Microphthalmia-associated transcription factor is required for mature myotube formation

Background: The roles of microphthalmia-associated transcription factor (Mitt) in the skeletal muscle and during myogenesis are unclear. Methods: Expression of Mitf in mouse tissues and during myogenesis was evaluated. Effects of Miff knockdown on myogenesis and gene expression related to myogenesis were subsequently explored. Furthermore, effects of p21, a cyclin-dependent kinase inhibitor, and integrin alpha 9 (Itga9) were examined. Results: Mitf was highly expressed in the skeletal muscle; Miff-A and -J were expressed. Mitf expression increased after differentiation stimulation in C2C12 myogenic cells. Down-regulation of Miff expression by transfection of siRNA for common Mitf inhibited myotube formation, which was reproduced by Miff-A knockdown. Morphometric analyses indicated that both multinucleated cell number and the proportion of myotubes with more than 6 nuclei were decreased in Mitf-knockdown cells, suggesting that Miff is required for not only the formation of nascent myotubes but also their maturation. Searching for genes positively regulated by Mitf revealed p21 and Itga9; decreasing Miff expression inhibited up-regulation of p21 expression after differentiation stimulation and blocked the induction of Itga9 expression in response to differentiation. Knockdown of p21 decreased the number of multinucleated cells, whereas Itga9 knockdown did not affect the myotube number. Both p21 knockdown and Itga9 knockdown decreased the proportion of myotubes with more than 6 nuclei. General significance: Miff positively regulates skeletal muscle formation; Miff is significantly expressed during myogenesis, and is required for efficient myotube formation through expression of p21 and Itga9. (C) 2011 Elsevier B.V. All rights reserved.