Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

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.