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. 2014 Jan 2;9(1):e84153.
doi: 10.1371/journal.pone.0084153. eCollection 2014.

Proteomic Analysis of C2C12 Myoblast and Myotube Exosome-Like Vesicles: A New Paradigm for Myoblast-Myotube Cross Talk?

Free PMC article

Proteomic Analysis of C2C12 Myoblast and Myotube Exosome-Like Vesicles: A New Paradigm for Myoblast-Myotube Cross Talk?

Alexis Forterre et al. PLoS One. .
Free PMC article

Erratum in

  • PLoS One. 2014;9(1). doi:10.1371/annotation/ecd1e074-2618-4ad0-95c0-efdb467c714b


Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Quality analysis of purified ELV preparations by Western-blot.
Equal protein amounts of extracts prepared from cells or exosomes were subjected to western blot analysis. The multivesicular body markers TSG101 and Alix (ALG2-interacting protein 1), and the tetraspanin CD81, were strongly enriched in exosome preparations compared with cell lysates.
Figure 2
Figure 2. Transmission electron microscopy images of purified nanovesicles from myoblasts (ELV-MB) or myotubes (ELV-MT) conditioned media.
Bar = 100 nm. Nanovesicles are labeled with anti-CD81 gold particles specifically expressed at exosome-like vesicle membranes.
Figure 3
Figure 3. Functional analysis of ELV-MB and ELV-MT common proteins using Babelomics 4.0.
Only significant Gene Ontology terms are indicated.
Figure 4
Figure 4. Western-blot analysis of proteins found differentially expressed between ELV-MB and ELV-MT (see Table S1).
ITGB5 (Itgb5) : Integrin beta-5; TGFBR2 (Tgfbr2) : Transforming growth factor, beta receptor II; TAGLN2 (Tagln2) : Transgelin-2; TSPAN8 (Tspan8) : Tetraspanin-8. *, only detected in ELV-MT but not selected as statistically more expressed in ELV-MB (see Table S1).
Figure 5
Figure 5. Effects of ELV-MT on myoblast proliferation.
(A)_Myoblasts were grown in 96-well plates either with DMEM 5% FBS or with DMEM 5% Exosome-Depleted serum (DED) in order to calculate the cell index doubling time, using the xCELLigence RTCA HT Software. The doubling time is the time required for cell index to double and thus represents time when whole cell population has performed at least one division. As shown, C2C12 myoblasts divided once in both control media, every 15 hours (replicates = 8). B_Myoblasts were incubated with DED supplemented either with 2 µg of ELV-MB or 2 µg of ELV-MT/ml of medium. C2C12 doubling time in each medium are shown (replicates = 8). (** = p-values<0.05, DED+ELV-MB vs DED+ELV-MT). C_24 h after treatment with ELVs, C2C12 myoblasts were trypsinized and resuspended in DED for size determination by the Scepter 2.0 handheld automated cell counter. Cells were diluted in 100 µl DED in order to analyze at least 10,000 cells/ml for each replicate as recommended by the supplier (replicates = 8).
Figure 6
Figure 6. Effects of ELV-MT on myoblast cyclin and myogenin expressions.
A_mRNA levels of CCND1 in C2C12 myoblasts grown in DED supplemented with ELV-MB or ELV-MT (n = 5 replicates). B_mRNA levels of Myogenin 48 h after the induction of C2C12 differentiation (n = 5 replicates). All qrt-PCR values are expressed as means ± SEM (* = p<0.05); C_ Myoblasts were pre-treated with ELVs during proliferation (2 µg/ml of medium). Then the percentage of C2C12 nuclei expressing myogenin was quantified by immunocyofluorescence, 48 h after the induction of C2C12 differentiation (n = 5 replicates). Chi-square test was used to determine whether the % of myogenin-positive nuclei was significantly different. (*) p-values <3.84 (considering 1 degree of freedom) are significant. D_ Representative pictures of the myogenin staining by using 2 µg ELVs.
Figure 7
Figure 7. ELV-MT can transfer cytoplasmic GFP proteins from myotubes to myoblasts.
A_ Differentiated myotubes infected with a non-replicative adenovirus expressing GFP protein. B_ Western-blot analysis to detect GFP protein in infected myotubes (1 µg), their released ELV-MT (1 µg), and in MB incubated with GFP-containing ELV-MT (60 µg). B_Myoblasts were incubated for 24 h with GFP-containing ELV-MT released from myotubes (2 µg/ml of medium). Arrows indicate the cells which express GFP in cytoplasm.

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Grant support

This work was supported by grants from Fondation pour la Recherche Médicale (FRM), Association Française de recherche sur les Myopathies (AFM), Association Française de Diabétologie (SDF/Rochediagnostics France) and INRA specific grant (ANSSD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.