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Skeletal muscle gene expression in space-flown rats ¹

TAKESHI NIKAWA^*,2, KAZUMI ISHIDOH, KATSUYA HIRASAKA^*, IBUKI ISHIHARA^*, MADOKA IKEMOTO, MIHOKO KANO^*, EIKI KOMINAMI, IKUYA NONAKA, TAKAYUKI OGAWA^||, GREGORY R. ADAMS^¶, KENNETH M. BALDWIN^¶, NATSUO YASUI^||, KYOICHI KISHI^* and SHIN’ICHI TAKEDA

^* Department of Nutrition, The University of Tokushima School of Medicine, Tokushima, Japan;
Department of Biochemistry, Juntendo University School of Medicine,Tokyo, Japan;
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry,Tokyo, Japan;
Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan;
^|| Department of Orthopaedics, University of Tokushima School of Medicine, Tokushima, Japan; and
^¶ Department of Physiology and Biophysics, University of California, Irvine, California, USA

²Correspondence: Department of Nutrition, The University of Tokushima School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail: nikawa{at}nutr.med.tokushima-u.ac.jp

SPECIFIC AIMS

Microgravity and simulated conditions cause muscle atrophy through a distinct transcriptional alteration of skeletal muscle cells. To explore this issue in more detail, we examined the expression of 26,000 genes in gastrocnemius muscles of neonatal rats exposed to spaceflight, tail suspension or denervation by DNA microarray analysis and found two spaceflight-specific gene expression patterns in the muscle as well as key genes for elucidating the mechanisms of muscle wasting in space.

PRINCIPAL FINDINGS

1. The transcriptional response of skeletal muscle cells to spaceflight was different from those of the simulated conditions
Scatter plot of DNA microarray analysis showed that the spaceflight markedly changed the gene expression in gastrocnemius muscle compared with tail suspension and denervation (Fig. 1 , full data are available at http://www.hosp.med.tokushima-u.ac.jp/university/servlet/index?&level=4&reference=0/10 002/5/30010/20032). The numbers of the genes that exhibited more than eightfold change in the expression level in response to spaceflight, tail suspension and denervation were 257 (up, 100 genes; down, 157 genes), 74 (up, 37 genes; down, 37 genes) and 154 (up, 106 genes; down, 48 genes), respectively. Spaceflight down-regulated gene expression in skeletal muscle cells more than tail suspension and denervation. Thus, the changes in muscle gene expression observed after spaceflight are unique and not just an extension of those seen in simulated models.

View larger version (29K): [in this window] [in a new window]   Figure 1. Gene expression in gastrocnemius muscle of rats exposed to spaceflight (A), tail suspension (B) or denervation (C). In the STS-90 mission, male Sprague-Dawley rats (8-day-old) stayed in space for 16 days in the space shuttle Columbia. During spaceflight, rats suckled from their dams in addition to eating food bars. About 2–4 h elapsed after the return to weightbearing conditions before the animals were killed. Isolated gastrocnemius muscles were weighed and immediately frozen in chilled isopentane and liquid nitrogen. The asynchronous ground control rats were housed with their dams in cage conditions simulating the shuttle environment. Another group of male Sprague-Dawley rats (8-day-old) was subjected to tail suspension or denervation for 16 days. For the tail suspension procedure, a piece of tape was attached on the tail, and the tape was connected to a swivel tied to a horizontal bar at the top of cage. For the denervation procedure, the left sciatic nerve was divided in the mid-thigh region, leading to denervation of hindlimb muscles. Tail-suspended or denervated pups were returned to their mothers every 6 h for 2 h of nursing. Respective asynchronous control rats were prepared. Gastrocnemius muscle wet weights per body weight of the spaceflight, tail-suspended and denervated rats decreased to 70, 86, and 58%, respectively, relative to control rats. Total RNA isolated from these atrophied gastrocnemius muscles was analyzed on DNA microarrays (Affymetrix, Santa Clara, CA, USA) with the rat genome U34 gene chip set including 26,000 genes. Gene expression of spaceflight, tail-suspended and denervated rats was compared with that of respective control rats prepared in parallel. Several genes with spaceflight-induced changes in expression levels that exceeded eightfold are represented by individual dots. Red dots, cytoskeletal genes (1, myosin light chain 1, X51531; 2, transgelin, M83107; 3, Rho interacting protein 3, D26154; 4, actin cross-linking family protein 7, AA943397; 5, A-kinase anchoring protein 12, AI233818; 6, -actin, AI175789; 7, cytoplasmic dynein, AI009806; 8, µ-crystallin, AI233209; 9, tropomyosin 5, AI170847; 10, similar to myosin binding C-protein, AI012249; 11, similar to SC65 synaptonemal complex protein, AA891769; 12, nexin, AI102497; 13, myosin heavy polypeptide 9, U31463; 14, similar to mouse villin 1, AI236596). Green dots, mitochondrial genes (1, similar to NADH-ubiquinone oxidoreductase, AA684929; 2, cytochrome b558 -subunit, U18729; 3, ribosomal protein L21, AI170353; 4, cytochrome b5, AA817685; 5, cytochrome P450IIE1, M20131; 6, pyruvate dehydrogenase kinase 4, AF034577; 7, uncoupling protein 3, AF030163). Blue dots, ubiquitin-related genes (1, low molecular mass polypeptide 2, immunoproteasome ß-subunit, D10757; 2, similar to RING finger protein 23, AI103266; 3, MuRF-1, AI639465; 4, ubiquitin conjugating enzyme 6, AI230131; 5, Siah-1A, AA923973; 6, Cbl-b, AI072631). Genes with similar expression lie on a line from the origin to the top right corner; the expression level is indicated by distance from the origin. The scale on each axis is log scale.

2. The spaceflight microarray data showed two spaceflight-specific gene expression patterns
The functional classification of the genes listed above revealed two spaceflight-specific gene expression patterns (Fig. 1) 1) imbalanced expression of mitochondrial genes with disturbed expression of 13 cytoskeletal molecules, including putative mitochondria-anchoring proteins, A-kinase anchoring protein and cytoplasmic dynein; and 2) up-regulated expression of ubiquitin ligase genes MuRF-1, Cbl-b, and Siah-1A, which are rate-limiting enzymes in ubiquitin-dependent proteolysis. These results were evaluated semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR). They are key genes for elucidating the mechanisms of muscle wasting in space.

3. Spaceflight caused abnormal distribution of mitochondria in skeletal muscles
We examined whether preferential disturbance of cytoskeletal gene expression by spaceflight alters the location of organelles in the cell, especially the mitochondria. Mitochondria in muscle fibers are normally found in four major locations: subsarcolemmal, intermyofibrillar at the para-Z-disc position, perinuclear, and at the endplate. SDH staining showed that mitochondria in gastrocnemius muscle cell of ground control rats were localized at the subsarcolemmal region more than at intermyofibrillar area (Fig. 2 A, B). Tail suspension did not change the pattern of SDH staining in gastrocnemius muscle, although it decreased the cross-sectional area of myofibrils (Fig. 2E, F ). In contrast, intermyofibrillar SDH activities in gastrocnemius muscle cells of spaceflight and denervated rats were higher than the subsarcolemmal activities (Fig. 2C, D, G, H ).

View larger version (90K): [in this window] [in a new window]   Figure 2. Spaceflight changes mitochondrial localization in gastrocnemius muscle. To examine distribution of mitochondria in skeletal muscle fibers, histochemical staining of SDH (an enzyme locating at inner membrane of mitochondria) was performed. The gastrocnemius muscles of spaceflight (C, D), tail-suspended (E, F) and denervated rats (G, H) were prepared as described in the text. Control muscles (A, B) were also prepared in parallel. Scale bar = 100 µm.

4. Hydrogen peroxide induced expression of Cbl-b and Siah-1A in skeletal muscle cells
To elucidate the involvement of oxidative stress in the gene expression in space, we examined the expression of oxidative stress/exercise-inducible muscle genes. The expression of hypoxia-inducible factor 1ß (aryl hydrocarbon receptor nuclear translocator) was highly up-regulated by the spaceflight, while denervation as well as spaceflight stimulated expression of metallothionein I. In L6 cells at 100% confluence treated with 0.1 or 0.25 mM hydrogen peroxide, expression of Cbl-b and Siah-1A was transiently up-regulated, similar to the effect of spaceflight. Our RT-PCR system did not detect MuRF-1 mRNA in L6 cells even after treatment with hydrogen peroxide.

CONCLUSIONS AND SIGNIFICANCE

Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional response of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of 26,000 gastrocnemius muscle genes in space-flown rats by DNA microarray analysis. We found that the transcriptional response to spaceflight is different from that of tail suspension or denervation, as described above. This is the first report that describes screening candidate molecular mediators of spaceflight-induced muscle atrophy. The results are relevant to our understanding of the distinct mechanisms of muscle wasting in space.

Cytoskeletal molecules along with associated motor proteins play a fundamental role in cellular functions, such as movement of organelles and cell migration. In this study, we focused on reduced expression of cytoskeletal molecules, such as A-kinase anchoring protein and cytoplasmic dynein, which are associated with anchoring and movement of mitochondria. Distinct SDH staining pattern in space muscle samples indicated that microgravity caused abnormal distribution of mitochondria in skeletal muscles. This result was consistent with the previous report describing a significant decrease in mitochondria occupying the subsarcolemmal area in flight (Cosmos 1887 biosatellite) muscle. Thus, it is conceivable that microgravity disrupts structural integrity of muscle mitochondria by inhibiting expression of mitochondria-anchoring proteins (Fig. 3 ).

View larger version (19K): [in this window] [in a new window]   Figure 3. Is muscle mitochondria structural integrity disrupted in space? The cytoskeleton maintains organelles in precise positions. Spaceflight preferentially disturbed expression of 13 cytoskeletal genes, such as mitochondrial anchoring protein, compared with tail suspension and denervation. Based on these findings, we propose that abnormal distribution of organelles, especially mitochondria, in space-flown skeletal muscle, has deleterious effects on muscle fibers, leading to muscle atrophy caused by low production of chemical energy and leak of free radical species.

Disturbance of structural integrity of muscle mitochondria has deleterious effects on muscle fiber. Indeed, trabecular (lacy) muscle fibers, which are characterized by a peculiar pattern of oxidative enzyme activity, are observed in various myopathies. In the present study, imbalanced expression of genes in the electron transport system and up-regulated expression of oxidative stress-inducible genes were observed in space-flown muscle. These anomalies in gene expression may result in insufficient energy provision for construction and leakage of reactive oxygen species from mitochondria (Fig. 3) .

Abnormal distribution of organelles, especially mitochondria, has been suggested as a candidate gravity sensing system in skeletal muscle cells, since mitochondria in skeletal muscles can undergo rapid and characteristic morphological and functional changes in response to environmental changes. Present microarray results imply that cytoskeletal molecules mediate reduced mechanical stress to mitochondrial location and functions in space. Identifying cytoskeletal protein(s) responsible for anchoring mitochondria is the next important step in elucidating the gravity-sensing system in muscle cells.

Consistent with our previous report that the ubiquitin-dependent proteolytic pathway plays an important role in muscle atrophy, spaceflight up-regulated the expression of ubiquitin ligases, MuRF-1, Siah-1A and Cbl-b, which are rate-limiting enzymes in the protein-ubiquitination system. However, mediators that trigger expression of ubiquitin ligase during spaceflight remain unknown. In the present study, treatment of skeletal muscle cells with hydrogen peroxide also induced expression of Cbl-b and Siah-1A, although it did not induce MuRF-1 expression in L6 cells. Oxidative stressors that leak from mitochondria may activate the ubiquitin system in skeletal muscle exposed to weightlessness. To further address this issue, our proposal for a space experiment using L6 cells on the International Space Station has been accepted.

Unfortunately, the Columbia tragedy denied us the opportunity to confirm "the mitochondria hypothesis" for a mechanism of muscle atrophy caused by spaceflight. Our microarray data suggest that space muscle samples are indispensable for developing the space myology research field. We look with anticipation for the early recommencement of space shuttle flight.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0419fje; doi: 10.1096/fj.03-0419fje

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