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

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.

Bmi1 enhances skeletal muscle regeneration through MT1-mediated oxidative stress protection in a mouse model of dystrophinopathy

“…Interestingly there is evidence that MT1 expression increases in human skeletal muscle 24 h after intense exercise (Urso et al, 2006); here, we show decreased expression of MT1 in human DMD muscle, raising the possibility that manipulation of Bmi1 expression could be a useful approach to enhance satellite cell-driven regeneration also in human primary myopathies. Identifying novel pharmacological agents to enhance and sustain muscle regeneration in DMD is highly desirable as they could have a tremendous impact on DMD therapy either alone or, most DMD patients (Fig.…”

Characterization of the Human Skeletal Muscle Proteome by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

“…They have been reported to bind to insulin receptor substrate (IRS)-1, IRS-2, and PDK1 and to modulate the activities of IRS-1 associated PI3-kinase and PDK1 (3). Extracellular Matrix (ECM) and Contractile Proteins-Muscle adaptation in response to a number of physiological and pathophysiological conditions involves changes in ECM and contractile proteins (13,14,45). Among ECM proteins, we found three ␣-subunits of type VI collagen, fibronectin, decorin, lumican, and prolargin.…”

β - catenin is central to DUX4 -driven network rewiring in facioscapulohumeral muscular dystrophy

“…TP53 [16], JUNB [20,17], HIF1A [20], WNT3 [4], LMO3 [20], ANXA4 [5] and HSPB1 [22]. Gene Set Enrichment Analysis (GSEA) [23] on the intersection also implicated many FSHD associated processes, such as myogenesis [17] and regulation of the actin cytoskeleton [15], and pathways, including, p53 [16], Wnt [4], and VEGF [5] To identify genes implicated as rewiring specifically in FSHD, we also ran InSpiRe on two datasets each describing skeletal muscle gene expression during ageing (GSE5086 [24] and GSE9676 [25]), disuse atrophy (GSE5110 [26] and GSE8872 [27]) and other muscle diseases involving inflammation and wasting (GSE3307 [19], where juvenile dermatomyositis and limb-girdle muscular dystrophy type 2A datasets were independently analysed). Genes rewiring in these non-FSHD datasets were considered as secondary rewiring.…”

“…We also used human skeletal muscle studies into gene expression in muscle diseases other than FSHD (GSE3307 [19]), ageing (GSE5086 [24] and GSE9676 [25]) and atrophy (GSE5110 [26] and GSE8872 [27]). …”