Metabolic phenotype of skeletal muscle in early critical illness.

Objectives: To characterise the sketetal muscle metabolic phenotype during early critical illness.

Methods: Vastus lateralis muscle biopsies and serum samples (days 1 and 7) were obtained from 63 intensive care patients (59% male, 54.7±18.

Isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56(+) and later as CD56(+)/desmin(+) cells and (ii) muscle-derived fibroblasts, identified as CD56(-) and TE-7(+).

An investigation into the relationship between age and physiological function in highly active older adults.

Key Points: The relationship between age and physiological function remains poorly defined and there are no physiological markers that can be used to reliably predict the age of an individual. This could be due to a variety of confounding genetic and lifestyle factors, and in particular to ill-defined and low levels of physical activity. This study assessed the relationship between age and a diverse range of physiological functions in a cohort of highly active older individuals (cyclists) aged 55-79 years in whom the effects of lifestyle factors would be ameliorated.

Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation.

We characterised the adherent cell types isolated from human skeletal muscle by enzymatic digestion, and demonstrated that even at 72 hours after isolation these cultures consisted predominantly of myogenic cells (CD56(+), desmin(+)) and fibroblasts (TE-7(+), collagen VI(+), PDGFRα(+), vimentin(+), fibronectin(+)). To evaluate the behaviour of the cell types obtained, we optimised a double immuno-magnetic cell-sorting method for the separation of myogenic cells from fibroblasts. This procedure gave purities of >96% for myogenic (CD56(+), desmin(+)) cells.

Primary human muscle precursor cells obtained from young and old donors produce similar proliferative, differentiation and senescent profiles in culture.

The myogenic behaviour of primary human muscle precursor cells (MPCs) obtained from young (aged 20-25 years) and elderly people (aged 67-82 years) was studied in culture. Cells were compared in terms of proliferation, DNA damage, time course and extent of myogenic marker expression during differentiation, fusion, size of the formed myotubes, secretion of the myogenic regulatory cytokine TGF-β1 and sensitivity to TGF-β1 treatment. No differences were observed between cells obtained from the young and elderly people.

An image analysis method for the precise selection and quantitation of fluorescently labeled cellular constituents: application to the measurement of human muscle cells in culture.

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture.

Sera from young and older humans equally sustain proliferation and differentiation of human myoblasts.

Using a human primary muscle cell culture model the behaviour of myoblasts (satellite cells) cultured in human serum obtained from either young or elderly individuals was studied. Serum was obtained from a total of 13 young (7 males and 6 females aged, 23-36 years) and 9 elderly (4 males and 5 females aged 69-84 years) subjects and used in a number of experiments. Myoblasts were extracted from human muscle biopsy samples taken from the vastus lateralis.

A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humans.

An integrated MS-based proteomic approach is described that combines MALDI-MS and LC-MS with artificial neural networks for the identification of protein and peptide biomarkers associated with recombinant human growth hormone (rhGH) administration. Serum from exercised males administered with rhGH or placebo was analysed using ELISA to determine insulin-like growth factor-I concentrations. Diluted serum from rhGH- and placebo-treated subjects was analysed for protein biomarkers by MALDI-MS, whereas LC-MS was used to analyse tryptically digested ACN-depleted serum extracts for peptide biomarkers.

Investigation of MGF mRNA expression in patients with amyotrophic lateral sclerosis using parallel in vivo and in vitro approaches.

In an animal model of ALS, intramuscular administration of MGF, the IGF-I Ec gene splice variant, improved muscle strength and increased both motor unit and motor neuron survival. Here we investigated whether there is a deficit in MGF production in the muscles of patients with ALS. We used complementary in vivo and in vitro techniques to study the IGF-I splice variant response of human muscle to exercise or mechanical stretch.

IGF-I and GH: potential use in gene doping.

Gene doping is the term given to the potential misuse of gene therapy for the purposes of enhancing athletic performance. Insulin like growth factor-I (IGF-I), the prime target of growth hormone action, is one candidate gene for improving performance. In recent years a number of transgenic and somatic gene transfer studies on animals have shown that upregulation of IGF-I stimulates muscle growth and improves function.

Serum IGF-I levels and IGF-I gene splicing in muscle of healthy young males receiving rhGH.

Objective: Elevated growth hormone (GH) levels lead to increased circulating insulin-like growth factor-I (IGF-I), but the effects on localised muscle IGF-I splice variant expression is not known. The effects of rhGH administration, with or without an acute bout of high resistance exercise, were measured on serum IGF-I and on the mRNA levels of IGF-I splice variants in the vastus lateralis muscle of healthy young men.

Design: The study was a randomised double blind trial with a crossover design.

Gene doping.

Gene doping is the misuse of gene therapy to enhance athletic performance. It has recently been recognised as a potential threat and subsequently been prohibited by the World Anti-Doping Agency. Despite concerns with safety and efficacy of gene therapy, the technology is progressing steadily.

The regenerative plasticity of isolated urodele myofibers and its dependence on MSX1.

The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture.