Protein profile and directed gene expression of developing C2C12 cells

Abstract

Myogenesis is a tightly regulated process resulting in unique structures called myotubes or myofibers, which compose skeletal muscle. Myotubes are multi-nucleated fibers containing a functional unit composed of cytoskeletal proteins called the sarcomere. The specific arrangement of these proteins in the sarcomere works to contract and relax muscles. During embryonic and post-embryonic development, fluctuations in expression of growth factors throughout the program account for the dramatic structural changes from cell to mature muscle fiber. In vivo, these growth factors are strictly spatiotemporally regulated according to a 'myogenic program.' In order to assess the dynamics of protein expression throughout this program, we conducted a time course study using the mouse myoblast cell line C2C12, in which cells were allowed to differentiate and insoluble protein fractions were collected at seven time points. The insoluble protein fraction accounts for cytoskeletal proteins, involved in the dramatic remodeling of cells. In order to discover influence of growth factors on protein expression, a similar time course was conducted with the addition of growth factors (serum) at specific time points after induction of myogenesis. The cells were allowed to differentiate through the time course and the detergent- insoluble protein fractions were collected. Proteins were separated according to their eletrophoretic mobility using SDS-PAGE and were identified using LC-MS/MS. Proteins were then functionally annotated using Gene Ontology (GO) terms. Analysis of protein expression during in vitro myogenesis shows progression of the myogenic program. Furthermore, a targeted gene expression study was conducted using reverse-transcriptase quantitative PCR. Expression of the sarcomeric proteins actin, myosin, and titin also fluctuated throughout the time course, indicative of a developmental program.