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Functional characterization of muscle fibers relies on ATPase activity and on differential measurements of metabolic proteins, including mitochondrial and glycolytic enzymes, glucose, lactate and lactic acid transporters, calcium cycling proteins and components of the contractile machinery. The recent introduction of microarray technology has enabled detailed gene expression studies under different physiological and pathological conditions, thus generating novel hypotheses on muscle function. However, microarray approaches are limited by the incomplete genome coverage of currently available chips, and by poor correlation between mRNA concentration and protein expression level. We have used 2-DE and MS to build a reference map of proteins from rat mixed gastrocnemius and soleus muscle, and to assess qualitative and quantitative differences in protein distribution between these two functionally dissimilar muscles. More than 800 spots on each gel were detected by silver staining, of which 167 were excised, digested in-gel with trypsin and analyzed by ESI-MS/MS. One hundred and twenty eight distinct gene products were identified, including metabolic, transport and contractile proteins. Forty one spots displayed differences in relative expression level between mixed gastrocnemius and soleus samples. These data not only enable differentiation of functionally distinct slow-twitch and fast-twitch fiber types, but also provide tools for investigating muscle plasticity in response to physiological and environmental conditions such as aging or hypoxia.

We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the expression of contractile and regulatory proteins in human vastus lateralis and deltoideus muscles, in order to understand protein turnover and isoform switching in muscles with the same fiber-type composition but different functional properties. We demonstrate a two- to six-fold overexpression of enzymes associated with glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and substrate transport in vastus lateralis compared to deltoideus. Expression levels of contractile protein isoforms correlated to the proportion of slow-twitch fibers in deltoideus compared to vastus lateralis are consistent with the different contractile properties of the two muscles. Two proteins involved in free radical homeostasis were differentially expressed, suggesting a direct relationship between radical scavenging and the muscle function. The application of 2-DE and MS to studies of muscle physiology thus offers a more comprehensive assessment of the molecular determinants of muscle function than traditional approaches.

The high iron demand associated with enhanced erythropoiesis during high-altitude hypoxia leads to skeletal muscle iron mobilization and decrease in myoglobin protein levels. To investigate the effect of enhanced erythropoiesis on systemic and muscle iron metabolism under nonhypoxic conditions, 8 healthy volunteers were treated with recombinant erythropoietin (rhEpo) for 1 month. As expected, the treatment efficiently increased erythropoiesis and stimulated bone marrow iron use. It was also associated with a prompt and considerable decrease in urinary hepcidin and a slight transient increase in GDF-15. The increased iron use and reduced hepcidin levels suggested increased iron mobilization, but the treatment was associated with increased muscle iron and L ferritin levels. The muscle expression of transferrin receptor and ferroportin was up-regulated by rhEpo administration, whereas no appreciable change in myoglobin levels was observed, which suggests unaltered muscle oxygen homeostasis. In conclusion, under rhEpo stimulation, the changes in the expression of muscle iron proteins indicate the occurrence of skeletal muscle iron accumulation despite the remarkable hepcidin suppression that may be mediated by several factors, such as rhEpo or decreased transferrin saturation or both.

Ageing induces a progressive morphological change and functional decline in muscles and in nerves. Light and electron microscopy, 2-D DIGE and MS, were applied to profile the qualitative and quantitative differences in the proteome and morphology of rat gastrocnemius muscle and sciatic nerve, in healthy 22-month-old rats. At muscle level, morphological changes are associated to fibre atrophy accompanied by myofibrillar loss and degeneration, disappearance of sarcomeres and sarcoplasmic reticulum dilatation, internal migration of nuclei, longitudinal fibre splitting, increment of subsarcolemmal mitochondria aggregates and increment of lipofuscin granules. Sciatic nerve shows myelin abnormalities like enfoldings, invaginations, onion bulbs, breakdowns and side axonal atrophy. Proteomic analysis identified changes correlated to morphological abnormalities in metabolic, contractile and cytoskeletal proteins, deregulation of iron homeostasis, change of Ca(2+) balance and stress response proteins, accompanied by a deregulation of myelin membrane adhesion protein and proteins regulating the neuronal caliber. By comparing proteomic results from the two tissues, 16 protein isoforms showed the same up and down regulation trend suggesting that there are changes implying a general process which may act as a signal event of degeneration. Only beta enolase and tropomyosin 1alpha were differentially expressed in the tissues.

Iron is essential for oxygen transport because it is incorporated in the heme of the oxygen-binding proteins hemoglobin and myoglobin. An interaction between iron homeostasis and oxygen regulation is further suggested during hypoxia, in which hemoglobin and myoglobin syntheses have been reported to increase. This study gives new insights into the changes in iron content and iron-oxygen interactions during enhanced erythropoiesis by simultaneously analyzing blood and muscle samples in humans exposed to 7 to 9 days of high altitude hypoxia (HA). HA up-regulates iron acquisition by erythroid cells, mobilizes body iron, and increases hemoglobin concentration. However, contrary to our hypothesis that muscle iron proteins and myoglobin would also be up-regulated during HA, this study shows that HA lowers myoglobin expression by 35% and down-regulates iron-related proteins in skeletal muscle, as evidenced by decreases in L-ferritin (43%), transferrin receptor (TfR; 50%), and total iron content (37%). This parallel decrease in L-ferritin and TfR in HA occurs independently of increased hypoxia-inducible factor 1 (HIF-1) mRNA levels and unchanged binding activity of iron regulatory proteins, but concurrently with increased ferroportin mRNA levels, suggesting enhanced iron export. Thus, in HA, the elevated iron requirement associated with enhanced erythropoiesis presumably elicits iron mobilization and myoglobin down-modulation, suggesting an altered muscle oxygen homeostasis.

We investigated the effects of hypoxia and intermittent hypoxia in rat muscle by quantitating the expression of genes encoding cytochrome c oxidase (CytOx) subunits I, II and IV, and ribosomal 12S RNA. The quantitative assessment was made by CE in a polymer network, laser being used to facilitate the detection of induced fluorescence. Constant and intermittent hypoxia influence gene expression and ribosomal activity to different degrees. We found constant hypoxia to be accompanied by an increment in the mitochondrial CytOx subunit transcripts II and I and 12S ribosomal subunit, whereas the nuclear subunit (IV) remained unchanged. No changes were observed in intermittent hypoxic rats. Despite the increment in messenger expression, the decrease in enzyme activity was accompanied by a decrease in citrate synthase activity, a marker of mitochondrial function. The increment in CytOx mitochondrial subunits messengers and ribosomal 12S RNA under prolonged chronic hypoxia could be a consequence of reduced protein synthesis that leads to messenger accumulation.

This study employed differential proteomic and immunoassay techniques to elucidate the biochemical mechanisms utilized by human muscle (vastus lateralis) in response to high altitude hypoxia exposure. Two groups of subjects, participating in a medical research expedition (A, n = 5, 19 d at 5300 m altitude; B, n = 6, 66 d up to 8848 m) underwent a ≈ 30% drop of muscular creatine kinase and of glycolytic enzymes abundance. Protein abundance of most enzymes of the tricarboxylic acid cycle and oxidative phosphorylation was reduced both in A and, particularly, in B. Restriction of α-ketoglutarate toward succinyl-CoA resulted in increased prolyl hydroxylase 2 and glutamine synthetase. Both A and B were characterized by a reduction of elongation factor 2 alpha, controlling protein translation, and by an increase of heat shock cognate 71 kDa protein involved in chaperone-mediated autophagy. Increased protein levels of catalase and biliverdin reductase occurred in A alongside a decrement of voltage-dependent anion channels 1 and 2 and of myosin-binding protein C, suggesting damage to the sarcomeric structures. This study suggests that during acclimatization to hypobaric hypoxia the muscle behaves as a producer of substrates activating a metabolic reprogramming able to support anaplerotically the tricarboxylic acid cycle, to control protein translation, to prevent energy expenditure and to activate chaperone-mediated autophagy.

In the present bed rest (BR) study, 23 volunteers were randomized into 3 subgroups: 60 d BR control (Ctr); BR with resistive exercise (RE; lower-limb load); and resistive vibration exercise (RVE; RE with superimposed vibration). The aim was to analyze by confocal and electron microscopy the effects of vibration on myofibril and filament integrity in soleus (Sol) and vastus lateralis (VL) muscle; differential proteomics of contractile, cytoskeletal, and costameric proteins (TN-C, ROCK1, and FAK); and expression of PGC1? and atrophy-related master genes MuRF1 and MuRF2. RVE (but not RE) preserved myofiber size and phenotype in Sol and VL by overexpressing MYBPC1 (42%, P

The aim of the present study was to assess age-dependent changes of proteins in the vastus lateralis muscle of physically active elderly and young subjects by a combination of two-dimensional difference gel electrophoresis, SDS-PAGE and ESI-MS/MS. The differences observed in the elderly group included down-regulation of regulatory myosin light chains, particularly the phosphorylated isoforms, a higher proportion of myosin heavy chain isoforms 1 and 2A, and enhanced oxidative and reduced glycolytic capacity.