Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation

Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent inhibition of differentiation was associated with MyoD degradation by the ubiquitin-proteasome pathway. MyoD overexpression in C2C12 myoblasts overrode the differentiation block imposed by hypoxic conditions. Thus, hypoxia by inducing MyoD degradation blocked accumulation of early myogenic differentiation markers such as myogenin and p21 and pRb, preventing both permanent cell cycle withdraw and terminal differentiation. Our study revealed a novel anti-differentiation effect exerted by hypoxia in myogenic cells and identified MyoD degradation as a relevant target of hypoxia.     

In vitro indeterminate teleost myogenesis appears to be dependent on Pax3

The zebrafish (Danio rerio) has been used extensively as a model system for developmental studies but, unlike most teleost fish, it grows in a determinate-like manner. A close relative, the giant danio (Devario cf. aequipinnatus), grows indeterminately, displaying both hyperplasia and hypertrophy of skeletal myofibers as an adult. To better understand adult muscle hyperplasia, a postlarval/postnatal process that closely resembles secondary myogenesis during development, we characterized the expression of Pax3/7, c-Met, syndecan-4, Myf5, MyoD1, myogenin, and myostatin during in vitro myogenesis, a technique that allows for the complete progression of myogenic precursor cells to myotubes. Pax7 appears to be expressed only in newly activated MPCs while Pax3 is expressed through most of the myogenic program, as are c-Met and syndecan-4. MyoD1 appears important in all stages of myogenesis, while Myf5 is likely expressed at low to background levels, and myogenin expression is enriched in myotubes. Myostatin, like MyoD1, appears to be ubiquitous at all stages. This is the first comprehensive report of key myogenic factor expression patterns in an indeterminate teleost, one that strongly suggests that Pax3 and/or Myf5 may be involved in the regulation of this paradigm. Further, it validates this species as a model organism for studying adult myogenesis in vitro, especially mechanisms underlying nascent myofiber recruitment.     

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.     

Transformation by activated ras or fos prevents myogenesis by inhibiting expression of MyoD1

Transformation of myoblasts by activated ras inhibits myogenic differentiation. We demonstrate that this oncogene inhibits expression of the muscle regulatory factors MyoD1 and myogenin. Expression of retroviral-encoded MyoD1 in ras-transformed myoblasts leads to the re-expression of both terminal differentiation markers and lineage markers expressed in proliferating myoblasts (including endogenous MyoD1 and myogenin), suggesting that ras inhibits myogenic differentiation in a manner dependent on the loss of MyoD1 expression. In addition, we show that fos transformation of myoblasts inhibits muscle differentiation by a similar mechanism.     

Expression of myogenic factors in denervated chicken breast muscle: isolation of the chicken Myf5 gene.

In this study, we have isolated and characterized the chicken Myf5 gene, and cDNA clones encoding chicken MyoD1 and myogenin. The chicken Myf5 and MRF4 genes are tandemly located on a single genomic DNA fragment, and the chicken Myf5 gene is organized into at least three exons. Using genomic and cDNA probes, we further analyzed the mRNA levels of four myogenic factors during chicken breast muscle development. This analysis revealed that myogenin expression is restricted to in ovo stages in breast muscle, and is not detectable in neonatal and adult stages. On the other hand, Myf5 expression is detectable until day 7 post-hatching, and is not found in adult muscle, whereas high levels of MyoD1 and MRF4 are detectable at all stages. To further understand the roles of innervation on muscle maturation, we analyzed the expression of the four myogenic factors in denervated adult breast muscle. We found that MyoD1, myogenin, and MRF4 are induced at high levels in denervated muscle, whereas no change occurs in the level of Myf5. These studies suggest that innervation controls the relative abundance and type of myogenic factors that are expressed in adult muscle, and that when nerve control is removed, the muscle reverts to a neonatal phenotype, with the enhanced expression of three myogenic factors (MyoD1, myogenin, and MRF4).     

Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf-5, two additional factors Myf-3 and Myf-4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf-3, Myf-4 and Myf-5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf-4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf-5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf-3 and Myf-4 mRNA but very little Myf-5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf-5 but no MyoD1. Myf-4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)