Metabolic Modulation Induced by Chronic Hypoxia in Rats Using a Comparative Proteomic Analysis of Skeletal Muscle Tissue S. De Palma,O,†,# M. Ripamonti,‡,# A. Vigano` ,† M. Moriggi,† D. Capitanio,† M. Samaja,§ G. Milano,| P. Cerretelli,† R. Wait,⊥ and C. Gelfi*,†,‡ Department of Surgical Sciences, University of Milano-Bicocca, Italy, Institute of Molecular Imaging and Physiology, National Research Council, Segrate, Italy, Department of Sciences and Biomedical Technologies, University of Milan, L.I.T.A., Segrate, Italy, Department of Medicine, Surgery and Dentistry, University of Milan, San Paolo Hospital, Milan, Italy, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, and Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College, London, United Kingdom Received November 20, 2006
Hypoxia-induced changes of rat skeletal muscle were investigated by two-dimensional difference ingel electrophoresis (2D-DIGE) and mass spectrometry. The results indicated that proteins involved in the TCA cycle, ATP production, and electron transport are down-regulated, whereas glycolytic enzymes and deaminases involved in ATP and AMP production were up-regulated. Up-regulation of the hypoxia markers hypoxia inducible factor 1 (HIF-1R) and pyruvate dehydrogenase kinase 1 (PDK1) was also observed, suggesting that in vivo adaptation to hypoxia requires an active metabolic switch. The kinase protein, mammalian target of rapamycin (mTOR), which has been implicated in the regulation of protein synthesis in hypoxia, appears unchanged, suggesting that its activity, in this system, is not controlled by oxygen partial pressure. Keywords: skeletal muscle • hypoxia • 2D-DIGE • mass spectrometry
Introduction Hypoxia occurs when the supply of oxygen to tissues is unable to meet cellular demand. Physiological and pathological instances of hypoxia include embryonic development, exposure to high altitude, muscular exercise, inflammation, anemia, infarction of the myocardium or the central nervous system, and in solid tumors.1,2 Tissue oxygen homeostasis is maintained by a sophisticated physiological network involving the capture, binding, transport, and delivery of molecular oxygen. Many of the mechanisms by which tissues sense and respond to low-oxygen partial pressures remain poorly understood. Both in vivo and in vitro hypoxic cells exhibit increased conversion of glucose to lactate, a process mediated by the transcriptional activator hypoxia inducible factor 1 (HIF-1R),3-8 which up-regulates glycolytic enzymes, increasing anaerobic ATP production to compensate for reduced mitochondrial oxidative phosphorylation.9 Oxidative phosphorylation gener* Corresponding author: Prof. Cecilia Gelfi, Department of Sciences and Biomedical Technologies, L.I.T.A., University of Milan, Via Fratelli Cervi 93, 20090, Segrate, Milan, Italy. E-mail:
[email protected]. O University of Milano-Bicocca. ‡ Institute of Molecular Bioimaging and Physiology, National Research Council. † Department of Sciences and Biomedical Technologies, University of Milan. # These authors contributed equally to this work. § Department of Medicine, Surgery and Dentistry, University of Milan. | Centre Hospitalier Universitaire Vaudois. ⊥ Imperial College.
1974
Journal of Proteome Research 2007, 6, 1974-1984
Published on Web 03/29/2007
ates about 90% of total cellular ATP, and these levels cannot be maintained by a switch to glycolysis. Moreover, activation of the anaerobic pathway under hypoxia paradoxically increases oxidative stress because of mitochondrial generation of toxic reactive oxygen species (ROS).10,11 Recently, pyruvate dehydrogenase kinase 1 (PDK1) has been shown to be crucial for both the maintenance of ATP levels and for the attenuation of ROS production in vitro.12,13 The mammalian target of rapamycin (mTOR) protein kinase may, according to its roles in control of protein synthesis, also be involved in the adaptive response to chronic hypoxia.14 Muscular tissue provides a good model of in vivo hypoxia adaptation, since its metabolic rate can increase by a 100-fold over basal levels, and it is subjected to fluctuating oxygen levels during exercise. We showed previously in a rat model that prolonged hypoxia increases levels of transcripts of mitochondrial cytochrome oxidase subunit I and II and 12S ribosomal subunits, whereas nuclear subunit IV remained unchanged.15 These changes were not observed when the animals were intermittently re-oxygenated. Despite the increased cytochrome oxidase mRNA expression, citrate synthase activity, a marker of mitochondrial number,16 was slightly, though not significantly, decreased. A study of Caucasian subjects exposed to severe chronic hypoxia, combined with vigorous exercise, showed increased glycolysis, decreased mitochondrial mass, and accumulation of lipofuscin, a marker for ROS exposure.7 However, a proteomic investigation of subjects native to high altitude revealed 10.1021/pr060614o CCC: $37.00
2007 American Chemical Society
research articles
Metabolic Modulation Induced by Chronic Hypoxia in Rats
Figure 1. Gastrocnemius profiling by 2D-DIGE. Typical 2-D pattern (Cy3, 50 µg of protein loaded) gel image of gastrocnemius protein extract using a NL pH 3-10 IPG strip in the first dimension and SDS gel (12% T, 2.5% C) in the second. Automated image analysis by DeCyder detected and matched 2200 protein spots in a single-gel images. Fifty-four spots were differentially expressed (t-test value was 1.1, and area < 200. The DeCyder DIA program uses a normal distribution model to determine the differentially expressed spots. The threshold was set to (2 standard deviations based on the assumption that 95% protein spots are not Journal of Proteome Research • Vol. 6, No. 5, 2007 1979
research articles
De Palma et al.
Figure 3. Mitochondria-enriched fractions profiled by 2D-DIGE. Typical 2-D pattern (Cy3, 50 µg of protein load) gel image of mitochondrial protein extract in a NL pH 3-10 IPG strip in the first dimension and SDS gel (12% T, 2.5% C) in the second dimension. Automated image analysis by DeCyder software detected and matched 1200 protein spots over all single-gel images. Twenty-three spots were differentially expressed; t-test value was
