In this study we found that ACL silencing is sufficient
In this study, we found that ACL silencing is sufficient to impair myoblast differentiation and that this effect is accompanied by a decrease in MYOD early in the myogenic process and by a subsequent decline in fast MyHC protein bcr-abl tyrosine kinase inhibitors at a later stage of differentiation (Figure 4J). Furthermore, overexpression of ACL enhances Myod, Myh1, and Myh4 levels, and it promotes myogenesis, demonstrating that an increase in ACL is sufficient to enhance myogenesis. These data demonstrate that ACL is necessary for myoblast differentiation and provides a modulatory step for promoting fast MyHC synthesis. The effect of ACL on fast myosins is at least in part MYOD dependent, since overexpression of MYOD partially rescued the decrease in Myh1 and Myh2 transcript abundance exerted by ACL silencing in human primary myoblasts. Interestingly, MYOD expression is higher in fast fibers, and it has been shown to activate the Myh4 promoter in an E-box-dependent manner (Wheeler et al., 1999). Several transcription factors have been shown to promote fast fiber identity via MYOD; for example, the co-transcriptional activator Eya1, a MyoD target (upregulated by ACL overexpression), is also enriched in type 2B and 2X fibers, and its forced expression into slow-twitch fibers together with Six1 induces a switch from fiber type 1 and 2A to 2x and 2B (Grifone et al., 2004). Myogenesis is dependent on transcriptional but also other mechanisms. In this study, we show that ACL regulates the net amount of acetyl groups available, and this leads to alterations in acetylation of H3(K9/14) and H3(K27) at the MYOD locus, thus increasing MYOD levels. Increased Myod expression is accompanied by the upregulation of genes that promote SC activation, differentiation, and, in turn, regeneration. ACL overexpression in murine tibialis muscle leads to improved regeneration after cardiotoxin-mediated damage, resulting in significantly increased muscle mass and no effect on fiber type composition. Bracha et al. (2010) recently reported that ACL silencing induces differentiation. The evidence was based mainly on analysis of an unspecified isoform of myosin heavy chain by immunofluorescence and qPCR in C2C12 myoblasts. This is a crucial point, since we found that ACL silencing selectively reduces fast and increases slow MyHC expression. Furthermore, in our study, we used several complementary approaches, in vitro and in vivo, to show that blockade or overexpression of ACL in muscle cells leads to significant perturbation of myogenesis and an increase in regeneration. Generation of skeletal muscle-specific ACL knockout mice would be valuable toward understanding the role of ACL in skeletal muscle development and function. Although AceCS1 (Hallows et al., 2006) as well as nuclear pyruvate dehydrogenase (Sutendra et al., 2014) also provide acetyl-CoA and, thus, regulate histone acetylation, our results clearly demonstrated that, in skeletal muscle, ACL catalyzes the rate-limiting step providing acetyl-CoA, which is further utilized by acetyltransferases to modulate gene expression. It is surprising that changes in acetyl-CoA lead to a particular increase in fast myosin isoforms; this shows a more granular level of control than was seen previously in proliferating cancer cells. H3K9/14/27 acetylation in particular is apparently limited by the availability of acetyl-CoA. However, mechanistic insights on the specificity of histone acetylation and modulation of specific genes by ACL remain elusive and warrant further investigation. Our study does not rule out the possibility that ACL activity might modulate other myogenic factors or myosins directly by regulating histone and/or protein acetylation. The mechanism by which IGF-1 induces differentiation has been long sought; its effect is striking, since in the early stages of myogenesis, IGF-1 induces proliferation, whereas later on it induces differentiation into multi-nuclear myotubes. In the present study, it is demonstrated that the pro-myogenic effect of IGF-1 is mediated, at least in part, by ACL and by promoting acetylation of H3 at the MYOD locus. Since IGF-1 is sufficient to activate ACL via Akt in skeletal muscle (Das et al., 2015), the findings in this study provide a previously unrecognized arm in the IGF-1-signaling pathway. Our data suggest that activation of IGF-1 signaling through anabolic exercise would support the required muscle regeneration that occurs during anabolism-induced breakdown, repair, and hypertrophy. These data also help to show why there is an increase in fast glycolytic myofiber size with anabolism, and perhaps they would indicate why there is a loss of fast fibers coincident with aging and decreases in IGF-1 signaling.