Skeletal muscle atrophy occurs as a consequence of injury, illness, surgery, and muscle disuse, impacting appreciably on health care costs and patient quality of life, particularly in the absence of appropriate rehabilitation. The molecular mechanisms that regulate muscle mass during atrophy and rehabilitation in humans have not been elucidated, despite several robust candidate pathways being identified. Here, we induced skeletal muscle atrophy in healthy volunteers using two weeks of limb immobilization, and then stimulated the restoration of muscle mass with six weeks of supervised exercise rehabilitation. We determined muscle mass and function and performed targeted gene expression analysis at prescribed time points during immobilization and rehabilitation. For the first time, we have identified novel changes in gene expression following immobilization-induced atrophy and during a program of rehabilitative exercise that restored muscle mass and function. Furthermore, we have shown that exercise performed immediately following immobilization induces profound changes in the expression of a number of genes in favor of the restoration of muscle mass, within 24 h. This information will be of considerable importance to our understanding of how immobilization and contraction stimulate muscle atrophy and hypertrophy, respectively, and to the development of novel therapeutic strategies aimed at maintaining or restoring muscle mass.

1 Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biologically active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be critical in determining the most effective sequence. 2 In the present study, we show by FACS analysis that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine ¼ transportan4antennapedia4 TAT. 3 However, examination of cellular toxicity showed that antennapediaoTATotransportanopolyarginine, with antennapedia-peptide conjugates having no significant toxicity even at 100 mM. 4 Confocal studies of the mechanism of antennapedia-and TAT-peptide uptake showed that the time course of uptake and their cellular distribution did not correlate with transferrin, a marker of clathrin-mediated endocytosis. In contrast, the peptides co-localised with a marker of lipid rafts domains, cholera toxin, which was attenuated following the disruption of these domains using methylb-cyclodextrin. 5 Overall, comparison of the uptake and toxicity suggests that antennapedia provides the optimal cell-penetrating peptide for peptide delivery in vitro and that both antennapedia-and TAT-mediated peptide delivery occurs predominantly via lipid raft-dependent but clathrin-independent endocytosis.

We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.

The therapeutic application of siRNA shows promise as an alternative approach to small molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the non-viral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line, showed that siRNA conjugated to cholesterol, TAT(48-60) and penetratin but not siRNA alone achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localisation within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6hrs. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown whilst penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression whilst the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA mediated p38 Figure S1, dose-dependent percentile p38 MAP kinase mRNA levels in vitro following lipofection with three mouse p38 MAP kinase siRNA or two mismatch controls; Figure S2, analytical HPLC and electrospray mass spectrograms of peptide-and cholesteryl-RNA constructs; Figure S3, analytical gels of CPP and cholesterol conjugate annealing products; Figure S4, cell viability following incubation with siRNA, CPP or siRNA conjugates; Figure S5, MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.