Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components

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Abstract
We examined the effects of 48 h of knee immobilization on alterations in mRNA and protein in human skeletal muscle. We hypothesized that 48 h of immobilization would increase gene expression and respective protein products for ubiquitin-proteasome pathway (UPP) components. Also, we used microarray analysis to identify novel pathways. Biopsies were taken from the vastus muscle of five men (20.4 ± 0.5 yr) before and after 48-h immobilization. Global changes in gene expression were analyzed by use of Affymetrix GeneChips. Candidate genes were confirmed via quantitative RT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes and to assess Akt pathway activation. Immunohistochemistry was used to localize proteins found to be altered when assessed via WB. The greatest percentage of genes showing altered expression with the GeneChip included genes involved in the UPP, metallothionein function, and extracellular matrix (ECM) integrity. Quantitative RT-PCR analysis confirmed increases in mRNA for UPP components [USP-6, small ubiquitin-related modifier (SUMO-1)] and the metallothioneins (MT2A, MT1F, MT1H, MT1X) and decreases in mRNA content for matrix metalloproteinases (MMP-28, TIMP-1) and ECM structural components [collagen III (COLIII) and IV (COLIV)]. Only phosphorylated Akt (Ser473, Thr308), COLIII and COLIV protein levels were significantly different postimmobilization (25, 10, 88, and 28% decrease, respectively). Immunohistochemistry confirmed WB showing decreased staining for collagens postimmobilization. Our results suggest that 48 h of immobilization increases mRNA content for components of the UPP and metallothionein function while decreasing mRNA and protein for ECM components as well as decreased phosphorylation of Akt.