Myogenic transcription factors with a basic helix-loop-helix (bHLH) such as MYOD, myogenin, MRF4, and MYF5 contribute to muscle differentiation and regulation. The MYF5 gene located on chromosome 12 encodes for myogenic factor 5 (MYF5), which has a role in skeletal and extraocular muscle development and rib formation. Variants in MYF5 were found to cause external ophthalmoplegia with rib and vertebral anomalies (EORVA), a rare recessive condition. To date, three homozygous variants in MYF5 have been reported to cause EORVA in six members of four unrelated families. Here, we present a novel homozygous MYF5 frameshift variant, c.596dupA p. (Asn199Lysfs*49), causing premature protein termination and presenting with external ophthalmoplegia, ptosis, and scoliosis in three siblings from a consanguineous family of Pakistani origin. With four MYF5 variants now discovered, genetic testing and paediatric assessment for extra-ocular features should be considered in all cases of congenital ophthalmoplegia.

Although it has long been known that growth media withdrawal is a prerequisite for myoblast differentiation and fusion, the underpinning molecular mechanism remains somewhat elusive. Using isolated porcine muscle satellite cells (SCs) as the model, we show elevated O-GlcNAcylation by O-GlcNAcase (OGA) inhibition impaired SC differentiation (D5 P < 0.0001) but had unnoticeable impacts on SC proliferation. To explore the mechanism of this phenotype, we examined the expression of the transcription factor myogenin, a master switch of myogenesis, and found its expression was downregulated by elevated O-GlcNAcylation. Because insulin/IGF-1/Akt axis is a strong promoter of myoblast fusion, we measured the phosphorylated Akt and found that hyper O-GlcNAcylation inhibited Akt phosphorylation, implying OGA inhibition may also work through interfering with this critical differentiation-promoting pathway. In contrast, inhibition of O-GlcNAc transferase (OGT) by its specific inhibitor had little impact on either myoblast proliferation or differentiation (P > 0.05). To confirm these in vitro findings, we used chemical-induced muscle injury in the pig as a model to study muscle regenerative myogenesis and showed how O-GlcNAcylation functions in this process. We show a significant decrease in muscle fiber cross sectional area (CSA) when OGA is inhibited (P < 0.05), compared to nondamaged muscle, and a significant decrease compared to control and OGT inhibited muscle (P < 0.05), indicating a significant impairment in porcine muscle regeneration in vivo. Together, the in vitro and in vivo data suggest that O-GlcNAcylation may serve as a nutrient sensor during SC differentiation by gauging cellular nutrient availability and translating these signals into cellular responses. Given the importance of nutrition availability in lean muscle growth, our findings may have significant implications on how muscle growth is regulated in agriculturally important animals.
Cells use a variety of post translational modifications (PTMs) as a mechanism to transduce extracellular signals and adapt their behaviors in response to intracellular nutrient abundance. O-GlcNAcylation, the addition of single sugars to a protein’s serine/threonine residues, has been established as a nutrient sensing PTM in a wide range of cell types. Here, we show the functional importance O-GlcNAcylation in porcine myogenesis. We used isolated porcine satellite cells as the model and pharmacological inhibitors to O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) as the tool to study the role of O-GlcNAcylation in porcine myogenesis. Our data show that although O-GlcNAcylation does not play a significant role in muscle cell proliferation, low level of O-GlcNAcylation is critical for muscle cell differentiation. We demonstrate that inhibition of OGA leads to higher level of O-GlcNAcylation and inhibition of myoblast fusion even though the growth medium (high nutrients) has been shifted to the differentiation medium (low nutrients). Together, these data show that porcine muscle cells use O-GlcNAcylation to sense the cellular nutrient levels and adjust their fate in accordance with the strength of the O-GlcNAcylation signals.

BACKGROUND: Current treatment options for cachexia, which impairs disease prognosis, are limited. Muscle-enriched microRNAs and protein acetylation are involved in muscle wasting including lung cancer (LC) cachexia. Poly(ADP-ribose) polymerases (PARP) are involved in muscle metabolism. We hypothesized that muscle-enriched microRNA, protein hyperacetylation, and expression levels of myogenic transcription factors (MTFs) and downstream targets, muscle loss and function improve in LC cachectic Parp-1(−/−) and Parp-2(−/−) mice.
METHODS: Body and muscle weights, grip strength, muscle phenotype, muscle-enriched microRNAs (miR-1, -133, -206, and -486), protein acetylation, acetylated levels of FoxO1, FoxO3, and PGC-1α, histone deacetylases (HDACs) including SIRT1, MTFs, and downstream targets (α-actin, PGC-1α, and creatine kinase) were evaluated in diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) wild type (WT), Parp-1(−/−) and Parp-2−/− mice.
RESULTS: Compared to WT cachectic animals, in both respiratory and limb muscles of Parp-1(−/−) and Parp-2(−/−) cachectic mice: downregulation of muscle-specific microRNAs was counterbalanced especially in gastrocnemius of Parp-1(−/−) mice; increased protein acetylation was attenuated (improvement in HDAC3, SIRT-1, and acetylated FoxO3 levels in both muscles, acetylated FoxO1 levels in the diaphragm); reduced MTFs and creatine kinase levels were mitigated; body and muscle weights, strength, and muscle fiber sizes improved, while tumor weight and growth decreased.
CONCLUSIONS: These molecular findings may explain the improvements seen in body and muscle weights, limb muscle force and fiber sizes in both Parp-1(−/−) and Parp-2(−/−) cachectic mice.
CONCLUSIONS: PARP-1 and -2 play a role in cancer-induced cachexia, thus selective pharmacological inhibition of PARP-1 and -2 may be of interest in clinical settings.

Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein expression of Myf-5 (FC: 1.57 GMAZA vs GM p≤0.05), MyoD (FC: 1.14 DM vs GM p≤0.05; FC: 1.47 DMAZA vs GM p≤0.05), p21 (FC: 1.36 GMAZA vs GM p≤0.01; FC: 1.49 DM vs GM p≤0.05; FC: 1.82 DMAZA vs GM p≤0.01) and MyHC (FC: 1.40 GMAZA vs GM p≤0.01; FC: 2.39 DM vs GM p≤0.05; FC: 3.51 DMAZA vs GM p≤0.01). Our results suggest that AZA-induced DNA demethylation can modulate cell cycle progression and enhance myogenesis. The effects of AZA may open novel clinical uses in the field of muscle injury research and treatment.