COMPARATIVE PROTEOMES OF C2C12 MYOBLASTS AND MYOTUBES

N. S.Tannu, V. K. Rao, R. M.Chaudhary, R. Raghow

Myogenesis is a step-wise process of cell lineage determination
and specialization by which pluripotent mesodermal cells of the
embryo are fated to become myoblasts. Mouse C2C12 cells can undergo
myogenic differentiation. When cultured in low serum-containing medium,
C2C12 cells withdraw from the cell cycle and form multi-nucleated myotubes
and thus mimic the final step of embryonal myogenesis elicited.

With a goal to identify differentially expressed proteins during myotube
formation, we separated total and membrane-enriched proteins from myoblasts
and myotubes by 2-dimensional polyacrylamide gel electrophoresis. The images
of silver nitrate stained gels were analyzed by image analysis software PDQuest
and key protein spots were identified by matrix-assisted laser
desortpion/ionization-time of flight mass spectrometry (MALDI-TOF-MS).
We analyzed more than 700 unique protein spots from undifferentiated and
differentiated C2C12 cells. Not surprisingly the vast majority of the common
proteins shared by myoblasts and myotubes represented either housekeeping genes
or constituents of cellular organelles. The levels of such proteins (e. g.,
60 KDa heat shock protein and chain of ATP synthase) remained unchanged
regardless of the state of differentiation. In contrast, formation
of myotubes was accompanied by enhanced expression of 16 proteins while the
levels of 19 proteins were reduced in these cells. Among the differentially
regulated proteins in the myotubes were includes signaling molecules
(dual specificity mitogen activated protein kinase kinase-3, -subunit of
type II cAMP-dependent protein kinase, Jagged-2 and insulin growth factor-1
receptor), transcription factors (TAF28 kD and Pax7), regulators of
cyto-skeletal assembly (and A-X actins, myosin heavy chain, microtubule-associated
APC-binding protein EB1 and LIM kinase1) and apoptosis (IAP-repeat containing
protein MIAP3, p53-regulated protein sestrin 1 and annexin 1).
We will experimental data to illustrate the utility and potential of the
proteomics-based strategies to decipher the mechanistic basis of
myogenic differentiation.

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 The Departments of Biomedical Engineering Pharmacology
University of Tennessee Health Science Center, Memphis,
and Veterans Affairs Medical Center, Memphis, TN