Effect of serum and mechanical stretch on skeletal α-actin gene regulation in cultured primary muscle cells

James Carson, Frank W. Booth

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39 Citations (Scopus)

Abstract

The purpose of this study was to determine whether mechanical stretch or serum availability alters pretranslational regulation of skeletal α-actin (SkA) in cultured striated muscle cells. Chicken primary skeletal myoblasts and cardiac myocytes were plated on collagenized Silastic membranes adherent to nylon supports and stretched 8-20% of initial length 96 h postplating. Serum dependence of SkA gene regulation was determined by maintaining differentiated muscle cells in growth/differentiation (G/D; skeletal myotubes, 10% horse serum-2% chick embryo extract; cardiac myocytes, 10% horse serum) or growth-limiting (G-L; 0.5% horse serum) medium. Skeletal myotubes had higher SkA mRNA and SkA promoter activity in G/D than in G-L medium. Cardiac myocyte SkA mRNA was higher in G-L than in G/D medium. Serum response factor (SRF) protein binding to serum response element 1 (SRE1) of SkA promoter increased in skeletal cultures in G/D compared with G-L medium. Western blot analysis demonstrated that increased SRF-SRE1 binding was due, in part, to increased SRF protein. Stretching skeletal myotubes in G,L medium reduced SkA mRNA and repressed SkA promoter activity. The first 100 bp of SkA promoter were sufficient for stretch-induced repression of SkA promoter activity, and an intact transcriptional enhancer factor 1 (TEF-1) binding site was necessary for this response. Serum and stretch appear to repress SkA promoter activity in skeletal myotubes through different DNA binding elements, the SRE1 and TEF-1 sites, respectively. Stretching increased SkA mRNA in cardiac myocytes in G-L medium but did not alter SkA mRNA level in cardiac cells in G/D medium. These results demonstrate that stretch and serum interact differently to alter SkA expression in cultured cardiac and skeletal muscle cells.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume275
Issue number6 44-6
StatePublished - Dec 1 1998

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Muscle Cells
Actins
Serum
Genes
Skeletal Muscle Fibers
Cardiac Myocytes
Serum Response Element
Serum Response Factor
Messenger RNA
Horses
Skeletal Myoblasts
Gastrin-Secreting Cells
Striated Muscle
Nylons
Chick Embryo
Growth
Protein Binding
Chickens
Skeletal Muscle
Western Blotting

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology

Cite this

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title = "Effect of serum and mechanical stretch on skeletal α-actin gene regulation in cultured primary muscle cells",
abstract = "The purpose of this study was to determine whether mechanical stretch or serum availability alters pretranslational regulation of skeletal α-actin (SkA) in cultured striated muscle cells. Chicken primary skeletal myoblasts and cardiac myocytes were plated on collagenized Silastic membranes adherent to nylon supports and stretched 8-20{\%} of initial length 96 h postplating. Serum dependence of SkA gene regulation was determined by maintaining differentiated muscle cells in growth/differentiation (G/D; skeletal myotubes, 10{\%} horse serum-2{\%} chick embryo extract; cardiac myocytes, 10{\%} horse serum) or growth-limiting (G-L; 0.5{\%} horse serum) medium. Skeletal myotubes had higher SkA mRNA and SkA promoter activity in G/D than in G-L medium. Cardiac myocyte SkA mRNA was higher in G-L than in G/D medium. Serum response factor (SRF) protein binding to serum response element 1 (SRE1) of SkA promoter increased in skeletal cultures in G/D compared with G-L medium. Western blot analysis demonstrated that increased SRF-SRE1 binding was due, in part, to increased SRF protein. Stretching skeletal myotubes in G,L medium reduced SkA mRNA and repressed SkA promoter activity. The first 100 bp of SkA promoter were sufficient for stretch-induced repression of SkA promoter activity, and an intact transcriptional enhancer factor 1 (TEF-1) binding site was necessary for this response. Serum and stretch appear to repress SkA promoter activity in skeletal myotubes through different DNA binding elements, the SRE1 and TEF-1 sites, respectively. Stretching increased SkA mRNA in cardiac myocytes in G-L medium but did not alter SkA mRNA level in cardiac cells in G/D medium. These results demonstrate that stretch and serum interact differently to alter SkA expression in cultured cardiac and skeletal muscle cells.",
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