At the recent “Making Muscle in the Embryo and Adult” meeting held at Columbia University, New York from May 28 - June 2, Marie-Catherine Le Bihan, post-doctorate from Gillian Butler-Brown’s laboratory, Therapy of Striated Muscle Disorders, UMR S 974, was awarded 1st prize for an innovative and exciting research topic “Are secreted microvesicles a novel mechanism of communication for skeletal muscle?.”
What is the objective of this research project?
Our initial objective was to characterize the secreted proteome or “secretome” of differentiating human myoblasts thus mimicking the behaviour of cells entering the process of muscle fibre regeneration. Secreted signalling molecules including growth factors and cytokines have been shown to regulate activation, proliferation and differentiation of the muscle progenitors known as satellite cells. Therefore they must serve important functions during skeletal muscle development, maintenance and repair.
In order to do this, we used a proteomic approach with the latest developments in mass spectrometry. This was done in collaboration with Professor Ole Jensen, from the Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark.
What results did you obtain?
We identified 965 non-redundant proteins secreted by human muscle cells. Among those, 257 proteins (27%) were secreted in the extracellular space via the classical ER/Golgi secretion pathway, 85 of which were extracellular matrix components. Several known secreted proteins such as Galectin-1, IGF-II and myostatin were also identified.
In addition to these extracellular “soluble” secreted proteins, 708 intracellular proteins from various origins (membrane, cytosolic, Golgi/ER, mitochondrial, nuclear, etc) were found in the conditioned medium of differentiating myoblasts.
What is the significance of these results?
This leakage of intracellular proteins into the media suggested that cell lysis had occurred subsequent to cell death. However, examination of the differentiating culture revealed no evidence of floating cells and the level of cell death in our serum free condition was minimal (~4.5%). This led to the hypothesis that these proteins could be transported outside the cell via secreted microvesicles.
The presence of these particles in the culture supernatant was confirmed by electron microscopy. We extensively characterized the protein cargo of those isolated microvesicles therefore confirming our initial hypothesis: more than 40% of the protein released in the extracellular space by differentiating culture was associated with membrane bound vesicles.
In addition we have demonstrated that these secreted microvesicles can dock and fuse with differentiating muscle cells.
We are thus able to postulate a novel mechanism of paracrine signaling for skeletal muscle cells, which may play an important functional role in skeletal muscle homeostasis, regeneration and myogenesis.
What are the next steps?
Only a thorough understanding of the secretory behaviour of human muscle cells will allow us to obtain insight into human muscle regeneration and ageing as well as disease (e.g. muscular dystrophy, diabetes etc), and into the putative cell interactions involved. Resolving the human skeletal muscle secretome will also provide new and potential targets for future therapeutic interventions.
Questions remaining to be answered concerning these secreted microvesicles: “Is this a novel mechanism of communication for skeletal muscle? Can they be used as a vector, “physiological liposome” for therapeutic purpose (packed with a “gene medicine”?)
Interview by Anne Berthomier, translation by Racquel N. Cooper