Cecilia Gelfi

New aspects of altitude adaptation in Tibetans: a proteomic approach

A prolonged sojourn above 5500 m induces muscle deterioration and accumulation of lipofuscin in Caucasians, probably because of overproduction of reactive oxygen species (ROS). Because Sherpas, who live at high altitude, have very limited muscle damage, it was hypothesized that Himalayan natives possess intrinsic mechanisms protecting them from oxidative damage. This possibility was investigated by comparing the muscle proteomes of native Tibetans permanently residing at high altitude, second‐generation Tibetans born and living at low altitude, and Nepali control subjects permanently residing at low altitude, using 2D gel electrophoresis and mass spectrometry. Seven differentially regulated proteins were identified: glutathione‐S‐transferase P1‐1, which was 380% and 50% overexpressed in Tibetans born and living at high and low altitude, respectively; Δ2‐enoyl‐CoA‐hydratase, which was up‐regulated in both Tibetan groups; glyceraldehyde‐3‐phosphate dehydrogenase and lactate dehydrogenase, which were both slightly down‐regulated in Tibetans born and living at high altitude; phosphoglycerate mutase, which was 50% up‐regulated in the native Tibetans; NADH‐ubiquinone oxidoreductase, slightly overexpressed in Tibetans born and living at high altitude; and myoglobin, which was overexpressed in both Tibetan groups. We concluded that Tibetans at high altitude, and to some extent, those born and living at low altitude, are protected from ROS‐induced tissue damage and possess specific metabolic adaptations.

Proteins modulation in human skeletal muscle in the early phase of adaptation to hypobaric hypoxia.

High altitude hypoxia is a paraphysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. In man, skeletal muscle, after prolonged exposure to hypoxia, undergoes mass reduction and alterations at the cellular level featuring a reduction of mitochondrial volume density, accumulation of lipofuscin, a product of lipid peroxidation, and dysregulation of enzymes whose time course is unknown. The effects of 7–9u2005days exposure to 4559u2005m (Margherita Hut, Monte Rosa, Italy) on the muscle proteins pattern were investigated, pre‐ and post‐exposure, in ten young subjects, by 2‐D DIGE and MS. Ten milligram biopsies were obtained from the mid part of the vastus lateralis muscle at sea level (control) and at altitude, after 7–9u2005days hypoxia. Differential analysis indicates that proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses were significantly (p<0.05) decreased in hypoxia. Parenthetically, hypoxia markers such as hypoxia inducible factor 1 α (HIF‐1α) and pyruvate dehydrogenase kinase 1 (PDK1) were still at the pre‐hypoxia levels, whereas the mammalian target of rapamycin (mTOR), a marker of protein synthesis, was reduced.

Comparative proteomic profile of rat sciatic nerve and gastrocnemius muscle tissues in ageing by 2-D DIGE

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 Ca2+ 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 β enolase and tropomyosin 1α were differentially expressed in the tissues.

A DIGE approach for the assessment of rat soleus muscle changes during unloading: effect of acetyl‐L‐carnitine supplementation

After hind limb suspension, a remodeling of postural muscle phenotype is observed. This remodeling results in a shift of muscle profile from slow‐oxidative to fast‐glycolytic. These metabolic changes and fiber type shift increase muscle fatigability. Acetyl‐L‐carnitine (ALCAR) influences the skeletal muscle phenotype of soleus muscle suggesting a positive role of dietary supplementation of ALCAR during unloading. In the present study, we applied a 2‐D DIGE, mass spectrometry and biochemical assays, to assess qualitative and quantitative differences in the proteome of rat slow‐twitch soleus muscle subjected to disuse. Meanwhile, the effects of ALCAR administration on muscle proteomic profile in both unloading and normal‐loading conditions were evaluated. The results indicate a modulation of troponin I and tropomyosin complex to regulate fiber type transition. Associated, or induced, metabolic changes with an increment of glycolytic enzymes and a decreased capacity of fat oxidation are observed. These metabolic changes appear to be counteracted by ALCAR treatment, which restores the mitochondrial mass and decreases the glycolytic enzyme expression, suggesting a normalization of the metabolic shift observed in unloaded animals. This normalization is accompanied by a maintenance of body weight and seems to prevent a switch of fiber type.

Changes in Muscle Cell Metabolism and Mechanotransduction Are Associated with Myopathic Phenotype in a Mouse Model of Collagen VI Deficiency

This study identifies metabolic and protein phenotypic alterations in gastrocnemius, tibialis anterior and diaphragm muscles of Col6a1−/− mice, a model of human collagen VI myopathies. All three muscles of Col6a1−/− mice show some common changes in proteins involved in metabolism, resulting in decreased glycolysis and in changes of the TCA cycle fluxes. These changes lead to a different fate of α-ketoglutarate, with production of anabolic substrates in gastrocnemius and tibialis anterior, and with lipotoxicity in diaphragm. The metabolic changes are associated with changes of proteins involved in mechanotransduction at the myotendineous junction/costameric/sarcomeric level (TN-C, FAK, ROCK1, troponin I fast) and in energy metabolism (aldolase, enolase 3, triose phosphate isomerase, creatine kinase, adenylate kinase 1, parvalbumin, IDH1 and FASN). Together, these change may explain Ca2+ deregulation, impaired force development, increased muscle-relaxation-time and fiber damage found in the mouse model as well as in patients. The severity of these changes differs in the three muscles (gastrocnemius

Specific protein changes contribute to the differential muscle mass loss during ageing

In the skeletal muscle, the ageing process is characterized by a loss of muscle mass and strength, coupled with a decline of mitochondrial function and a decrease of satellite cells. This profile is more pronounced in hindlimb than in forelimb muscles, both in humans and in rodents. Utilizing light and electron microscopy, myosin heavy chain isoform distribution, proteomic analysis by 2D‐DIGE, MALDI‐TOF MS and quantitative immunoblotting, this study analyzes the protein levels and the nuclear localization of specific molecules, which can contribute to a preferential muscle loss. Our results identify the molecular changes in the hindlimb (gastrocnemius) and forelimb (triceps) muscles during ageing in rats (3‐ and 22‐month‐old). Specifically, the oxidative metabolism contributes to tissue homeostasis in triceps, whereas respiratory chain disruption and oxidative‐stress‐induced damage imbalance the homeostasis in gastrocnemius muscle. High levels of dihydrolipoyllysine‐residue acetyltransferase (Dlat) and ATP synthase subunit alpha (Atp5a1) are detected in triceps and gastrocnemius, respectively. Interestingly, in triceps, both molecules are increased in the nucleus in aged rats and are associated to an increased protein acetylation and myoglobin availability. Furthermore, autophagy is retained in triceps whereas an enhanced fusion, decrement of mitophagy and of regenerative potential is observed in aged gastrocnemius muscle.

Proteomic signature of reversine-treated murine fibroblasts by 2-D difference gel electrophoresis and MS: Possible associations with cell signalling networks

Recent advances in stem cell biology have demonstrated that terminally differentiated adult cells can be induced to de‐differentiate into progenitor cells (induced stem cells) upon proper stimuli. This has been achieved by the induced expression of key regulatory genes by retro‐ or lenti‐viral systems. On the other hand, synthetic “small molecules” can also induce de‐differentiation and may represent a potentially safer approach as compared with genetic manipulation. Along this line, a synthetic purine called “reversine” has been shown to induce the de‐differentiation of fibroblasts into mesenchymal stem‐cell‐like progenitors, which can be successively induced to differentiate into skeletal muscle, smooth muscle and bone cells. The mechanism whereby reversine is able to achieve de‐differentiation has yet to be clarified. In this context, we defined the protein changes induced by reversine treatment in murine fibroblasts by 2‐D difference gel electrophoresis, coupled with MS. Proteins involved in cytoskeletal and cell shape remodeling, RNA export, degradation, folding, stress control and ATP production were found to be remarkably changed after reversine treatment. Ingenuity pathway analysis (IPA) predicted that these protein pattern changes enabled to propose that about 40 proteins might be associated to several biological functional networks, including cellular assembly, cell signaling and cell death. Altogether our data confirm the intrinsic complexity of the de‐differentiation process induced by reversine and suggest more selected approaches to investigate the action mechanism of this small molecule.