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Myoblasts generated by lentiviral mediated MyoD transduction of myotonic dystrophy type 1 (DM1) fibroblasts can be used for assays of therapeutic molecules
Jan Larsen, Olof J Pettersson, Maria Jakobsen, Rune Thomsen, Christina B Pedersen, Jens M Hertz, Niels Gregersen, Thomas J Corydon, Thomas G Jensen
BMC Research Notes , 2011, DOI: 10.1186/1756-0500-4-490
Abstract: Transduced fibroblasts show a multinuclear phenotype and express the differentiation marker myogenin. Furthermore, fluorescence in situ hybridization (FISH) analysis revealed a statistical significant increase in the amount of nuclear foci in DM1 patient fibroblasts after myogenesis. Finally, no nuclear foci were found after treatment with oligonucleotides targeting the repeat expansions.The abundance of nuclear foci in DM1 patient fibroblasts increase following myogenesis, as visualized by FISH analysis. Foci were eradicated after treatment with antisense oligonucleotides. Thus, we propose that the current cell model is suitable for testing of novel treatment modalities.Myotonic dystrophy 1 (DM1) is a multisystemic dominant disease and it is the most common muscular dystrophy in adults [1]. The symptoms include muscle wasting (muscular dystrophy), cataract, heart conduction defects, insulin resistance, and myotonia. The current treatment is insufficient, ranging from muscle exercise to breathing assistance. The genetic cause of DM1 is a (CTG)n repeat in the 3'-untranslated region of the dystrophia myotonica protein kinase gene, DMPK [2] . Current evidence supports an RNA-gain-of-function pathogenesis [1]. Indeed, mutant DMPK mRNA localizes to distinct foci in the nucleus and sequesters multiple proteins, among these the alternative splicing regulator muscleblind-like protein 1 (MBNL1). This results in a depletion of MBNL1 in the nucleus, leading to multiple events of aberrant splicing. Other factors affected by the accumulation of foci include CUG-binding protein 1 (CUG-BP1) which is another alternative splicing regulator. Both MBNL1 and CUG-BP1 were recently shown to regulate the alternative splicing of numerous genes [3-5]. The importance of the nuclear foci has been underlined by the discovery that reduction of the number of foci is associated with normalized splice patterns in DM1 cells [6,7]. Foci abundancy and brightness has been reported to increase during m
Identification of valid reference genes during the differentiation of human myoblasts
Jens Stern-Straeter, Gabriel A Bonaterra, Karl H?rmann, Ralf Kinscherf, Ulrich R Goessler
BMC Molecular Biology , 2009, DOI: 10.1186/1471-2199-10-66
Abstract: Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked.RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP.The real-time polymerase chain reaction (qRT-PCR) has revolutionized the field of gene expression analysis in living organisms. In comparison to classical semi-quantitative reverse transcription-PCR (sqRT-PCR), the main advantages of qRT-PCR are its higher sensitivity, specificity, and broad quantification range [1,2]. Despite being an extremely powerful technique, qRT-PCR suffers from certain pitfalls, the most important being the normalization with a reliable reference gene, habitually called 'housekeeping gene (HKG)'. The housekeeping term was initially given to genes that are necessary for cell function and being constitutively expressed in each cell type [3] but obviously it would be more precise to call it 'reference gene (RG)'. RG are often taken from the literature and used across a variety of experimental conditions, some of which may induce differences in the RG own expression under certain conditions [3]. Thus, experimental results are highly dependent on the RG gene chosen [4]. If unrecognized, unexpected changes in RG expression could result in erroneous conclusions about real biological effects such as respon
Stress-Induced C/EBP Homology Protein (CHOP) Represses MyoD Transcription to Delay Myoblast Differentiation  [PDF]
Joel Alter, Eyal Bengal
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0029498
Abstract: When mouse myoblasts or satellite cells differentiate in culture, the expression of myogenic regulatory factor, MyoD, is downregulated in a subset of cells that do not differentiate. The mechanism involved in the repression of MyoD expression remains largely unknown. Here we report that a stress-response pathway repressing MyoD transcription is transiently activated in mouse-derived C2C12 myoblasts growing under differentiation-promoting conditions. We show that phosphorylation of the α subunit of the translation initiation factor 2 (eIF2α) is followed by expression of C/EBP homology protein (CHOP) in some myoblasts. ShRNA-driven knockdown of CHOP expression caused earlier and more robust differentiation, whereas its constitutive expression delayed differentiation relative to wild type myoblasts. Cells expressing CHOP did not express the myogenic regulatory factors MyoD and myogenin. These results indicated that CHOP directly repressed the transcription of the MyoD gene. In support of this view, CHOP associated with upstream regulatory region of the MyoD gene and its activity reduced histone acetylation at the enhancer region of MyoD. CHOP interacted with histone deacetylase 1 (HDAC1) in cells. This protein complex may reduce histone acetylation when bound to MyoD regulatory regions. Overall, our results suggest that the activation of a stress pathway in myoblasts transiently downregulate the myogenic program.
Analyses of the differentiation potential of satellite cells from myoD-/-, mdx, and PMP22 C22 mice
Marion M Schuierer, Christopher J Mann, Heidi Bildsoe, Clare Huxley, Simon M Hughes
BMC Musculoskeletal Disorders , 2005, DOI: 10.1186/1471-2474-6-15
Abstract: Single extensor digitorum longus muscle fibres were cultured from mdx and PMP22 mice and age- and genetic background-matched controls. Mice at several ages were compared with regard to the differentiation of satellite cells, assayed as the proportion of desmin-expressing cells that accumulated sarcomeric myosin heavy chain.Satellite cells of 2 month, 6 month, and 12 month old mdx mice were capable of differentiating to a similar extent to age-matched wild type control animals in an in vitro proliferation/differentiation model. Strikingly, differentiation efficiency in individual 6 month and 12 month old mdx animals varies to a much higher extent than in age-matched controls, younger mdx animals, or PMP22 mice. In contrast, differentiation of myoblasts from all myoD null mice assayed was severely impaired in this assay system. The defect in satellite cell differentiation that occurs in some mdx animals arises from a delay in differentiation that is not overcome by IGF-1 treatment at any phase of cultivation.Overall, a defect in satellite cell differentiation above that arising through normal ageing does not occur in mdx or PMP22 mouse models of human disease. Nonetheless, the impaired differentiation of satellite cells from some mdx animals suggests that additional factors, environmental or epigenetic, may lead to deteriorating muscle repair through poor differentiation of satellite cells in genetically predisposed individuals.Like many muscle diseases, Duchenne muscular dystrophy (DMD) is characterised by a gradual loss of muscle function with age. Patients are initially ambulatory and have mild muscle pathology, despite ongoing degeneration and repair. In later stages of DMD, patients experience progressively more severe muscular changes, accompanied by loss of function, physical dependency, and ultimately, death [1].DMD patients generally lack the cytoskeletal protein dystrophin, a member of the spectrin-like superfamily of actin binding proteins. Functional dystr
Integrated Functions of Pax3 and Pax7 in the Regulation of Proliferation, Cell Size and Myogenic Differentiation  [PDF]
Charlotte A. Collins, Viola F. Gnocchi, Robert B. White, Luisa Boldrin, Ana Perez-Ruiz, Frederic Relaix, Jennifer E. Morgan, Peter S. Zammit
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004475
Abstract: Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.
MyoD-dependent regulation of NF-κB activity couples cell-cycle withdrawal to myogenic differentiation
Maura H Parker, Julia von Maltzahn, Nadine Bakkar, Ban Al-Joubori, Jeff Ishibashi, Denis Guttridge, Michael A Rudnicki
Skeletal Muscle , 2012, DOI: 10.1186/2044-5040-2-6
Abstract: Primary myoblasts isolated from wild type and MyoD-/- mutant mice were examined by microarray analysis and further characterized by cell and molecular experiments in cell culture.We found that NF-κB, a key regulator of cell-cycle withdrawal and differentiation, aberrantly maintains nuclear localization and transcriptional activity in MyoD-/- myoblasts. As a result, expression of cyclin D is maintained during serum withdrawal, inhibiting expression of muscle-specific genes and progression through the differentiation program. Sustained nuclear localization of cyclin E, and a concomitant increase in cdk2 activity maintains S-phase entry in MyoD-/- myoblasts even in the absence of mitogens. Importantly, this deficit was rescued by forced expression of IκBαSR, a non-degradable mutant of IκBα, indicating that inhibition of NF-κB is sufficient to induce terminal myogenic differentiation in the absence of MyoD.MyoD-induced cytoplasmic relocalization of NF-κB is an essential step in linking cell-cycle withdrawal to the terminal differentiation of skeletal myoblasts. These results provide important insight into the unique functions of MyoD in regulating the switch from progenitor proliferation to terminal differentiation.Cell survival and differentiation is regulated by NF-κB, a family of ubiquitously expressed transcription factors comprising RelA/p65, c-Rel, RelB, p50 (processed form of p105), and p52 (processed form of p100) [1]. NF-κB proteins function as homo- or heterodimers, the most common of which is the p50/p65 heterodimer. All family members contain a DNA-binding domain, a protein-protein dimerization domain, and a nuclear localization sequence (NLS). However, only RelA/p65, c-Rel, and RelB have a transactivation domain [2].Sub-cellular localization of NF-κB is regulated by ‘inhibitor of κB’ proteins: IκBα, IκBβ, and IκB? [3]. IκB proteins bind NF-κB, mask the nuclear localization signal, and sequester NF-κB in the cytoplasm as an inactive protein. Upon induction,
Increased Angiogenesis and Improved Left Ventricular Function after Transplantation of Myoblasts Lacking the MyoD Gene into Infarcted Myocardium  [PDF]
Yasuhiro Nakamura, Yoko Asakura, Bryan A. Piras, Hiroyuki Hirai, Christopher T. Tastad, Mayank Verma, Amanda J. Christ, Jianyi Zhang, Takanori Yamazaki, Minoru Yoshiyama, Atsushi Asakura
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0041736
Abstract: Skeletal myoblast transplantation has therapeutic potential for repairing damaged heart. However, the optimal conditions for this transplantation are still unclear. Recently, we demonstrated that satellite cell-derived myoblasts lacking the MyoD gene (MyoD?/?), a master transcription factor for skeletal muscle myogenesis, display increased survival and engraftment compared to wild-type controls following transplantation into murine skeletal muscle. In this study, we compare cell survival between wild-type and MyoD?/? myoblasts after transplantation into infarcted heart. We demonstrate that MyoD?/? myoblasts display greater resistance to hypoxia, engraft with higher efficacy, and show a larger improvement in ejection fraction than wild-type controls. Following transplantation, the majority of MyoD?/? and wild-type myoblasts form skeletal muscle fibers while cardiomyocytes do not. Importantly, the transplantation of MyoD?/? myoblasts induces a high degree of angiogenesis in the area of injury. DNA microarray data demonstrate that paracrine angiogenic factors, such as stromal cell-derived factor-1 (SDF-1) and placental growth factor (PlGF), are up-regulated in MyoD?/? myoblasts. In addition, over-expression and gene knockdown experiments demonstrate that MyoD negatively regulates gene expression of these angiogenic factors. These results indicate that MyoD?/? myoblasts impart beneficial effects after transplantation into an infarcted heart, potentially due to the secretion of paracrine angiogenic factors and enhanced angiogenesis in the area of injury. Therefore, our data provide evidence that a genetically engineered myoblast cell type with suppressed MyoD function is useful for therapeutic stem cell transplantation.
FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation.  [PDF]
Dmitriev P. V.,Barat A. L.,Cochet E.,Ogryzko V. V.
Biopolymers and Cell , 2011,
Abstract: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been shown previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to normal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myoblasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vimentin could be an easily detectable marker of FSHD myotubes
MyoD- and nerve-dependent maintenance of MyoD expression in mature muscle fibres acts through the DRR/PRR element
Sophie B Chargé, Andrew S Brack, Stéphanie A Bayol, Simon M Hughes
BMC Developmental Biology , 2008, DOI: 10.1186/1471-213x-8-5
Abstract: In culture, MD6.0-lacZ expresses in myotubes but not myogenic cells, unlike endogenous MyoD. Reporter expression in vivo is in muscle fibre nuclei and is reduced in MyoD null mice. The MD6.0-lacZ reporter is down-regulated both in adult muscle fibres by denervation or muscle disuse and in cultured myotubes by inhibition of activity. Activity induces and represses MyoD through the DRR and PRR, respectively. During the postnatal period, accumulation of β-galactosidase correlates with maturation of innervation. Strikingly, endogenous MyoD expression is up-regulated in fibres by complete denervation, arguing for a separate activity-dependent suppression of MyoD requiring regulatory elements outside the DRR/PRR.The data show that MyoD regulation is more complex than previously supposed. Two factors, MyoD protein itself and fibre activity are required for essentially all expression of the 6 kb proximal enhancer/promoter (DRR/PRR) of MyoD in adult fibres. We propose that modulation of MyoD positive feedback by electrical activity determines the set point of MyoD expression in innervated fibres through the DRR/PRR element.Myogenic regulatory transcription factors (MRFs) are essential for skeletal myogenesis during embryonic development and for proper muscle regeneration [1-6]. Myf5 and MyoD are expressed in proliferating myoblasts, whereas myogenin and MRF4 are important in terminal differentiation [4,5,7,8]. In the absence of MyoD, muscle regeneration is impaired [9] possibly due to delayed differentiation of muscle precursor cells [5,7,10]. However, MyoD is also expressed in adult muscle fibres, albeit at low levels [11,12]. Conditions that damage muscle or change muscle phenotype often lead to changes of MyoD expression [13-15]. Nevertheless, when changes in MyoD expression occur, it is unclear how much is in fibres, myogenic cells or both [3,16]. Therefore, the activity and regulation of MyoD in normal muscle fibres is unknown.The role of MyoD within muscle fibres is un
Maged1, a new regulator of skeletal myogenic differentiation and muscle regeneration
Tuan HN Nguyen, Mathieu JM Bertrand, Christiane Sterpin, Younes Achouri, Olivier RY De Backer
BMC Cell Biology , 2010, DOI: 10.1186/1471-2121-11-57
Abstract: We show that Maged1 is expressed at very low levels in normal adult muscle but is strongly induced after injury, during the early phase of myoblast differentiation. By comparing in vitro differentiation of myoblasts derived from wild-type or Maged1 knockout mice, we observed that Maged1 deficiency results in reduced levels of p21CIP1/WAF1, defective cell cycle exit and impaired myotube maturation. In vivo, this defect results in delayed regeneration of injured muscle.These data demonstrate for the first time that Maged1 is an important factor required for proper skeletal myoblast differentiation and muscle healing.Skeletal muscle contains quiescent mononucleated myogenic cells, called satellite cells, that are able to proliferate in response to injury and give rise to new functional myofibers. Soon after a lesion, inflammatory cells such as neutrophiles and macrophages are recruited at the damaged site where they release growth factors and cytokines that induce satellite cells to proliferate. The activated satellite cells divide asymmetrically to (1) reconstitute the stock of stem cells and (2) provide myoblasts that will first proliferate and then undergo myogenic differentiation and maturation to form new muscle fibers. The new cells can fuse between themselves or with existing fibers. Muscle differentiation involves a cascade of muscle-specific gene activation coordinated with irreversible withdrawal from the cell cycle. The commitment of cells to the muscle lineage and to progression through differentiation requires the activation of a limited set of muscle-specific transcription factors. These key factors are the basic helix-loop-helix (bHLH) transcription factors MyoD, Myf5, myogenin (MyoG) and MRF4, which are collectively called myogenic regulatory factors (MRFs). The MRFs activate their target genes by binding to the E-box CANNTG consensus sequence [1-4]. Primary cultures of newborn or adult skeletal muscles or established muscle cell lines like C2C12 can be
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