Role of the transcription factor MRF4 in adult skeletal muscle

Myogenesis is a dynamic process in which mononucleated undifferentiated myoblasts first proliferate, then withdraw from the cell cycle and finally differentiate and fuse to form the multinucleated mature muscle fibers. This process is controlled by members of a family of muscle-specific basic helix-loop-helix (bHLH) proteins that, in concert with members of the ubiquitous E2A and myocytes enhancers factor 2 (MEF2) families, activate the differentiation program by inducing transcription of regulatory and structural muscle specific gene. The MRF proteins contains one or two transactivation domain, a conserved basic DNA-binding domain essential for sequence-specific DNA binding, and an HLH motif required for heterodimerization. MRFs specific knockout studies suggest that MyoD and Myf5 are required for commitment to the myogenic lineage, whereas myogenin plays a critical role in the expression of the terminal muscle phenotype; Mrf4 partly subserves both roles. In the adult skeletal muscle, the expression of MRFs considerably changes: Myf5 is not expressed in adult fibers, MyoD and myogenin are expressed at low levels and respectively in fast muscle and in slow muscle, Mrf4 is the only muscle regulatory factors expresses at high levels in adult skeletal muscle, but its role is still unknown. The aim of this work is study the expression and the physiological role of Mrf4 in the adult skeletal muscle. We demonstrate that Mrf4 is similarly expressed at mRNA and protein levels in the slow soleus muscle and in the fast EDL muscle, but this transcription factor has a predominantly nuclear localization in soleus and a predominantly cytosolic localization in EDL. We also demonstrate that Mrf4 expression is activity dependent using two experimental models: elettrostimulation and denervation (inactivity condition). When EDL muscles were stimulates with a slow pattern, Mrf4 translocates to the nucleus, whereas with a fast pattern Mrf4 remains in the cytoplasm. Moreover, after denervation Mrf4 accumulates in to the nucleus. This observation suggests that MRF4 may undergo nucleo-cytoplasmic shuttling, as in the case of other transcription factors, but not describes for the MRFs. To get further insight about the function of Mrf4 in adult muscle we examined the effect of Mrf4 overexpression and knockdown using an in vivo transfection approach in adult rat skeletal muscles. We evaluated if Mrf4 is involved in the regulation of two features of muscle phenotype: the muscle growth and the fiber type specification. Muscle growth: we demonstrate that Mrf4 silencing in adult and regenerating muscles induces hypertrophy. On the other hand, the overexpression of Mrf4 cDNA in regenerating muscles, but not in the adult muscles, causes a decrease of cross sectional area of transfected fibers. Moreover the MRF4 knockdown prevents denervation atrophy. This data suggest that Mrf4 acts as a negative regulator of muscle fiber growth. Fiber type specification: we used two luciferase reporter under control of the MyHC slow (MyHC slow-Luc) and the MyHC 2B (MyHC 2B-Luc) promoter. We demonstrate that Mrf4 knockdown in adult skeletal muscle inhibits the activity of MyHC slow-Luc and induces the activity of MyHC 2B-Luc; on the other hand, this reporter is activated by Mrf4 overexpression. We have also study the effect of Mrf4 silencing on endogenous gene: we demonstrate that Mrf4 knockdown blocks the expression of endogenous MyHC slow. This data suggest that Mrf4 is involved in the induction and in the maintenance of slow gene program.