Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by miR-1/133a-Mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster

Abstract

Muscle stem cells undergo a dramatic metabolic switch to oxidative phosphorylation during differentiation, which is achieved by massively increased mitochondrial activity. Since expression of the muscle-specific miR-1/133a gene cluster correlates with increased mitochondrial activity during muscle stem cell (MuSC) differentiation, we examined the potential role of miR-1/133a in metabolic maturation of skeletal muscles in mice. We found that miR-1/133a downregulate Mef2A in differentiated myocytes, thereby suppressing the Dlk1-Dio3 gene cluster, which encodes multiple microRNAs inhibiting expression of mitochondrial genes. Loss of miR-1/133a in skeletal muscles or increased Mef2A expression causes continuous high-level expression of the Dlk1-Dio3 gene cluster, compromising mitochondrial function. Failure to terminate the stem cell-like metabolic program characterized by high-level Dlk1-Dio3 gene cluster expression initiates profound changes in muscle physiology, essentially abrogating endurance running. Our results suggest a major role of miR-1/133a in metabolic maturation of skeletal muscles but exclude major functions in muscle development and MuSC maintenance.

miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro

“…Furthermore, treatment of these clones with the mitochondrial uncoupling agent BAM15, which produces maximal oxygen consumption, revealed an even greater difference in OCR between control and tKO clones (Figure 2F). These results suggest that these three microRNA are required to maintain mitochondrial respiratory capacity, and are consistent with the findings from miR-1a-/-133a-/- skeletal muscle specific double KO animals that showed impairment of mitochondrial function (Wüst et al, 2018). Thus, although the tKO C2C12 clones differentiate, their differentiation is slightly impaired compared to the WT cells and the cells suffer a downregulation of mitochondria function.…”

“…We also did not see a change in number of nuclei visible per fiber cross-section (Figure 5F). Moreover, we do not observe mitochondrial aggregates in the center of fibers or granulate-like patterns in tKO skeletal muscle fibers, phenotypes that were seen in miR-1a/-133a knockouts (Wüst et al, 2018). The size of heart in the triple KO animals is comparable to that in wild type (Figure S4A).…”

“…Even though the quadriceps size is very similar in all experimental mice (Figure 5A), the H&E staining shows that the average fiber cross-sectional area in the tKO animals lacking miR-206 and miR-1a is significantly smaller than in wild type mice (Figure 5B, 5C). Based on previous literature report (Wüst et al, 2018) we decided to check the mitochondria content and organization in muscle fibers. Staining with anti-mitochondria antibody (Figure 5D) does not reveal any differences in fiber type content (Figure 5E).…”

“…A conditional skeletal muscle specific knockout of Dicer in mice leads to global loss of miRNAs in developing skeletal muscle, resulting in widespread apoptosis and abnormal myofiber morphology (O’Rourke et al, 2007). Over the last 14 years miR-206 , miR-1a-1 and miR-1a-2 have been hypothesized to not only be very important for muscle differentiation, but also essential for this process (Anderson et al, 2006; Chen et al, 2006; Chen et al, 2010; Dey et al, 2011; Gagan et al, 2012; Goljanek-Whysall et al, 2012; Heidersbach et al, 2013; Hirai et al, 2010; Kim et al, 2006; Koutsoulidou et al, 2011; Kwon et al, 2005; Mishima et al, 2009; Rao et al, 2006; Sokol and Ambros, 2005; Sweetman et al, 2008; Vergara et al, 2018; Wystub et al, 2013; Wüst et al, 2018; Yuasa et al, 2008; Zhao et al, 2007; Zhao et al, 2005). miR-206 , miR-1a-1 and miR-1a-2 are members of the myomir family and are expressed from bicistronic loci.…”

“…Moreover, knockout of two bicistronic loci ( miR-1a-1/133a-2 and miR-1a-2/133a-1 dKO ) showed complete embryonic lethality at embryonic stage E11.5 (Wystub et al, 2013). The miR-1a/133a skeletal muscle specific dKO mice were alive, but had metabolic problems caused by incorrect functioning of mitochondria (Wüst et al, 2018).…”