. The binding capacity of CREB with promoter sequences was analyzed by EMSA and Ch IP assay. These results suggested that the CRE of the My HC 2a promoter was capable of binding to both unphosphorylated CREB and p CREB, but the binding capacity to unphosphorylated CREB was weaker than that of p CREB and the knockdown of FHL3 decreased the binding capacity of p CREB the My HC 2a promoter.Taken together, we concluded that FHL3 up-regulated the expression of My HC 2a and down-regulated the expression of My HC 1/slow; FHL3 inhibited the expression of My HC 1/slow by inhibiting Myo D transcriptional activity and promoted the expression of My HC 2a through interaction with p CREB and p300. Our study provided a new molecular mechanism for the regulation of muscle fiber type composition, and also provided an important theoretical basis for improving meat quality traits in animal production.