Proteome Dynamics during C2C12 Myoblast Differentiation

Mouse-derived C2C12 myoblasts serve as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development.To examine the biochemical adaptations associated with myocyte formation comprehensively, we used large scale gel-free tandem mass spectrometry to monitor global proteome alterations throughout a time course analysis of the myogenic C2C12 differentiation program.The relative abundance of ϳ1,800 high confidence proteins was tracked across multiple time points using capillary scale multidimensional liquid chromatography coupled to high throughput shotgun sequencing.Hierarchical clustering of the resulting profiles revealed differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across the early, mid, and late stages of differentiation.Several hundred previously uncharacterized proteins were likewise detected in a stage-specific manner, suggesting novel roles in myogenesis and/or muscle function.These proteomic data are complementary to recent microarraybased studies of gene expression patterns in developing myotubes and provide a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development. Molecular & Cellular Proteomics 4:887-901, 2005.Skeletal muscle consists of tightly bundled cylindrical multinucleated myocytes, each packed with ordered tandem arrays of actin-myosin contractile filaments, which together account for nearly 40% of the total mass of the human body.These highly differentiated cells originate late in embryogenesis from somite-derived mesenchymal myoblast precursor cells in a multistep pathway initiated in response to inductive physiological cues (1).Activation of the myogenic program is normally tightly coupled to a network of signal transduction pathways that act to repress or stimulate cell proliferation and differentiation during embryonic development (1).These pathways function, at least in part, by altering the levels of critical target regulatory proteins through both transcriptional and post-transcriptional mechanisms (1).However, the developmental transitions responsible for myoblast commitment, myocyte specification, and possible developmental plasticity are not fully understood and are just now beginning to be described in integrated molecular terms (2).Cultured undifferentiated fibroblast-like mouse and human myoblast cell lines serve as excellent model systems for investigating the complex biochemical adaptations that underlie the formation of functional myocytes.In the absence of mitogenic stimuli, proliferating myoblasts synchronously withdraw from the cell cycle, elongate, adhere, and finally fuse together to form myotubes exhibiting most, if not all, of the principle mechanobiochemical adaptations associated with contractility (1).Molecular genetic studies of this myogenic program have provided fundamental insight into key regulatory events associated with this profound resetting of cell physiology (2).These include the discovery of critical intracellular signaling pathways and their target sequence-specific transcriptional factors that control cascading waves of gene expression in terminally differentiating myoblasts, leading to large scale biological reorganization and the formation of functional myofibrils (3, 4).Nonetheless knowledge of the full range of biochemical adaptations associated with myocyte formation remains incomplete, masking the complexity that is likely to exist in vivo.Genome scale molecular profiling studies offer a unique opportunity to investigate the molecular hierarchy and biochemical logic that governs muscle cell development and physiology.To this end, several groups have reported the use of DNA-based microarray technology to examine global patterns of transcription in cultured myoblasts during the transition from mitotic cell proliferation to terminal differentiation (5-7).These studies have provided evidence of significant changes in gene expression patterns during myogenesis.However, because mRNA transcript levels do not always correlate with corresponding cognate protein levels (8), determination of the full spectrum of biochemical alterations associated with skeletal muscle formation might be best ascertained by monitoring alterations in protein abundance and turnover directly (9, 10).Systematic comparison of changes in the proteome composition of mitotic and differentiating myoblasts should also provide insight into the various mechanisms and pathways that underlie the formation of skeletal muscle.

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