Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

Marie-Catherine Le Bihan, Inigo Barrio, Tenna Pavia Mortensen, Jeanette Henningsen, Søren Skov Jensen, Anne Bigot, Blagoy Blagoev, Gillian Butler-Browne, Irina Kratchmarova

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

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.

OriginalsprogEngelsk
TidsskriftJournal of Proteome Research
Vol/bind14
Udgave nummer8
Sider (fra-til)3348-3361
ISSN1535-3893
DOI
StatusUdgivet - 7. aug. 2015

Fingeraftryk

Proteome
Muscle
Proteins
Muscles
Cells
Isotope Labeling
Muscle Development
Myogenic Regulatory Factors
Muscular Diseases
Proteolysis
Cell Adhesion
Computer Simulation
Statistical Factor Analysis
Cell Cycle
Cell adhesion
Maintenance
Stem cells
RNA
Cell culture
Metabolism

Citer dette

Le Bihan, Marie-Catherine ; Barrio, Inigo ; Mortensen, Tenna Pavia ; Henningsen, Jeanette ; Jensen, Søren Skov ; Bigot, Anne ; Blagoev, Blagoy ; Butler-Browne, Gillian ; Kratchmarova, Irina. / Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts. I: Journal of Proteome Research. 2015 ; Bind 14, Nr. 8. s. 3348-3361.
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Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts. / Le Bihan, Marie-Catherine; Barrio, Inigo; Mortensen, Tenna Pavia; Henningsen, Jeanette; Jensen, Søren Skov; Bigot, Anne; Blagoev, Blagoy; Butler-Browne, Gillian; Kratchmarova, Irina.

I: Journal of Proteome Research, Bind 14, Nr. 8, 07.08.2015, s. 3348-3361.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

AU - Le Bihan, Marie-Catherine

AU - Barrio, Inigo

AU - Mortensen, Tenna Pavia

AU - Henningsen, Jeanette

AU - Jensen, Søren Skov

AU - Bigot, Anne

AU - Blagoev, Blagoy

AU - Butler-Browne, Gillian

AU - Kratchmarova, Irina

PY - 2015/8/7

Y1 - 2015/8/7

N2 - 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.

AB - 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.

U2 - 10.1021/acs.jproteome.5b00397

DO - 10.1021/acs.jproteome.5b00397

M3 - Journal article

VL - 14

SP - 3348

EP - 3361

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 8

ER -