In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts
Open accessAbstract
Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling proteins, including 43 previously implicated in myogenesis, while others are known to modulate various cell types thus implying a much broader role for myoblasts in muscle remodeling. We also isolated and characterized two types of secreted membrane-derived vesicles: nanovesicles harboring typical exosomal features and larger, morphologically distinct, microvesicles. While they share some common features, their distinct protein and RNA cargos suggest independent functions in myogenesis. We further demonstrate that both types of microvesicles can dock and fuse with adjacent muscle cells but also deliver functional protein cargo. Thus, the intercellular signaling networks invoked during muscle differentiation and regeneration may employ conventional soluble signaling molecules acting in concert with muscle derived microvesicles delivering their cargos directly into target cells.
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MPs were enriched in genes coding for proteins involved in protein synthesis, folding, and trafficking, RNA post-transcriptional modifications, translation modifications, and amino acid metabolism.
“MPs were enriched in genes coding for proteins involved in “protein synthesis, folding, and trafficking,” “RNA post-transcriptional modifications,” “translation modifications,” and “amino acid metabolism.””
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
A similar result was obtained for human muscle cells.
“A similar result was obtained for human muscle cells (Le Bihan et al., 2012).”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
Muscle cells release extracellular vesicles into the extracellular milieu, which represent new paracrine and endocrine signals.
“During the last decade, it has been shown that muscle cells also release extracellular vesicles (EVs) into the extracellular milieu, which represent new paracrine and endocrine signals that have modified our conceptual basis to explain how muscles communicate to other organs (Guescini et al., 2010; Le Bihan et al., 2012; Romancino et al., 2013; Forterre et al., 2014a).”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
Gene enrichment analyses of proteins commonly identified in ELV-MT from human and murine SkM cells further confirmed that ELV-MT are enriched in proteins involved in exosome formation and intracellular trafficking.
“Gene enrichment analyses of the proteins commonly identified in ELV-MT from human (Le Bihan et al., 2012) and murine (Forterre et al., 2014a) SkM cells further confirmed that ELV-MT are enriched in proteins involved in exosome formation and intracellular trafficking (e.g., endocytosis, vesicle fusion, and transport, and signaling pathways involved in vesicle cellular localization) (Figure 4B).”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
Compared to MPs, human ELV-MT were enriched in proteins for free-radical scavenging and cell-to-cell signaling and interactions.
“Compared to MPs, human ELV-MT were enriched in proteins for “free-radical scavenging” and “cell-to-cell signaling and interactions.””
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
MPs were mainly cytosolic proteins, located in endoplasmic reticulum, Golgi vesicles, mitochondria, and cytoskeleton.
“They were mainly cytosolic proteins, located in “endoplasmic reticulum,” “Golgi vesicles,” “mitochondria,” and “cytoskeleton.””
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
ELV-MT proteins were localized in subplasma-membrane cytoplasmic vesicles, endosomes, lysosome, and plasma membrane.
“Conversely to MP proteins, ELV-MT proteins were localized in “subplasma-membrane cytoplasmic vesicles,” “endosomes,” “lysosome,” and “plasma membrane” (Le Bihan et al., 2012)”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
Myotubes release small exosome-like vesicles (ELV-MT).
“Myotubes also release small ELVs (ELV-MT) (Figure 1D; Le Bihan et al., 2012; Romancino et al., 2013; Forterre et al., 2014a,b)”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
MPs released from human myotubes were enriched in tetraspanin CD81 and in CLIC1 and galectin-1 (LGALS1) proteins and did not contain tetraspanins CD63, CD82, and CD9, which were found associated with ELVs from human myotubes.
“MPs released from human MT were enriched in tetraspanin CD81 and in CLIC1 and galectin-1 (LGALS1) proteins and did not contain tetraspanins CD63, CD82, and CD9 which were found associated with ELVs from human MT (Figures 1D, 3; Le Bihan et al., 2012).”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
Proteomic analyses have identified two specific subsets of proteins for MPs and ELVs released in the conditioned medium from human myotubes.
“Proteomic analyses have identified two specific subsets of proteins for MPs and ELVs, released in the conditioned medium from human myotubes (Figure 4A; Le Bihan et al., 2012).”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
For human muscle cells, iodixanol gradients with ELV-MT resulted in the identification of five potential ELV-MT subpopulations with specific tetraspanin enrichments.
“For human muscle cells, iodixanol gradients have been performed with ELV-MT and resulted in the identification of five potential ELV-MT subpopulations with specific tetraspanin enrichments (Figure 3; Le Bihan et al., 2012).”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
Messenger RNAs were detected by using DNA microarrays in ELVs and MPs released from human SkM cells.
“Messenger RNAs were also detected by using DNA microarrays, in ELVs and MPs released from human SkM cells (Le Bihan et al., 2012).”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
ELVs were significantly enriched in transcripts encoding receptors and ionic channels, and regulators of transcription, in particular zinc-finger transcription factors.
“ELVs were significantly enriched in transcripts encoding receptors and ionic channels, and regulators of transcription in particular zinc-finger transcription factors (Le Bihan et al., 2012).”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
Hierarchical clustering showed that the mRNA cargos of ELVs and MPs were more closely related to each other than to the expression pattern of the secreting myotubes.
“Hierarchical clustering showed that the mRNA cargos of ELVs and MPs were more closely related to each other than to the expression pattern of the secreting myotubes.”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
Respectively, 185 and 4,431 transcripts were identified in ELVs and MPs.
“Respectively, 185 and 4,431 transcripts were identified in ELVs and MPs.”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
The abundance of the 185 ELV mRNAs correlated with their abundance in myotubes, indicating that myotubes release a specific subset of transcripts in ELVs, and not only the most concentrated one.
“The abundance of the 185 ELV mRNAs correlated with their abundance in myotubes, indicating that myotubes release a specific subset of transcripts in ELVs, and not only the most concentrated one.”
From Skeletal Muscle-Released Extracellular Vesicles: State of the Art (2019) · cites this paper as Le Bihan et al., 2012
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- Authors
- Le Bihan, M.-C., Bigot, A., Jensen, S. S., Dennis, J. L., Rogowska-Wrzesinska, A., Lainé, J., Gache, V., Furling, D., Jensen, O. N., Voit, T., Mouly, V., Coulton, G. R., & Butler-Browne, G.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- University of Southern Denmark (DK)
- St George's, University of London (GB)
- Inserm (FR)
- Corresponding author
- Butler-Browne, Gillian
- ORCID
- Venue
- Journal of Proteomics
- Publisher
- Elsevier BV
- Published
- 2012-12
- Volume
- 77
- Pages
- 344-356
- ISSN
- 1874-3919 (ISSN-L)
- OpenAlex
- W1980465387
- Cited by
- 153
- References
- 53
- Citations by year
- 2026: 8 · 2025: 10 · 2024: 10 · 2023: 13 · 2022: 16 · 2021: 12 · 2020: 14 · 2019: 13 · 2018: 4 · 2017: 15 · 2016: 8 · 2015: 12 · 2014: 9 · 2013: 9
- FWCI
- 3.85
- Type
- journal article
- Language
- en
- Open access
- green
- zenodo.org
- Licence
- Locations
- Journal of Proteomics (publishedVersion)
- PubMed (publishedVersion)
- University of Southern Denmark Research Portal (University of Southern Denmark) (publishedVersion)
- Zenodo (CERN European Organization for Nuclear Research) (submittedVersion, other-oa)
- Funders
- European Commission
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Caveolin-1 and cellular processes
- Keywords
- secretome, myogenesis, muscle regeneration, exosomes, microvesicles, membrane vesicles, intercellular communication, muscle remodeling, vesicle fusion, RNA cargo
- MeSH terms
- Cell Differentiation, Cells, Cultured, Female, Humans, Infant, Newborn, Muscle Proteins, Proteome, Satellite Cells, Skeletal Muscle, Proteomics, Cell-Derived Microparticles, Secretory Pathway
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