Formation Of Multinucleated Myotubes Research Articles

Arsenic Inhibits Myogenic Differentiation and Muscle Regeneration

BackgroundThe incidence of low birth weights is increased in offspring of women who are exposed to high concentrations of arsenic in drinking water compared with other women. We hypothesized that effects of arsenic on birth weight may be related to effects on myogenic differentiation.ObjectiveWe investigated the effects of arsenic trioxide (As2O3) on the myogenic differentiation of myoblasts in vitro and muscle regeneration in vivo.MethodsC2C12 myoblasts and primary mouse and human myoblasts were cultured in differentiation media with or without As2O3 (0.1–0.5 μM) for 4 days. Myogenic differentiation was assessed by myogenin and myosin heavy chain expression and multinucleated myotube formation in vitro; skeletal muscle regeneration was tested using an in vivo mouse model with experimental glycerol myopathy.ResultsA submicromolar concentration of As2O3 dose-dependently inhibited myogenic differentiation without apparent effects on cell viability. As2O3 significantly and dose-dependently decreased phosphorylation of Akt and p70s6k proteins during myogenic differentiation. As2O3-induced inhibition in myotube formation and muscle-specific protein expression was reversed by transfection with the constitutively active form of Akt. Sections of soleus muscles stained with hematoxylin and eosin showed typical changes of injury and regeneration after local glycerol injection in mice. Regeneration of glycerol-injured soleus muscles, myogenin expression, and Akt phosphorylation were suppressed in muscles isolated from As2O3-treated mice compared with untreated mice.ConclusionOur results suggest that As2O3 inhibits myogenic differentiation by inhibiting Akt-regulated signaling.

RNA‐binding proteins Rbm38 and Rbm24 regulate myogenic differentiation via p21‐dependent and ‐independent regulatory pathways

Skeletal muscle differentiation entails organized sequential events, including cell cycle arrest of proliferating myoblast cells and cell fusion, which lead to the formation of multinucleated myotubes. This process involves both transcriptional and post-transcriptional regulation of the gene expression of myogenic proteins, as well as cell-cycle related proteins. RNA-binding proteins bind to specific sequences of target RNA and regulate gene expression in a post-transcriptional manner. However, few tissue-specific RNA binding proteins have been identified. Herein, we report that the RNA binding proteins Rbm24 and Rbm38 were found to be preferentially expressed in muscle during differentiation in vitro. Further, knockdown of either by RNA interference suppressed cell-cycle arrest and delayed myogenic differentiation in C2C12 cells. In contrast, over-expression of Rbm24 or Rbm38 induced cell cycle arrest, and then had a positive effect on myogenic differentiation. Immunoprecipitation-RT-PCR analysis using tagged Rbm proteins indicated that Rbm38 binds to the p21 transcript in vivo. Consistent with this, differentiation of Rbm38 knockdown cells was rescued by over-expression of p21. Together, our results suggest that Rbm38 plays a crucial role in cell cycle arrest and myogenic differentiation via its binding to p21.

Transduction of MyoD protein into myoblasts induces myogenic differentiation without addition of protein transduction domain

Protein transduction is a new technology with the potential for controlling cellular functions. Addition of tissue-specific transcription factors into cells could induce a specific differentiation pathway. In this paper, MyoD protein, a muscle-specific transcription factor, was introduced into the mouse myoblast cell line C2C12 to induce myogenic differentiation. Addition of fluorescently labeled MyoD protein revealed that MyoD can permeate the cell membrane without the addition of a protein transduction domain (PTD). Addition of MyoD protein induced formation of multi-nucleated myotubes. These results showed that MyoD protein has a PTD in its primary sequence and that myogenic differentiation can be induced by addition of MyoD protein.

Engineering a multi‐nucleated myotube, the role of the actin cytoskeleton

Myoblasts in vitro form characteristic arrays of bipolar-shaped cells prior to fusion. We have shown that the actin cytoskeleton re-organizes in these fusing cells and that the interaction of non-muscle myosin 2A with actin at the plasma membrane helps to generate the bipolar shape of myoblasts, which is key for fusion. Here we discuss how fusion occurs, and in particular how the actin cytoskeleton is involved. Myoblast fusion is essential to form the multi-nucleated muscle fibres that make up the skeletal muscle. Skeletal muscle fibres contain many nuclei, roughly one nucleus to every 15 sarcomeres (35 microm) in adult muscle, although this varies with muscle type (Bruusgaard et al., 2006). Thus a muscle fibre 30 cm long contains about 8000 nuclei and is formed by the fusion of about 8000 cells during development. The formation of multi-nucleated myotubes has been intensively studied for many years using a number of different systems. Many different proteins have been identified using Drosophila as a model system (e.g. see reviews by Taylor, 2000, 2002) that have given an insight into what happens in mammals. However, the process of fusion of mammalian cells is less well understood, and this paper will cover some of the aspects of mammalian myoblast fusion, with a particular focus on the role of the actin cytoskeleton.

Regulation of Myoblast Motility and Fusion by the CXCR4-associated Sialomucin, CD164

Myoblast fusion is fundamental to the development and regeneration of skeletal muscle. To fuse, myoblasts undergo cell-cell recognition and adhesion and merger of membranes between apposing cells. Cell migration must occur in advance of these events to bring myoblasts into proximity, but the factors that regulate myoblast motility are not fully understood. CD164 is a cell surface sialomucin that is targeted to endosomes and lysosomes via its intracellular region. In hematopoietic progenitor cells, CD164 forms complexes with the motility-stimulating chemokine receptor, CXCR4, in response to the CXCR4 ligand, CXCL12/SDF-1 (Forde, S., Tye, B. J., Newey, S. E., Roubelakis, M., Smythe, J., McGuckin, C. P., Pettengell, R., and Watt, S. M. (2007) Blood 109, 1825-1833). We have previously shown that CD164 stimulates myotube formation in vitro. We report here that CD164 is associated with CXCR4 in C2C12 myoblasts. Cells in which CD164 levels are increased or decreased via overexpression or RNA interference-mediated knockdown, respectively, show enhanced or reduced myotube formation and cell migration, the latter both basally and in response to CXCL12/SDF-1. Furthermore, expression of CD164 cytoplasmic tail mutants that alter the endosome/lysosome targeting sequence and, consequently, the subcellular localization in myoblasts, reveals a similar correlation between cell motility and myotube formation. Finally, Cd164 mRNA is expressed in the dorsal somite (the early myogenic compartment of the mouse embryo) and in premuscle masses. Taken together, these results suggest that CD164 is a regulator of myoblast motility and that this property contributes to its ability to promote myoblast fusion into myotubes.

Twist induces reversal of myotube formation

Mammals possess reduced ability to regenerate lost tissue, compared with other vertebrates, which can regenerate through differentiation of precursor cells or de-differentiation. Mammalian multinucleated myotube formation is a differentiation process, which arises from the fusion of mononucleated myoblasts and is thought to be an irreversible process toward muscle formation. By overexpressing the Twist gene in terminally differentiated myotubes, we managed to induce reversal of cell differentiation. More specifically, following expression of the Twist gene, myotubes underwent morphological changes that caused them to cleave. This was accompanied by a reduction in the expression of certain myogenic markers. Interestingly, Twist overexpression also caused a reduction in the muscle transcription factor MyoD. Further experiments showed an increase in the cell cycle entry molecule, cyclin D1 and initiation of DNA synthesis, due to Twist overexpression. The exploitation of Twist-mediated reversal of differentiation and the study of its specific mechanism would be important in order to study mammalian cellular de-differentiation and determine its potential in muscle regeneration.

Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts

Tissue engineering of skeletal muscle from cultured cells has been attempted using a variety of synthetic and natural macromolecular scaffolds. Our study describes the application of artificial scaffolds (collagen sponges, CS) consisting of collagen-I with parallel pores (width 20–50 μm) using the permanent myogenic cell line C2C12. CS were infiltrated with a high-density cell suspension, incubated in medium for proliferation of myoblasts prior to further culture in fusion medium to induce differentiation and formation of multinucleated myotubes. This resulted in a parallel arrangement of myotubes within the pore structures. CS with either proliferating cells or with myotubes were grafted into the beds of excised anterior tibial muscles of immunodeficient host mice. The recipient mice were transgenic for enhanced green fluorescent protein (eGFP) to determine a host contribution to the regenerated muscle tissue. Histological analysis 14–50 days after surgery showed that donor muscle fibres had formed in situ with host contributions in the outer portions of the regenerates. The function of the regenerates was assessed by direct electrical stimulation which resulted in the generation of mechanical force. Our study demonstrated that biodegradable CS with parallel pores support the formation of oriented muscle fibres and are compatible with force generation in regenerated muscle.

Microenvironments Engineered by Inkjet Bioprinting Spatially Direct Adult Stem Cells Toward Muscle- and Bone-Like Subpopulations

In vivo, growth factors exist both as soluble and as solid-phase molecules, immobilized to cell surfaces and within the extracellular matrix. We used this rationale to develop more biologically relevant approaches to study stem cell behaviors. We engineered stem cell microenvironments using inkjet bioprinting technology to create spatially defined patterns of immobilized growth factors. Using this approach, we engineered cell fate toward the osteogenic lineage in register to printed patterns of bone morphogenetic protein (BMP) 2 contained within a population of primary muscle-derived stem cells (MDSCs) isolated from adult mice. This patterning approach was conducive to patterning the MDSCs into subpopulations of osteogenic or myogenic cells simultaneously on the same chip. When cells were cultured under myogenic conditions on BMP-2 patterns, cells on pattern differentiated toward the osteogenic lineage, whereas cells off pattern differentiated toward the myogenic lineage. Time-lapse microscopy was used to visualize the formation of multinucleated myotubes, and immunocytochemistry was used to demonstrate expression of myosin heavy chain (fast) in cells off BMP-2 pattern. This work provides proof-of-concept for engineering spatially controlled multilineage differentiation of stem cells using patterns of immobilized growth factors. This approach may be useful for understanding cell behaviors to immobilized biological patterns and could have potential applications for regenerative medicine.

Static magnetic fields enhance skeletal muscle differentiation in vitro by improving myoblast alignment

Static magnetic field (SMF) interacts with mammal skeletal muscle; however, SMF effects on skeletal muscle cells are poorly investigated. The myogenic cell line L6, an in vitro model of muscle development, was used to investigate the effect of a 80 +/- mT SMF generated by a custom-made magnet. SMF promoted myogenic cell differentiation and hypertrophy, i.e., increased accumulation of actin and myosin and formation of large multinucleated myotubes. The elevated number of nuclei per myotube was derived from increased cell fusion efficiency, with no changes in cell proliferation upon SMF exposure. No alterations in myogenin expression, a modulator of myogenesis, occurred upon SMF exposure. SMF induced cells to align in parallel bundles, an orientation conserved throughout differentiation. SMF stimulated formation of actin stress-fiber like structures. SMF rescued muscle differentiation in the presence of TNF, a muscle differentiation inhibitor. We believe this is the first report showing that SMF promotes myogenic differentiation and cell alignment, in the absence of any invasive manipulation. SMF-enhanced parallel orientation of myotubes is relevant to tissue engineering of a highly organized tissue such as skeletal muscle. SMF rescue of muscle differentiation in the presence of TNF may have important therapeutic implications.

Muscle Development in the Embryo and Hatchling

Muscle cell proliferation, migration, adhesion, and fusion are processes involved with the formation of multinucleated myotubes that will further differentiate into mature muscle fibers. The process of muscle fiber development is nearly complete at the time of hatch. Muscle growth during the embryonic period of development is characterized by an increase in myoblast cell number through hyperplasia. Posthatch muscle fiber growth occurs through muscle fiber enlargement by the process of hypertrophy, which results from the recruitment of satellite cell nuclei. Hyperplasia and hypertrophy are regulated by factors extrinsic to the cell. These extrinsic elements include growth factors and the extracellular matrix. The growth factors, hepatocyte growth factor, fibroblast growth factor 2, transforming growth factor-β, insulin-like growth factor, and myostatin stimulate or inhibit myoblast and satellite cell proliferation and differentiation. Some of these growth factors like fibroblast growth factor 2 must interact with a low affinity extracellular matrix macromolecule to bind to their high affinity receptor necessary for cell signaling. It is probable that the expression of extracellular matrix proteins involved in growth factor signaling will affect muscle growth properties during hyperplasia and hypertrophy. As signaling pathways associated with muscle growth mechanisms are further understood, the poultry industry may find it beneficial to include the expression of key genes in their selection strategies.

Enhancer of Polycomb1, a Novel Homeodomain Only Protein-binding Partner, Induces Skeletal Muscle Differentiation

Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.

Conversion of C2C12 Myoblast into Adipoblast with Thiazolidinediones - A Possible Basis for Intramuscular Fat Generation in Meat Animals

Thiazolidinediones (TZDs) act as potent activators of the adipose differentiation program in established preadipose cell lines. TZD's have also been investigated in diabetic patients and reported to act as PPAR-y ligands. In this report, the effects of TZDs on the differentiation pathway of myoblasts have been investigated. C2C12 mouse myoblasts were grown in Dulbecco's Modified Eagles medium for 4-5 days until they reached almost 100% confluency. Post-confluent cells (day 0) were further exposed to adipogenic induction medium along with TZDs for 48 hours. Thereafter, cells were exposed only to TZDs every 48 h until day 10. The control was provided with differentiation medium without any treatment. Alterations in the cells during the differentiation programme were analyzed on the basis of fusion index, oil-red-o staining, adipocyte index, adipocyte stain uptake measurement, immuno-histochemistry and western blotting. Exposure of C2C12 mouse myoblasts to TZDs prevented the expression of myosin heavy chain with parallel increase in the expression of C/EBP-a and PPAR-y and acquisition of adipocyte morphology, thus abolishing the formation of multinucleated myotubes. TZDs exert their adipogenic effects only in non-terminally differentiated myoblasts; myotubes were insensitive to the compound. Continuous exposure (at least 4-5 doses) to inducers after the growth arrest was essential to provide a sustained environment to the cells converting to fully matured adipoctyes. The results indicate that TZDs specifically converted the differentiation pathway of myoblasts into that of adipoblasts.

C-Abl acetylation by histone acetyltransferases regulates its nuclear-cytoplasmic localization.

c-Abl function is strictly dependent on its subcellular localization. Using an in vitro approach, we identify c-Abl as a new substrate for p300, CBP (CREB-binding protein) and PCAF (p300/CBP-associated factor) histone acetyltransferases. Remarkably, acetylation markedly alters its subcellular localization. Point mutagenesis indicated that Lys 730, located in the second nuclear localization signal, is the main target of p300 activity. It has previously been reported that c-Abl accumulates in the cytoplasm during myogenic differentiation. Here, we show that c-Abl protein is acetylated at early stages of myogenic differentiation. Indeed, acetylation on Lys 730 drives c-Abl accumulation in the cytoplasm and promotes differentiation. Thus, Lys 730 acetylation is a novel post-translational modification of c-Abl and a novel mechanism for modulating its subcellular localization that contributes to myogenic differentiation.

Tumor Necrosis Factor-like Weak Inducer of Apoptosis Inhibits Skeletal Myogenesis through Sustained Activation of Nuclear Factor-κB and Degradation of MyoD Protein

In this study we have investigated the effect and the mechanisms by which tumor necrosis factor-like weak inducer of apoptosis (TWEAK) modulates myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK inhibited their differentiation evident by a decrease in the expression of creatine kinase, myosin heavy chain-fast twitch, myogenin, and the formation of multinucleated myotubes. TWEAK also inhibited the differentiation of mouse primary myoblasts. Conversely, the proliferation of C2C12 myoblasts and the expression of a cell-cycle regulator cyclin D1 were increased in response to TWEAK treatment. Inhibition of cellular proliferation using hydroxyurea only partially reversed the inhibitory effect of TWEAK on myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK resulted in the activation of nuclear factor-kappaB (NF-kappaB), the (IkappaB) IkappaB kinase (IKK) complex, and the phosphorylation and degradation of IkappaBalpha protein. Inhibition of NF-kappaB activity by overexpression of a dominant negative mutant of IkappaBalpha (IkappaBalphaDeltaN) significantly increased the myogenic differentiation in TWEAK-treated C2C12 cultures. Furthermore, overexpression of a dominant negative mutant of IKKbeta (IKKbetaK44A) but not IKKalpha (IKKalphaK44M) reversed the inhibitory effect of TWEAK on myogenesis. TWEAK inhibited the expression of myogenic regulatory factors MyoD and myogenin and also induced the degradation of MyoD protein. Finally, inhibition of NF-kappaB activation through overexpression of IKKbetaK44A prevented the degradation of MyoD protein. Overall, our data suggest that TWEAK inhibits myogenesis through the activation of NF-kappaB signaling pathway and degradation of MyoD protein.

Myogenic Transdifferentiation of Autologous Cardiac Fibroblasts

Ventricular muscle cells of adult mammals are terminally differentiated cells that lack regenerative capability. The goal of this study is to induce myocardial regeneration by using myogenic cardiac fibroblasts to replace, repair, maintain, and enhance the cardiac function of an injured heart. Mongrel dogs (18) were randomly assigned into three groups (n=6/group) to have myocardial infarction (MI), MI + autologous cardiac fibroblasts, and MI + myogenic autologous cardiac fibroblasts. Cardiac fibroblasts were isolated from a biopsy sample of the heart after enzymatic treatment and proliferated into significant quantity in culture. Myogenic conversion was induced by 0.3 mM of 5-aza-2′-deoxycytidine treatment in culture 3 days before usage. Myogenic conversion of the treated cardiac fibroblasts was documented by the formation of multinucleated myotubes in differentiation medium. A dramatic reduction of scar area (11 ± 2 vs. 23 ± 4) replaced by viable muscle tissue and a significant increase in % thickening fraction (4.1 ± 0.6 vs. −0.6 ± 0.5) were observed at six weeks after implantation of autologous myogenic cardiac fibroblasts as compared to the other groups. Autologous cardiac fibroblasts did not produce any significant changes as compared to the MI group (injected with serum free culture medium). These results suggested that intra-coronary infusion or intra-myocardial injection of 5-aza-2′-deoxycytidine into the myocardial infraction could be used to induce myocardial regeneration by myogenic transdifferentiation of the existing cardiac fibroblasts. Supported by NIH grant HL72138 and AHA grant 0255009B

Effect of Transforming Growth Factor-β on Decorin and β1 Integrin Expression During Muscle Development in Chickens

Myoblast-extracellular matrix interactions play a pivotal role in skeletal muscle development. Transforming growth factor-β (TGF-β) is a key regulator of muscle cell proliferation and differentiation. The level of TGF-β expressed will affect the concentration of the extracellular matrix proteoglycan decorin and the cell surface β1 integrin subunit. The decorin proteoglycan is a regulator of cell growth as well as the organization of the extracellular matrix. The β1 integrin plays a role in muscle cell attachment, migration, and the formation of multinucleated myotubes. In the current study, chicken myogenic satellite cells isolated from the pectoralis major muscle from the chicken genetic muscle weakness, low score normal (LSN), and normal pectoralis major muscle were used to investigate TGF-β expression as it relates to decorin and β1 integrin mRNA expression. The LSN muscle defect is characterized by altered myotube formation and sarcomere structure, and the satellite cells have reduced proliferation and differentiation. The mRNA expression was measured by real-time quantitative reverse transcription PCR. The LSN condition has elevated expression of TGF-β2 and TGF-β4 with increased expression of decorin and decreased β1 integrin during myogenic satellite cell proliferation and differentiation. Normal satellite cell cultures were treated with the addition of exogenous TGF-β during differentiation to determine if the altered expression of LSN decorin and β1 integrin was associated with TGF-β expression. The addition of exogenous TGF-β decreased decorin expression during differentiation and reduced β1 integrin expression at 24 and 48 h of differentiation. These results suggested that alteration of decorin expression in the LSN myogenic satellite cells may occur by a mechanism involving factors in addition to TGF-β, but the addition of exogenous TGF-β did affect both decorin and β1 integrin expression. These data, therefore, suggested that TGF-β might play a pivotal role in chicken skeletal muscle formation through modulation of the expression of both extracellular matrix molecules and cellular receptors important in the control of cell migration and growth regulation.

Effect of phase limited inhibition of MyoD expression on the terminal differentiation of bovine myoblasts: No alteration of Myf5 or myogenin expression

To investigate the roles played by MyoD in the terminal differentiation of satellite cell-derived myoblasts, the effect of antisense inhibition of MyoD expression was examined in bovine adult myoblast culture, in which inhibition treatment was limited to the terminal differentiation phase. MyoD antisense oligonucleotide DNA (AS-mD) suppressed the formation of multinucleated myotubes in the cell culture. Myotube formation was suppressed even when AS-mD treatment was limited to the period preceding the onset of myotube formation. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that treatment with AS-mD suppressed the expression of myosin heavy chain embryonic isoform and troponin T isoforms at 4 days after the induction of differentiation. AS-mD also suppressed the expression of MRF4, but did not alter the expression of either Myf5 or myogenin, in contrast to previous results using mouse cells possessing MyoD(-/-) genetic background. These findings suggest that MyoD controls myogenesis but not Myf5 or myogenin mRNA expression during the terminal differentiation phase. Furthermore, among the alpha4, alpha5, alpha6, and alpha7 integrins, alpha4, alpha5, and alpha7 integrin expression was suppressed by AS-mD treatment, in parallel with the suppression of myotube formation, which suggests that MyoD controls myotube formation by regulating the expression of alpha4, alpha5, and alpha7 integrins.

Cholesterol depletion by methyl-?-cyclodextrin enhances myoblast fusion and induces the formation of myotubes with disorganized nuclei

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-beta-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric alpha-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and beta-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.

Marked reduction of focal adhesion kinase, serum response factor and myocyte enhancer factor 2C, but increase in RhoA and myostatin in the hindlimb dy mouse muscles.

Laminin alpha2 (merosin)-deficient congenital muscular dystrophy (CMD) patients show progressive muscle fiber necrosis and ineffective muscle regeneration. This is probably due to decreased formation of multi nucleated myotubes resulting from a myoblast fusion defect. When receiving a mechanical signal from muscle membranes, a cascade of RhoA, focal adhesion kinase (FAK), and serum response factor (SRF) positively regulates myogenesis and muscle hypertrophy associated with functional overload. In contrast, myostatin, a potent negative regulator of skeletal muscle hypertrophy, appears to be up-regulated in the muscles of mdx mice, an animal model for Duchenne muscular dystrophy. Using Western blot and immunohistochemical analyses, we investigated the levels of RhoA, FAK, SRF, and myostatin in the skeletal muscles of dy mice. The amount of RhoA protein was increased in the hindlimb muscles of dy mice aged 12 weeks. At 12 weeks, FAK immunoreactivity was observed in the myonuclei and/or satellite cells of normal mice, but not of dy mice. SRF protein levels decreased markedly in the gastrocnemius and rectus femoris muscles of dy mice at 2 and 12 weeks. Several muscle fibers in normal mice possessed uniform SRF immunoreactivity in the cytoplasm. An SRF immunostaining pattern in muscle was not detected in dy mice. Western blot and the densitometric analysis showed a decreased amount of myocyte enhancer factor 2C (MEF2C) in hindlimb muscles of dy mice. Although slight myostatin immunoreactivity was observed in the nuclei of some normal mice, marked myostatin immunoreactivity was observed in the cytoplasm of mature dy mice myonuclei and/or satellite cells. A low expression of FAK, SRF and MEF2C in muscles of dy mice may inhibit postnatal muscle hypertrophy by fusing satellite cells with existing fibers. Enhancing myostatin protein would result in further atrophy and degeneration of muscle fiber in dy mice.

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.