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1 Exercise Science, University of Massachusetts, Amherst, Massachusetts, United States
2 Military Nutrition, US Army Research Institute of Environmental Medicine, Natick, Massachusetts, United States
3 Childrens National Medical Center, Research Center for Genetic Medicine, Washington, District of Columbia, United States
4 Division of Cardiology, Henry Low Heart Center, Hartford Hospital, Hartford, Connecticut, United States
* To whom correspondence should be addressed. E-mail: murso{at}excsci.umass.edu.
We examined the effects of 48h of knee immobilization on alterations in mRNA and protein in human skeletal muscle. We hypothesized that 48h 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 years) before and after 48h immobilization. Global changes in gene expression were analyzed using Affymetrix GeneChips. Candidate genes were confirmed via quantitative real time polymerase chain reaction (qRT-PCR). Western blotting (WB) was used to quantify protein products of candidate genes and to assess Akt pathway activation. Immunohistochemistry (IHC) 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. qRT-PCR analysis confirmed increases in mRNA for UPP components (USP-6, 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 (Ser 473, Thr 308), COLIII and COLIV protein levels were significantly different post-immobilization (25%, 10%, 88% and 28% decrease, respectively. IHC confirmed WB showing decreased staining for collagens post-immobilization. Our results suggest that 48h of immobilization increases mRNA content for components of the UPP and MT function while decreasing mRNA and protein for ECM components as well as decreased phosphorylation of Akt.
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