title: Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts.
abstract: Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies.
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Marie-Catherine
authors.initials: MC
authors.lastname: Le Bihan
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Inigo
authors.initials: I
authors.lastname: Barrio-Hernandez
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Tenna Pavia
authors.initials: TP
authors.lastname: Mortensen
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Henningsen
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Søren Skov
authors.initials: SS
authors.lastname: Jensen
authors.affiliation: ‡Center for Research in Myology, Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS975, CNRS FRE3617, 75013 Paris, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Bigot
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Blagoy
authors.initials: B
authors.lastname: Blagoev
authors.affiliation: ‡Center for Research in Myology, Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS975, CNRS FRE3617, 75013 Paris, France.
authors.forename: Gillian
authors.initials: G
authors.lastname: Butler-Browne
authors.affiliation: †Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Irina
authors.initials: I
authors.lastname: Kratchmarova
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D016000:Cluster Analysis; D006801:Humans; D007150:Immunohistochemistry; D007231:Infant, Newborn; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D020543:Proteome / Q000145:classification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D000596:Amino Acids; D020543:Proteome
keywords: Cell differentiation; SILAC; humans; muscle regeneration; primary cell culture; quantitative proteomics; skeletal muscle