Patterning, prestress and adhesion dynamics of murine skeletal muscle cells: Studies with the C2C12 cell line and both normal and γ-sarcoglycan knockout primary cells

Maureen A. S Griffin, University of Pennsylvania

Abstract

The adhesion of a tissue cell to a substrate or matrix is increasingly understood to be in a dynamic balance against contractile tensions, or prestress, exerted by the cell on its substrate, but how this balance affects muscle differentiation, and how these factors contribute to the development of diseases such as muscular dystrophy (MD) are still unknown. One type of MD in particular, limb-girdle MD, is caused by the absence of γ-sarcoglycan, but the functions of γ-sarcoglycan in healthy muscle is not yet understood. With this in mind, micropatterned substrates were developed to grow isolated myotubes (or a stacked pair of myotubes) from myoblasts in order to study the differentiation, adhesion, and contractile prestress of individual myotubes, and ultimately to examine the effects of γ-sarcoglycan on these cellular functions. Micropipette peeling of normal murine muscle cells of C2C12 lineage demonstrated that cell adhesion is heterogeneous, with myotubes remaining stuck to the substrate at discrete points along their length ∼80% of the time. Peeling also revealed a buildup of prestress in myotubes which was further investigated through the use of cytoskeletal antagonists. The inhibition of myosin-II decreased the prestress by ∼60%; in contrast, F-actin was found to make broader structural contributions to both cell adhesion and contractility. Myotube-on-myotube results for both C2C12's and mouse primary cells suggest the importance of the substrate compliance to both myotube differentiation and myotube prestress relative to adhesion: At one week, 53% of cells grown on a compliant layer of other cells formed sarcomeres compared to 0% of cells grown on rigid glass, and cells-on-cells exhibited a ∼140% increase in prestress compared to cells-on-glass. Finally, γSG deficiency studied with knockout primary cells revealed no effect on cell adhesion but did lead to an enhanced prestress (by ∼100%) in cultured myotubes. These cells also exhibited a 17-fold increase in early apoptosis in vitro, consistent with in vivo studies of others. In sum, the in vitro methods and results here further an understanding of the adhesion-contractile balance in both normal differentiation of muscle and in one prototypical muscle disease.

Subject Area

Biophysics|Biomedical research|Cellular biology

Recommended Citation

Griffin, Maureen A. S, "Patterning, prestress and adhesion dynamics of murine skeletal muscle cells: Studies with the C2C12 cell line and both normal and γ-sarcoglycan knockout primary cells" (2003). Dissertations available from ProQuest. AAI3109181.
https://repository.upenn.edu/dissertations/AAI3109181

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