Skip to main content
SpringerLink
Log in

Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

Skeletal muscle's regenerative ability is vital for maintaining muscle function, but chronic diseases like Duchenne muscular dystrophy can deplete this capacity. Muscle satellite cells, quiescent in normal situations, are activated during muscle injury, expressing myogenic regulatory factors, and producing myogenic progenitor cells. It was reported that muscle stem cells in primary culture and reserve cells in C2C12 cells express anti-apoptotic protein Bcl-2. Although the role of Bcl-2 expressed in myogenic cells has been thought to be to enhance cell viability, we hypothesized that Bcl-2 may promote the formation of reserve cells. The expression pattern analysis showed the expression of Bcl-2 in undifferentiated mononucleated cells, emphasizing its usefulness as a reserve cell marker and reminding us that cells expressing Bcl-2 have low proliferative potential. Silencing of Bcl-2 by transfection with siRNA decreased cell viability and the number of reserve cells, while overexpression of Bcl-2 not only increases cell viability but also inhibits muscle differentiation and proliferation. These results emphasize dual roles of Bcl-2 in protecting cells from apoptosis and contributing to reserve cell formation by regulating myoblast proliferation and/or differentiation. Overall, the study sheds light on the multifaceted role of Bcl-2 in the maintenance of skeletal muscle regeneration.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.

Similar content being viewed by others

Data Availability

The data used in the current research are available from the corresponding author upon reasonable request.

References

  • Blau HM, Chiu CP, Webster C (1983) Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cell 32:1171–1180

    Article  CAS  PubMed  Google Scholar 

  • Bonnefoy-Berard N, Aouacheria A, Verschelde C, Quemeneur L, Marcais A, Marvel J (2004) Control of proliferation by Bcl-2 family members. Biochim Biophys Acta 1644:159–168

    Article  CAS  PubMed  Google Scholar 

  • Carnac G, Fajas L, L’Honore A, Sardet C, Lamb NJ, Fernandez A (2000) The retinoblastoma-like protein p130 is involved in the determination of reserve cells in differentiating myoblasts. Curr Biol 10:543–546

    Article  CAS  PubMed  Google Scholar 

  • Chae HJ, Kang JS, Byun JO, Han KS, Kim DU, Oh SM, Kim HM, Chae SW, Kim HR (2000) Molecular mechanism of staurosporine-induced apoptosis in osteoblasts. Pharmacol Res 42:373–381

    Article  CAS  PubMed  Google Scholar 

  • Cornelison DD, Wold BJ (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191:270–283

    Article  CAS  PubMed  Google Scholar 

  • De Angelis L, Borghi S, Melchionna R, Berghella L, Baccarani-Contri M, Parise F, Ferrari S, Cossu G (1998) Inhibition of myogenesis by transforming growth factor beta is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm. Proc Natl Acad Sci USA 95:12358–12363

    Article  PubMed  PubMed Central  Google Scholar 

  • Dominov JA, Dunn JJ, Miller JB (1998) Bcl-2 expression identifies an early stage of myogenesis and promotes clonal expansion of muscle cells. J Cell Biol 142:537–544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dominov JA, Houlihan-Kawamoto CA, Swap CJ, Miller JB (2001) Pro- and anti-apoptotic members of the Bcl-2 family in skeletal muscle: a distinct role for Bcl-2 in later stages of myogenesis. Dev Dyn 220:18–26

    Article  CAS  PubMed  Google Scholar 

  • Fukada S, Uezumi A, Ikemoto M, Masuda S, Segawa M, Tanimura N, Yamamoto H, Miyagoe-Suzuki Y, Takeda S (2007) Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells (dayton, Ohio) 25:2448–2459

    Article  CAS  PubMed  Google Scholar 

  • Gabellini C, Trisciuoglio D, Del Bufalo D (2017) Non-canonical roles of Bcl-2 and Bcl-xL proteins: relevance of BH4 domain. Carcinogenesis 38:579–587

    Article  CAS  PubMed  Google Scholar 

  • Greider C, Chattopadhyay A, Parkhurst C, Yang E (2002) BCL-x(L) and BCL2 delay Myc-induced cell cycle entry through elevation of p27 and inhibition of G1 cyclin-dependent kinases. Oncogene 21:7765–7775

    Article  CAS  PubMed  Google Scholar 

  • Grounds MD (1999) Muscle regeneration: molecular aspects and therapeutic implications. Curr Opin Neurol 12:535–543

    Article  CAS  PubMed  Google Scholar 

  • Haughn L, Hawley RG, Morrison DK, von Boehmer H, Hockenbery DM (2003) BCL-2 and BCL-XL restrict lineage choice during hematopoietic differentiation. J Biol Chem 278:25158–25165

    Article  CAS  PubMed  Google Scholar 

  • Hill M, Wernig A, Goldspink G (2003) Muscle satellite (stem) cell activation during local tissue injury and repair. J Anat 203:89–99

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hochreiter-Hufford AE, Lee CS, Kinchen JM, Sokolowski JD, Arandjelovic S, Call JA, Klibanov AL, Yan Z, Mandell JW, Ravichandran KS (2013) Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion. Nature 497:263–267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kitzmann M, Carnac G, Vandromme M, Primig M, Lamb NJ, Fernandez A (1998) The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol 142:1447–1459

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kitzmann M, Fernandez A (2001) Crosstalk between cell cycle regulators and the myogenic factor MyoD in skeletal myoblasts. Cell Mol Life Sci 58:571–579

    Article  CAS  PubMed  Google Scholar 

  • Luz MA, Marques MJ, Santo Neto H (2002) Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells. Braz J Med Biol Res 35:691–695

    Article  CAS  PubMed  Google Scholar 

  • Marvel J, Perkins GR, Lopez Rivas A, Collins MK (1994) Growth factor starvation of bcl-2 overexpressing murine bone marrow cells induced refractoriness to IL-3 stimulation of proliferation. Oncogene 9:1117–1122

    CAS  PubMed  Google Scholar 

  • Mohan A, Asakura A (2017) CDK inhibitors for muscle stem cell differentiation and self-renewal. J Phys Fit Sports Med 6:65–74

    Article  PubMed  PubMed Central  Google Scholar 

  • Murach KA, Peck BD, Policastro RA, Vechetti IJ, Van Pelt DW, Dungan CM, Denes LT, Fu X, Brightwell CR, Zentner GE, Dupont-Versteegden EE, Richards CI, Smith JJ, Fry CS, McCarthy JJ, Peterson CA (2021) Early satellite cell communication creates a permissive environment for long-term muscle growth. iScience 24:102372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nagata Y, Honda Y, Matsuda R (2010) FGF2 induces ERK phosphorylation through Grb2 and PKC during quiescent myogenic cell activation. Cell Struct Funct 35:63–71

    Article  CAS  PubMed  Google Scholar 

  • Nagata Y, Kobayashi H, Umeda M, Ohta N, Kawashima S, Zammit PS, Matsuda R (2006a) Sphingomyelin levels in the plasma membrane correlate with the activation state of muscle satellite cells. J Histochem Cytochem 54:375–384

    Article  CAS  PubMed  Google Scholar 

  • Nagata Y, Ohashi K, Wada E, Yuasa Y, Shiozuka M, Nonomura Y, Matsuda R (2014) Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation. Exp Cell Res 326:112–124

    Article  CAS  PubMed  Google Scholar 

  • Nagata Y, Partridge TA, Matsuda R, Zammit PS (2006b) Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling. J Cell Biol 174:245–253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Omura S, Iwai Y, Hirano A, Nakagawa A, Awaya J, Tsuchya H, Takahashi Y, Masuma R (1977) A new alkaloid AM-2282 OF Streptomyces origin. Taxonomy, fermentation, isolation and preliminary characterization. J Antibiot (tokyo) 30:275–282

    Article  CAS  PubMed  Google Scholar 

  • Otsuka T, Kan HM, Mengsteab PY, Tyson B, Laurencin CT (2024) Fibroblast growth factor 8b (FGF-8b) enhances myogenesis and inhibits adipogenesis in rotator cuff muscle cell populations in vitro. Proc Natl Acad Sci USA 121:e2314585121

    Article  CAS  PubMed  Google Scholar 

  • Reed JC (1997) Double identity for proteins of the Bcl-2 family. Nature 387:773–776

    Article  CAS  PubMed  Google Scholar 

  • Schöneich C, Dremina E, Galeva N, Sharov V (2014) Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes. Apoptosis 19:42–57

    Article  PubMed  PubMed Central  Google Scholar 

  • Seale P, Rudnicki MA (2000) A new look at the origin, function, and stem-cell status of muscle satellite cells. Dev Biol 218:115–124

    Article  CAS  PubMed  Google Scholar 

  • Trisciuoglio D, Gabellini C, Desideri M, Ragazzoni Y, De Luca T, Ziparo E, Del Bufalo D (2011) Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells. Cell Death Differ 18:1024–1035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsuchiya Y, Kitajima Y, Masumoto H, Ono Y (2020) Damaged Myofiber-Derived Metabolic Enzymes Act as Activators of Muscle Satellite Cells. Stem Cell Reports 15:926–940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tzachanis D, Freeman GJ, Hirano N, van Puijenbroek AA, Delfs MW, Berezovskaya A, Nadler LM, Boussiotis VA (2001) Tob is a negative regulator of activation that is expressed in anergic and quiescent T cells. Nat Immunol 2:1174–1182

    Article  CAS  PubMed  Google Scholar 

  • Vairo G, Soos TJ, Upton TM, Zalvide J, DeCaprio JA, Ewen ME, Koff A, Adams JM (2000) Bcl-2 retards cell cycle entry through p27(Kip1), pRB relative p130, and altered E2F regulation. Mol Cell Biol 20:4745–4753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wosczyna MN, Konishi CT, Perez Carbajal EE, Wang TT, Walsh RA, Gan Q, Wagner MW, Rando TA (2019) Mesenchymal Stromal Cells Are Required for Regeneration and Homeostatic Maintenance of Skeletal Muscle. Cell Rep 27(2029–2035):e2025

    Google Scholar 

  • Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725–727

    Article  CAS  PubMed  Google Scholar 

  • Yin H, Price F, Rudnicki MA (2013) Satellite cells and the muscle stem cell niche. Physiol Rev 93:23–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yin XM, Oltvai ZN, Korsmeyer SJ (1994) BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 369:321–323

    Article  CAS  PubMed  Google Scholar 

  • Yoshida N, Yoshida S, Koishi K, Masuda K, Nabeshima Y (1998) Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates “reserve cells.” J Cell Sci 111:769–779

    Article  CAS  PubMed  Google Scholar 

  • Zhu P, Zhang C, Gao Y, Wu F, Zhou Y, Wu WS (2019) The transcription factor Slug represses p16(Ink4a) and regulates murine muscle stem cell aging. Nat Commun 10:2568

    Article  PubMed  PubMed Central  Google Scholar 

  • Zinkel S, Gross A, Yang E (2006) BCL2 family in DNA damage and cell cycle control. Cell Death Differ 13:1351–1359

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by JSPS KAKENHI Grant Number JP21K11488. We would like to thank Daiho Imai and Masayo Kurokawa at Okayama University of Science for technical assistance. The monoclonal antibodies, MF20, F5D were obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa.

Author information

Authors and Affiliations

  1. Department of Bioscience, Faculty of Life Science, Okayama University of Science, 1-1 Ridaicho, Kita-Ku, Okayama-Shi, 700-0005, Japan

    Yosuke Nagata

  2. Department of Life Science, Faculty of Science, Okayama University of Science, Okayama, Japan

    Yosuke Nagata & Jun Tomimori

  3. Department of Natural Science, Graduate School of Science and Engineering, Okayama University of Science, Okayama, Japan

    Yosuke Nagata & Tomoharu Hagiwara

Authors
  1. Yosuke Nagata
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jun Tomimori
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomoharu Hagiwara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yosuke Nagata.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nagata, Y., Tomimori, J. & Hagiwara, T. Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells. In Vitro Cell.Dev.Biol.-Animal (2024). https://doi.org/10.1007/s11626-024-00905-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11626-024-00905-3

Keywords

Search

Navigation