MEF2A regulates the Gtl2-Dio3 microRNA mega-cluster to modulate WNT signaling in skeletal muscle regeneration

Abstract

SUMMARYUnderstanding the molecular mechanisms of skeletal muscle regeneration is crucial to exploiting this pathway for use in tissue repair. Our data demonstrate that the MEF2A transcription factor plays an essential role in skeletal muscle regeneration in adult mice. Injured Mef2a knockout mice display widespread necrosis and impaired myofiber formation. MEF2A controls this process through its direct regulation of the largest known mammalian microRNA (miRNA) cluster, the Gtl2-Dio3 locus. A subset of the Gtl2-Dio3 miRNAs represses secreted Frizzled-related proteins (sFRPs), inhibitors of WNT signaling. Consistent with these data, Gtl2-Dio3-encoded miRNAs are downregulated in regenerating Mef2a knockout muscle, resulting in upregulated sFRP expression and attenuated WNT activity. Furthermore, myogenic differentiation in Mef2a-deficient myoblasts is rescued by overexpression of miR-410 and miR-433, two miRNAs in the Gtl2-Dio3 locus that repress sFRP2, or by treatment with recombinant WNT3A and WNT5A. Thus, miRNA-mediated modulation of WNT signaling by MEF2A is a requisite step for proper muscle regeneration, and represents an attractive pathway for enhancing regeneration of diseased muscle.

MEF2 Transcription Factors Regulate Distinct Gene Programs in Mammalian Skeletal Muscle Differentiation

“…As previously reported by us and others, MEF2A depletion resulted in impaired myotube formation and differentiation (Fig. 2, A-C) (11,12). By contrast, individual depletion of the other MEF2 proteins failed to show any obvious impairment in myotube formation or differentiation (Fig.…”

“…These shRNAs were designed to target all mRNA transcripts generated from each Mef2 gene. Previously, we described the specific and robust knockdown of MEF2A and MEF2C using shRNA adenoviruses (11,15). We subsequently generated shRNAs to specifically target MEF2B (Fig.…”

“…Knockdown of MEF2 Proteins in C2C12 Myoblasts-The four mammalian MEF2 factors display different temporal expression patterns in C2C12 differentiation (11,21). Therefore, we focused our analysis of the individual MEF2 isoform knockdowns on differentiation day 3, because this reflects the time at which the four MEF2 proteins are co-expressed in myotubes.…”

“…We and others have previously shown that MEF2A deficiency results in impaired differentiation of C2C12 skeletal myoblasts and primary myoblasts isolated from injured skeletal muscle of adult MEF2A knock-out mice (11,12). In contrast, differentiation is not impaired when MEF2A is deleted specifically in adult satellite cells in the context of muscle injury but is perturbed when MEF2A, -C, and -D are deleted simultaneously in these cells (13).…”

Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 Increases Neovascularization and Blood Flow Recovery After Ischemia

“…Knockout of the cluster, via knockout of the Meg3 locus, 17,18 led to overexpression of several proangiogenic genes, including Vegfa and Dll4, and drastically increased vascular capillary density in mouse embryos. 19 A recent study showed that the cluster is under transcriptional control of a binding site for the myocyte enhancer factor 2A (MEF2A), 20 a transcription factor known to influence vascular remodeling. 21,22 We show here that inhibition of individual 14q32 microRNAs leads to improvements in postischemic blood flow recovery in vivo.…”