FHL1 is multifunctional and serves as a modular protein binding interface to mediate protein-protein interactions. In skeletal muscle, FHL1 is involved in sarcomere assembly, differentiation, growth, and biomechanical stress. Muscle abnormalities may play a major role in congenital clubfoot (CCF) deformity during fetal development. Thus, identifying the interactions of FHL1 could provide important new insights into its functional role in both skeletal muscle development and CCF pathogenesis. Using proteins derived from rat L6GNR4 myoblastocytes, we detected FHL1 interacting proteins by immunoprecipitation. Samples were analyzed by liquid chromatography mass spectrometry (LC-MS). Dynamic gene expression of FHL1 was studied. Additionally, the expression of the possible interacting proteins gamma-actin and non-muscle myosin IIB, which were isolated from the lower limbs of E14, E15, E17, E18, E20 rat embryos or from adult skeletal muscle was analyzed. Potential interacting proteins isolated from E17 lower limbs were verified by immunoprecipitation, and co-localization in adult gastrocnemius muscle was visualized by fluorescence microscopy. FHL1 expression was associated with skeletal muscle differentiation. E17 was found to be the critical time-point for skeletal muscle differentiation in the lower limbs of rat embryos. We also identified gamma-actin and non-muscle myosin IIB as potential binding partners of FHL1, and both were expressed in adult skeletal muscle. We then demonstrated that FHL1 exists as part of a complex, which binds gamma-actin and non-muscle myosin IIB.
Previous studies of the role of 17β-estradiol (E2) in myoblast differentiation have produced conflicting data. Therefore, this work aimed to determine the role of E2 on myoblast differentiation and specific myofiber formation. Murine C2C12 myoblasts were cultured in proliferation medium or differentiation medium/10 nM E2. The role of E2 on specific myosin heavy chain (MyHC) or estrogen receptor (ER) expression was examined using real-time quantitative RT-PCR (RT-qPCR). Transcriptome studies of E2 on myoblast differentiation were accomplished by microarray analyses. The expression levels of candidate genes from microarrays and four and a half LIM domains 1 (Fhl1) were detected with RT-qPCR. E2 in differentiation medium significantly up-regulated MyHC I expression, but exerted the opposite effects on MyHC II a, MyHC II b, and MyHC II d. Both ER-α and ER-β were decreased in differentiated C2C12, and E2 partially restored ER-β expression. Sixty-two up-regulated and 116 down-regulated genes treated by E2 were identified, and RT-qPCR validation results showed seven cytoskeletal genes (Myh8, Cenpe, Jak3, Obscn, Ldb3, Mybpc2, Col4a3bp), three genes related to ion channels (Kcnq1, Lrrc26, P2rx3) and Fhl1 transcript 2 were associated with the effects of E2 on myoblast differentiation. These findings suggested E2 helped slow type MyH I fiber formation and impeded fast 2A, 2X/D, and 2B fiber formation.
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Muscle abnormality could be a key reason for congenital clubfoot (CCF) deformity, which manifests itself during fetal development. FHL1 down-regulated expression is involved in the formation of skeletal muscle abnormalities in CCF and FHL1 gene mutations contribute to the development of some kinds of myopathies. Therefore, detecting dynamic expression of Fhl1 and other molecules (Hgf, MyoD1, Myogenin, and Myh4) that control limb muscle development in hind limbs of different gestational age will provide a foundation for further research on the molecular mechanism involves in the myopathies or CCF. The dynamic gene expression levels of Fhl1, Hgf, MyoD1, Myogenin, and Myh4 in the lower limbs of E16, E17, E19, and E20 rat embryos were examined by real-time RT-PCR. Immunofluorescence was used to detect formation of specific muscle fibers (fast or slow fibers) in distal E17 hind limbs. The expression levels of Fhl1, Hgf, MyoD1, Myogenin, and Myh4 were varying in hind limbs of different gestational age. Real-time PCR results showed that all the genes that control skeletal muscle development except for Fhl1 exhibited a peak in E17 lower limbs. Immunofluorescence results showed obviously positive fast-myosin in the distal E17 lower limbs and meanwhile slow-myosin had no apparently signals. E17 was a critical time point for terminal skeletal muscle differentiation in the lower limbs of rat embryos.
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