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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 20, 2004 as doi:10.1096/fj.03-1077fje. |
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* Institut of Molecular Bioimaging and Physiology, CNR, Segrate, Italy;
Department of Pharmacological Sciences, University of Milano, Italy;
Institut of Rheumatology, Imperial College of Medicine, London, UK;
Royal Nepal Academy of Science and Technology, Kathmandu, Nepal;
|| Institut of Anatomy, Faculty of Medicine, Bern, Switzerland; and
Department of Sciences and Biomedical Technologies, Faculty of Medicine, University of Milano, Italy
2Correspondence: Institute of Molecular Bioimaging and Physiology, CNR, Via Fratelli Cervi, 93, 20090 SEGRATE, Milano, Italy. E-mail: cecilia.gelfi{at}ibfm.cnr.it
SPECIFIC AIMS
Caucasian lowlanders exposed for 8 to 12 wk to altitudes above 5500 m undergo muscle cytological changes and accumulation of lipofuscin, a hallmark of overproduction of reactive oxygen species (ROS). This is not the case for Tibetan populations. The aim of this work was to gain insight into the mechanisms protecting muscle from oxidative damage. The differential concentration of enzymes catalyzing cytoprotective and metabolic reactions, of base propenal protein adducts generated by ROS and of myoglobin was determined by proteome analysis. Three groups of subjects with different genetic heritage and phenotypic characteristics were investigated: 1) first generation Tibetans (Tib1; nine males aged 18–23 years) born and living between 3500 and 4500 m until 5–30 days before the measurements; 2) second generation Tibetan migrants (Tib 2, six males aged 18–20 years), born and resident at 1300 m altitude; and 3) Nepali control subjects (N, nine males aged 18–20 years) resident to 1300 m.
PRINCIPAL FINDINGS
1. Differential protein expression in muscle
Seven differentially expressed proteins were identified by 2 DE and mass spectrometry in the vastus lateralis muscle: glutathione-S-transferase p (GST P1-1, EC 2.5.1.18), an enzyme catalyzing detoxifying and cytoprotective reactions; 
2-enoyl-CoA-hydratase (ECH, EC 4.2.1.17); glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12); lactate dehydrogenase (LDH, EC 1.1.1.27); phosphoglycerate mutase (muscle form, PGA, EC 5.4.2.1); and NADH-ubiquinone oxidoreductase (NUGM, EC 1.6.5.3); and myoglobin.
The results of quantitative analysis appear in Fig. 1
. GST P1-1 is 
380% and 50% more highly expressed in Tib 1 and in Tib 2, respectively, than in Nepali controls. ECH is greatly up-regulated in Tib 1 (400%) and Tib 2 (250%) compared with Nepali. Among glycolytic enzymes, GAPDH and LDH are down-regulated in Tib 1 and are similar in Tib 2 and Nepali, whereas PGA is 50% more highly expressed in Tib 1 compared with Tib 2 and Nepali. NUGM appears to be 30% overexpressed in Tib 1 compared with Tib 2. Myoglobin is detected as three isoforms corresponding to well-known amino acid substitutions. The form with pI of 7.29 is >100% more abundant in Tib 1 compared with Nepali and is 30% higher in Tib 2 than in Nepali.
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2. Characterization of protein expression changes
The RNA transcript of GST P1-1 was 80% higher in Tib 1 than in Nepali, whereas no significant differences were detected for ECH and myoglobin despite significantly increased protein expression. The RNA transcripts of HIF-1
, e-NOS, and n-NOS, the main genes involved in adaptation to hypoxia, behave differentially in Tib 1 and Nepali. In the high altitude Tibetan group, HIF-1 appears to be 60% down-regulated, n-NOS is 40% up-regulated, whereas e-NOS is unchanged compared with the Nepali.
3. Base propenal protein adducts accumulation
To establish the functional significance of the up-regulation of GST P1-1 in high altitude Tibetans, we compared levels of 4-hydroxynonenal (4-HNE) protein adducts in this group and in the Nepali controls. A comparison of the proteins modified by 4-HNE after electrotransfer and specific antigen-antibody reactions is shown in Fig. 2
. Despite the different hypoxic exposure history, it appears that the two investigated groups are characterized by the same amount of protein 4-HNE adducts. These results are compatible with the observation of a reduced accumulation of lipofuscin in Sherpas.
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CONCLUSIONS AND SIGNIFICANCE
Enzyme changes
The increase in GSTs, particularly the fourfold increase of the P1-1 isoform, in high altitude Tibetans compared with Nepali is the most striking result from the present study. This increase in GST protein was accompanied by an up-regulation of its mRNA transcript. By conjugating reduced glutathione (GSH) to a range of electrophilic acceptors, this enzyme plays an important role in cellular detoxification. The P1-1 isoform of GST was also 50% higher in the low altitude Tibetan group compared with Nepali controls and may represent a persistent genetic trait.
Another original result, and closely associated with the one described above, is that the muscle level of base propenal protein adducts (4-HNE), which are generated by hydroxyl radicals and ROS attack of lipid membranes, is similar in Tib1 and Nepali.
The outcome of the present investigation is the important metabolic role of muscle, the largest protein store of the body, in high altitude acclimatization. This conclusion is substantiated by the different strategy adopted by lowlanders and mountain populations to cope with oxidative stress, which is the most harmful consequence of chronic hypoxia.
The body reacts to hypoxia by NO-modulated HIF-1 expression of hypoxia-inducible genes in order to reestablish an adequate O2 flux to the tissues and to optimize metabolic processes. The erythropoietic response of lowlanders and natives with a relatively short history of hypoxia or intermarriage with lowlanders (e.g., Andean populations) leads to an excess of O2 carrying capacity which balances hemodynamic adaptations such as decreased maximal cardiac output and peripheral blood flow. By contrast, both Tibetan groups adapted or acclimatized to hypoxia are characterized by lower hemoglobin concentrations than Caucasians and Andeans, higher HbO2% saturation at maximum exercise in hypoxia, maximal heart rate levels close to those of lowlanders at sea level, and relatively high 
O2 max values similar to or even higher than those of sea level age, gender, and exercise practice-matched Caucasians. Higher O2 max levels in natives might also be the consequence of improved mitochondrial energy generation modulated by NO buffering. With regard to anaerobic metabolism, altitude natives are characterized by relatively low maximal blood lactate levels, different from Caucasians who, after a transient drop of [Lab]max, tend to resume prealtitude higher [Lab]max levels. The latter change has been attributed to a transient impairment of oxidative metabolism, possibly due to muscle wasting, which does not seem to affect altitude natives or lowlanders after several years of exposure.
As indicated above, one of the most striking differences between acclimatized lowlanders and altitude natives, particularly Sherpas, is the extent of muscle damage from reactive oxygen species (ROS). This occurs essentially in the former, and its main features are a drastic reduction of mitochondrial mass, accompanied by accumulation of lipofuscin, changes virtually absent in altitude natives. The observation in the present study that the GST P1-1 concentration is fourfold higher in high altitude Tibetans than in Nepali lowlanders but that levels of aldehydes (4-HNE) are similar in both indicates that high altitude populations may detoxify ROS more efficiently than acclimatized lowlanders.
The muscular deterioration observed in hypoxia-exposed lowlanders presents analogies with aging-induced muscle wasting and therefore may be a useful model system. However, significantly higher GST P1-1 levels are also found in low altitude Tibetans who have never experienced chronic hypoxia, which suggests that this higher enzyme activity may have a genetic basis. Experiments aimed at identifying origin and characteristics of such metabolic adjustments in altitude natives at the genomic level are in progress.
Thus, altitude natives might have activated a detoxifying system preventing and/or limiting tissue damage, allowing them to live and thrive in hostile environments such as extreme altitudes.
Another novel result from is the increased activity of ECH in both Tibetan groups compared with the Nepali. Thus, it would appear that fat metabolism in highlanders is enhanced rather than suppressed, as hypothesized by one of us (H.H.). A switch to lipid metabolism could optimize the use of body energy stores during sustained physical activities such as mountaineering and may aid thermoregulation.
Myoglobin
An interesting finding is the up-regulation of the pI = 7.29 isoform of myoglobin in both Tibetan groups. Apart from its role of O2 carrier, myoglobin is known to interact with intracellular NO, possibly sequestering it in the tissue. Low NO concentration stimulates mitochondrial biogenesis and aerobic metabolism. The extremely low mitochondrial volume density found in the muscle of highlanders (Sherpas and Andeans) living at altitude could be an adaptive feature to a permanent lack of oxygen. In fact, a reduction of the mitochondrial mass in hypoxia could limit muscle damage via decreased oxidative metabolism and ROS production. A finding compatible with the above hypothesis is that Caucasian mountaineers, after 8 to 12 wk exposure to extreme altitude, undergo a striking reduction of mitochondrial volume density. About one-third of the increase of Mb is still present in Tib 2, so that the same consideration as for GST P1-1 can be made with regard to the possibility that a genetic factor be involved in this adaptive process.
The proteomic approach to the study of adaptation to hypoxia in humans represents a breakthrough in altitude research. Thanks to its plasticity, the muscle of altitude natives appears to adapt to hypoxia by preventing tissue damage from ROS and, by optimizing metabolic control, making active life possible even in extreme conditions. The present results may also have important bearing on human physiopathology, particularly on aging.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1077fje 
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