MEF2A regulation of a miRNA mega‐cluster is required for proper skeletal muscle regeneration

Abstract

Skeletal muscle has the ability to regenerate, in response to disease or injury, through the activation of muscle stem cells and their subsequent differentiation into myofibers. This process is controlled by a hierarchy of just a few transcription factors. The role of the MEF2 transcription factor family in skeletal muscle regeneration has not been studied. We performed RNAi‐mediated knockdown of MEF2A in C2C12 cells, an established line of proliferating myoblasts isolated from injured muscle. Unlike developmental myogenesis, MEF2A is the first MEF2 factor expressed in this model. Knockdown of MEF2A in myoblasts resulted in markedly impaired differentiation. Given the source of C2C12 myoblasts we investigated the potential function of MEF2A in regenerative myogenesis in vivo. Cardiotoxin‐induced muscle injury in MEF2A knockout mice resulted in widespread necrosis and reduced myofiber cross‐sectional area indicating defective regeneration. Mechanistically, microarray analysis revealed a downregulation of more than 40 microRNAs (miRNAs); all of which are located in a large miRNA gene cluster. These miRNAs are not related to any of the well‐characterized skeletal muscle‐specific miRNAs. We demonstrate that a single, upstream promoter is bound and activated by MEF2A, suggesting its ability to regulate the entire miRNA domain. Furthermore, miRNA target gene prediction analysis revealed that a cohort of these miRNAs targets inhibitors of Wnt signaling, a key pathway promoting skeletal muscle regeneration. Consistent with the miRNA downregulation these Wnt inhibitors are massively upregulated in injured MEF2A‐deficient muscle. Thus, miRNA‐mediated modulation of Wnt signaling by MEF2A is a requisite step for proper muscle regeneration. Supported by NIH grant HL73304 to FJN.