KLF15Regulates Slow Myosin Heavy Chain Expression Through NFATc1in C2C12Myotubes

A comprehensive understanding of genetic and environmental factors that control skeletalmuscle fiber type specification and transformation is essential not only in sports science, butalso in myopathy and metabolic disorders. Krüppel-like factors (KLFs) are a subfamily of thezinc-finger class of transcription factors, which are involved in the development, homeostasis,and pathology of cardiovascular systems. Compared to cardiac and smooth muscles, the roleof KLFs in skeletal muscle is much less understood. In this study, overexpression andknockdown KLF15were used to analyze the mechanism between KLF15and myosin heavychain slow. The authors also studied the mechanism between KLF15and CaN-NFATsignaling. The main results were as follows:1, The expression of endogenous Klf15gene in skeletal muscle and cultured muscle cells.To investigate the role of KLF15in myogenesis, its transcription profile during myogenicdifferentiation was first analyzed using C2C12cells. The results showed that Klf15mRNAexpression in C2C12cells increased by about2.4-fold after two days of differentiation (P <0.01). This increase in Klf15expression persisted afterwards. At the same time, theendogenous expression of Klf15was analyzed in adult mouse skeletal muscle. The resultsshow that Klf15has a high expression in skeletal muscle and Klf15mRNA level was3-foldhigher in the mouse slow, oxidative soleus muscle (SL) than in the fast, glycolytic tibialisanterior muscle (TA)(P<0.01).2, KLF15regulates slow myosin heavy chain expression in C2C12myotubes. To test ifKLF15regulates myogenic differentiation of muscle cells, Klf15in C2C12myoblasts wassilenced by siRNA and the cells were then induced to differentiate for4days. The resultsshow that, inhibition of Klf15did not alter the expression of the myogenic regulatory factormyogenin. At the same time, the knockdown of Klf15had no effect on myogenicdifferentiation of C2C12myoblasts as evidenced by the lack of difference in the number ofmyogenin-and MHC-positive nuclei between mock and Klf15knockout cells. And the furtherstudy show that, KLF15can significant up-regulate the expression of Myhc-slowgene(P<0.05), but the study also showed that, KLF15cannot influence the expression of MYHC.3, KLF15regulation of slow myosin heavy chain depends on NFAT signaling. Toexplore the mechanism by which KLF15upregulates slow myosin heavy chain, NFATsignaling was analyzed in both Klf15knocked down and KLF15overexpressing C2C12myotubes by measuring the expression of Nfatc1target gene, modulatory calcineurininteracting protein exon4isoform (Mcip1.4) and Nfatc1gene. The results show that, KLF15can significant up-regulate the expression of Mcip1.4and Nfatc1gene mRNA andprotein(P<0.05). At the same time, inhibition of NFATc1abolished KLF15overexpressioninduced upregulation of MHC-β/slow showing that KLF15-mediated upregulation ofMHC-β/slow is dependent on NFATc1signaling.4, KLF15regulates NFATc1expression by direct targeting of the promoter region. Toexplore the mechanism of regulation of NFATc1by KFL15, the promoter structure of Nfatc1gene (-1554—+333) was analyzed and nine potential KLF15binding sites were identified.A luciferase reporter vector using the Nfatc1promoter (pGL3-Basic-Nfatc1) was constructedand co-transfected with PcDNA3.1(Control) or PcDNA3.1-KLF15into HEK293T cells for48h. co-transfection of the luciferase reporter vector with PcDNA3.1-KLF15induced asignificant4-fold increase in the cell lysate luciferase activity (P <0.01) when compared tothe control showing that KLF15upregulates NFATc1through direct activation of its promoteractivity(P<0.01).