Identification of gene expression modifications in myostatin-stimulated myoblasts

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Abstract

Myostatin belongs to the transforming growth factor beta superfamily and has been shown to function as an inhibitor of skeletal muscle proliferation and differentiation. To gain insight into the molecular mechanisms of myostatin function during myogenesis, differential display reverse transcription PCR was employed to identify altered gene expressions associated with myostatin inhibitory function in chicken fetal myoblasts (CFMs). In this work, we have identified seven up-regulated and 12 down-regulated genes in myostatin stimulated CFMs. Those genes are involved in myogenic differentiation, cell architecture, energy metabolism, signal transduction, and apoptosis. The down-regulation of muscle creatine kinase B, troponin C, and myosin regulatory light chain is in agreement with the myostatin negative role in myocyte differentiation. In addition, the expression alteration of skeletal muscle-specific cardiac ankyrin repeat protein and the bcl-2 related anti-apoptotic protein Nr-13 suggests possible unique roles for myostatin in regulating myogenesis by controlling cofactors participated transcriptional regulation and apoptosis.

Section snippets

Materials and methods

Cell culture and treatment. Chicken fetal myoblasts (CFMs) were prepared from the pectoralis muscles of 10-day-old White Leghorn chicken embryos. Briefly, the pectoralis muscles were removed and dissected in order to remove bone and skin. The muscle tissue was minced and incubated in growth medium, GM (DMEM supplemented with 10% fetal bovine serum, 2% chicken serum, and penicillin/streptomycin) at 37 °C in a 5% CO2 atmosphere, with intermittent shaking for 20 min. The dissociated cells were

Identification of expression-altered genes in myostatin stimulated CFMs

It has been reported that the mature portion of endogenous myostatin localized in the C-terminus from amino acid 266 to 376 [18]. Therefore, we expressed and purified the C-terminal truncated recombinant myostatin (Lys193-Ser375) as described [22]. To verify the biological function of the recombinant protein, a cytometry analysis and Northern blot were performed to determine CFMs responses to myostatin stimulation. Myostatin efficiently induced CFMs cell cycle arrest at G1 phase and decreased

Discussion

As a growth and differentiation factor, myostatin has been shown to determine muscle size by controlling its growth. However, molecular mechanism of myostatin action still remains to be explicated. The identification of gene expression alteration in response to myostatin stimulation could provide important information for understanding the molecular mechanisms of myostatin function for controlling skeletal muscle size. In the present study, we identified a number of genes whose expression was

Acknowledgments

This research was supported by the following grants: The National High Technology Research and Development Program of China (“863” Program, No. 2001AA222031), and the National Science Fund for Distinguished Young Scholars of China (No. 300250). We thank Sara Zhu for assistance with preparation of the manuscript.

References (39)

  • R. Jeyaseelan et al.

    A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes

    J. Biol. Chem.

    (1997)
  • C.C. Witt et al.

    Induction and myofibrillar targeting of CARP, and suppression of the Nkx2.5 pathway in the MDM mouse with impaired titin-based signaling

    J. Mol. Biol.

    (2004)
  • D. Joulia et al.

    Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin

    Exp. Cell Res.

    (2003)
  • M.E. Pownall et al.

    Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos

    Annu. Rev. Cell Dev. Biol.

    (2002)
  • R.L. Perry et al.

    Molecular mechanisms regulating myogenic determination and differentiation

    Front. Biosci.

    (2000)
  • A.C. McPherron et al.

    Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member

    Nature

    (1997)
  • L. Grobet et al.

    A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle

    Nat. Genet.

    (1997)
  • R. Kambadur et al.

    Mutations in myostatin (GDF8) in double-muscled belgian blue and piedmontese cattle

    Genome Res.

    (1997)
  • K.R. Wagner et al.

    Loss of myostatin attenuates severity of muscular dystrophy in mdx mice

    Ann. Neurol.

    (2002)
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    These authors contributed equally.

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