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(The FASEB Journal. 2001;15:684-692.)
© 2001 FASEB

Characterization of control and immobilized skeletal muscle: an overview from genetic engineering

JONNY ST-AMAND1, KOJI OKAMURA2, KEITARO MATSUMOTO, SEIICHI SHIMIZU and YOSHIRO SOGAWA

Saga Research Institute, Otsuka Pharmaceutical Company, Higashi-sefuri, Kanzaki, Saga, 842-0195, Japan

1Correspondence: Human Genomics Laboratory, Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Rd., Baton Rouge, LA 70808, USA. E-mail: StAmandJ{at}pbrc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
To elucidate the molecular basis of muscle atrophy, we have performed the serial analysis of gene expression (SAGE) method with control and immobilized muscles of 10 rats. The genes that expressed >0.5% in muscle are involved in the following three functions: 1) contraction (troponin I, C and T; myosin light chain 1–3; actin; tropomyosin; and parvalbumin), 2) energy metabolism (cytochrome c oxidase I and III, creatine kinase, glyceraldehyde-3-phosphate-dehydrogenase, phosphoglycerate mutase, ATPase 6, and aldolase A), and 3) housekeeping (lens epithelial protein). Muscle atrophy appears to be caused by changes in mRNA levels of specific regulators of proteolysis, protein synthesis, and contractile apparatus assembling, such as polyubiquitin, elongation factor 2, and nebulin. Immobilization has produced a decrease more than threefold in gene expression of enzymes involved in energy metabolism, especially ATPase, cytochrome c oxidase, NADH dehydrogenase, and protein phosphatase 1. Differential gene expressions of selenoprotein W and uroporphyrinogen decarboxylase, which can be involved in oxidative stress, were also observed. Other genes with various functions, such as cholesterol metabolism and growth factors, were also differentially expressed. Moreover, novel genes regulated by immobilization were discovered. Thus, the current study allows a better understanding of global muscle characteristics and the molecular mechanisms of sedentarity and sarcopenia.—St-Amand, J., Okamura, K., Matsumoto, K., Shimizu, S., Sogawa, Y. Characterization of control and immobilized skeletal muscle: an overview from genetic engineering.


Key Words: muscle atrophy • mRNA • serial analysis of gene expression (SAGE) • sarcopenia • gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
SKELETAL MUSCLES PLAY important roles in living organisms. Indeed, skeletal muscles allow the movements and accomplishment of daily physical tasks as well as sport performances. Sarcopenia is a health issue in elderly people characterized by a decrease in muscle mass (1) . Oxidative stress (2 , 3) , increased proteolysis by ubiquitination (4) , decreased energy metabolism (5) , and neuronal injury (5) have been suggested to play a role in muscle atrophy. Immobilization is considered to be an appropriate model to study mechanisms responsible for the muscle atrophy because factors of etiological importance can be expected to be differentially expressed during the early phases of plaster casting (6) .

However, the components of skeletal muscle have never been characterized simultaneously and globally. The molecular mechanisms responsible for muscle loss and sarcopenia are also poorly understood because of the limitations of the techniques available. Most of current biochemistry and molecular biology techniques only allow the characterization of known proteins (7 8 9) . However, less than half of the ~80,000 genes and proteins constituting the human body are known (10) . Genetic studies have improved the knowledge of pathophysiology and have led to the discovery of genes (11 12 13 14 15 16) . However, the efficiency of identifying the genes related to complex diseases is still low because the current methods could not investigate quantitatively, simultaneously, globally, and differentially the expression of all genes to proteins.

Recently, a powerful strategy known as the serial analysis of gene expression (SAGE) has been developed to accurately measure the expression of thousands of genes, previously known or not, and to find the genes related to a disease or the effects of stimuli (17 18 19 20) . The SAGE strategy uses many genetic-engineering techniques to isolate short expressed sequence tags (EST) specific for each transcript and to ligate them into long concatemers. Sequencing of cloned concatemers indicates the relative level of expression for each gene matched to the tag. Thus, in an attempt to characterize the most-expressed genes in skeletal muscle and to understand the molecular basis of sarcopenia, as well as to elucidate the regulatory mechanisms controlling muscle atrophy, we have used the SAGE strategy to characterize the skeletal muscle of control leg compared to immobilized leg in rat.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Sample preparation
Ten male Wistar rats, 12 wk of age, were purchased from Charles River Japan (Yokohama, Japan). Animals were provided a standard AIN93-G diet twice daily and water ad libitum. Before immobilization, body weights were 373 ± 22 g (mean ± SD). The immobilization was performed as described previously (2) , with some modifications. Under i.p. anesthesia with sodium-pentobarbital (50 mg/kg), the right leg was immobilized with casting tape (type 8002-J; Sumitomo 3M, Tokyo, Japan) to keep the ankle joint in fully extended position. No intervention was performed on the contralateral leg used as control. After 12 days of immobilization, the animals were killed under diethyl ether. Body weights were 358 ± 26 g at the time of death. The gastrocnemius muscle was atrophied from 2.0 ± 0.1 to 1.3 ± 0.2 g after the immobilization and reached 65% of the control leg. The gastrocnemius muscles of both legs were immediately dissected, minced, weighed, frozen with tongs cooled in liquid nitrogen, and stored at -80°C until analysis.

Global gene expression profile
The SAGE method was performed to quantify the global gene expression profile according to the strategy described by Velculescu et al. (18 , 19) and the modification of Kenzelmann and Muhlemann (17) , as well as some other optimizations. Polyadenylated RNA was purified with mRNA direct kit (Dynal, Oslo, Norway) from pooled gastrocnemius muscles of immobilized or control legs to eliminate any individual variation and to extract sufficient quantities of mRNA. After the annealing of biotin-5'T18-3' primer, the mRNA was converted to cDNA with Life Technologies (Rockville, Md.) synthesis kit and cleaved with NlaIII. The 3' restriction fragment was isolated with streptavidin-coated magnetic beads (Dynal) and ligated to one of two annealed linker pairs. After extensive washing to remove unligated linkers, adjacent tags were released from the magnetic beads by cleavage with BsmFI. The blunting kit of Takara shuzo (Otsu, Japan) was used for the blunting and ligation of tags because both reactions could be performed consecutively without phenol chloroform extraction and precipitation of short tags. The produced ditags were amplified by shortened PCR with an initial denaturation step of 1 min at 95°C, followed by 22 cycles consisting of 20 s at 94°C, 20 s at 60°C, and 2 s at 72°C using longer primers 5'-GGATTTGCTGGTGCAGTACAACTAGGC-3' and 5'CTGCTCGAATTCAAGCTTCTAACGATG-3'. The PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE) and digested with NlaIII. The band containing the ditags was excised and self-ligated to produce long concatemers. The concatemers ranging from 500 bp to 2 kb were isolated by agarose gel and extracted with GeneClean Spin (BIO 101, CEDEX, France). These products were cloned into SphI site of pUC19. White colonies were screened by PCR to select long inserts for automated sequencing. To identify the genes, the sequences of 15-bp SAGE tags (NlaIII site plus the adjacent 11 bp) were matched with GenBank database.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Most-expressed genes in skeletal muscle
One of the great accomplishments of the SAGE method is the capacity to quantify the expression frequency of all the genes known or unknown. Table 1 presents all the genes expressed >0.5% in gastrocnemius muscle. The relative frequency of a given tag was calculated by dividing the observed tag count by the total count of 2,256 tags sequenced in the control leg and 2,267 tags in immobilized gastrocnemius multiplied by 100. A total of 19 tags were expressed above the critical 0.5% threshold. The most-expressed mRNA in gastrocnemius muscle is coding for cytochrome c oxidase I because 59 tags (2.6%) had the sequence GATGCCCCCCA, which matched specifically with the last NlaIII restriction site of its gene (Accession no. X14848). The other genes, found between 1 and 2% of all the transcipts expressed in muscular cell, were coding for fast troponins I, C, and T; creatine kinase; myosin light chain (MLC) 1–3; cytochrome c oxidase III; glyceraldehyde-3-phosphate-dehydrogenase (GAPDH); actin; {alpha}-tropomyosin; and phosphoglycerate mutase, whereas the relative frequencies of cytochrome b, ATPase subunit 6, lens epithelial protein, aldolase A, ß-tropomyosin, and parvalbumin transcripts were between 1 and 0.5%. The tags expressed at >0.5% had a similar pattern of expression in control and immobilized legs.


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  		Table 1. All the genes expressed >0.5% in gastrocnemius leg muscle

Differential gene expression after immobilization
Among >2,400 specific transcripts identified by >4,500 tags in total, 40 specific transcripts showed a more than threefold differential expression between control and immobilized muscle. Table 2 shows the components of protein metabolism differentially expressed in control and immobilized gastrocnemius muscle. Polyubiquitin transcript, which is involved in proteolysis, was increased from one tag in control leg to five tags in immobilized leg, giving a ratio of fivefold up-regulation. In contrast, the gene expression of elongation factor-2 (EF2), which is necessary for peptide elongation, was decreased in immobilized leg. The ribosomal protein L22 had its gene expression decreased after the immobilization, whereas six other ribosomal proteins were up-regulated.


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  		Table 2. Components of protein metabolism with differential gene expression in control and immobilized gastrocnemius muscle

Table 3 presents the components of the contractile apparatus and energy metabolism differentially expressed in control and immobilized gastrocnemius muscle. In the contractile apparatus, the genes coding for myosin heavy chain (MHC) 2B and fast-type myosin-binding protein C were more expressed in immobilized leg than in control leg, whereas nebulin was down-regulated. Among the genes involved in the energy metabolism, ATPase, cytochrome c oxidase, NADH dehydrogenase, mitochondrial phosphoprotein MIPP65, and protein phosphatase 1 were all down-regulated, whereas the subunit {alpha} of ATPase and heart subunit VIa of cytochrome c oxidase were up-regulated.


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  		Table 3. Components of the contractile apparatus and energy metabolism differentially expressed in control and immobilized muscle

The level of gene expression in many other types of proteins were also differentially regulated, as shown in Table 4 . The gene expression of selenoprotein W, neuroendocrine-specific protein-like 1, high-density lipoprotein (HDL)-binding protein, cyclin G, and up stream of N-ras (unr) were decreased after immobilization. On the other hand, uroporphyrinogen decarboxylase and p35srj were up-regulated.


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  		Table 4. Other genes differentially expressed in control and immobilized muscle

When the tag sequence did not match with the genes in the rat nonredundant (nr) data bank of GenBank, we attempted to match them with the EST data bank, as shown in Table 5 . Thus, eight up-regulated tags and one down-regulated tag have matched with previously detected but not characterized genes. These EST with usual length of 500 bp were not homologous to any rat, mouse, or human genes. The sequence tag that matched with the EST no. AA819140 was the tag that showed the highest differential expression of 10-fold increase in immobilized muscle. Because the SAGE method can detect all the genes expressed in a cell, even if they have not been previously cloned, three down-regulated tags did not match with any previously reported genes contained in the rat and mouse nr data bank or EST data bank. We have named the uncharacterized and novel genes up-regulated by immobilization Immou 1–8, whereas the uncharacterized and novel genes down-regulated by immobilization were named Immod 1–4.


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  		Table 5. Uncharacterized and novel genes up- and down-regulated by immobilization


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Characterization of muscle cell
The power of the SAGE strategy to characterize comprehensively the gene expression in a cell or in pathological models as well as to elucidate the molecular mechanisms responsible for a pathology or physiological stimuli has been demonstrated by all the studies using the SAGE method (18 19 20 21 22 23 24) . The validity and accuracy of the SAGE method has also been examined in detail. Results from the SAGE method have been confirmed by many other methods such as cDNA hybridization, cloning, sequencing, northern blotting, RT-PCR, and western blotting (18 19 20 21 22 23) . The estimation of gene expression by the SAGE method and Northern blot analysis also highly correlate (r2=0.97) (19) . The threefold differentially expressed genes identified by the SAGE method have also been shown to agree well with additional analyses revealing the primordial role of the gene (21) . Finally, obvious support for the SAGE method comes from agreement of the gene expression profiles in cells such as pancreas (18) , yeast (19) , cancer cells (20) , neurons (21) , mast cells (22) , and brain (23) , with results expected from physiological knowledge.

In the current study, we characterized for the first time the global gene expression profile in gastrocnemius muscle. Similar to the pancreatic cell, which has 23 genes expressed >0.5% (18) , the muscle cell is constituted of 19 genes expressed >0.5%. In their study characterizing the pancreatic cell with the SAGE method, Velculescu et al. have shown that the most-expressed genes have well-known pancreatic function (18) . In the current study, none of these pancreatic genes were detected in the gastrocnemius muscle. In contrast, most of the highly expressed genes are well known to be necessary to the functions of skeletal muscle (7) . Indeed, 8 of the 12 genes expressed >1% were coding for myofibrillar proteins and their expression of isoforms is in agreement with the current knowledge of fast-type muscle (25) . Recently, Welle et al. reported on the abundance of mRNAs in vastus lateralis muscle from young men using the SAGE method (26) . Almost all the genes expressed >0.5% in rat gastrocnemius muscle were also expressed >0.5% in human vastus lateralis, with the exception of parvalbumin. Cytochrome c oxidase I, MLC3, cytochrome b, and {alpha}-tropomyosin were expressed at >0.25% in human vastus lateralis. More genes characteristic of the slow muscle were expressed in vastus lateralis compared with gastrocnemius muscle. Indeed, human vastus lateralis has a preponderance of type I fiber (9) , whereas rat gastrocnemius muscle consists predominantly of type II fiber (27) . Other studies have reported that actin is the most abundant nonmitochondrial transcript in human skeletal muscle (26 , 28 , 29) . Welle et al. observed that actin transcipt was expressed at 1.9% in human vastus lateralis (26) , whereas we estimated its expression at 1.3% in rat gastrocnemius muscle. On the other hand, a number of troponin transcripts were expressed more highly in the present study. Troponin transcripts are also more abundant than actin in human heart (30) . The relative expression of actin may vary in different muscles, or the abundance of certain myofibrillar transcripts may differ in rat compared with human.

Contractile apparatus
Most of the well-recognized components of the contractile apparatus including actin; MLC 1–3; troponin I, C, and T; and tropomyosin {alpha} and ß were identified in the current study (7 , 25) , and each one constituted >0.5% of the transcripts expressed in the muscle cell. GenBank does not contain any sequence for troponin C in rat. However, the rat EST no. AA90587 matching with the tag TGACAGACGAG shows an almost perfect match with the cloned mouse fast skeletal troponin C sequence (accession no. M57590). The accuracy of the matching is supported by the unique matching with only the fast skeletal troponin C gene but not with other genes, even the troponin C slow/cardiac isoform. Indeed, the fast-type troponin C isoform is well known to be expressed specifically in type II muscle (25) . The current study also underlines the importance of the rarely mentioned parvalbumin (7) because the last gene expressed >0.5% was coding for parvalbumin. Parvalbumin is a high-affinity Ca2+-binding protein that functions to facilitate the rate of muscle relaxation (31 , 32) . Thus, the fast-twitch muscle will need high expression of parvalbumin. Therefore, at least 14% of all the transcripts in skeletal muscle is dedicated to the expression of the contractile apparatus.

For some very closely related proteins, it can be difficult to distinguish between their transcripts, especially if they are derived by differential splicing. The multiple tags matching for troponin, tropomyosin, and MLC can be caused by multiple unexpected and complex mechanisms such as multiple genes, alternatively spliced exons, differential transcription, alternative promoters, overlapping genes, multiple poly(A) addition sites, and mRNA-editing enzyme, as well as the multifunction of a given DNA sequence (33 34 35) . These multiple transcripts can be translated into different protein isoforms with specialized functions. On the other hand, each isoform may be tissue-specific and developmentally regulated. The super gene families composing the contractile apparatus are particularly famous for this unusual transcription and processing of mRNAs to produce the different characteristics observed in striated muscle fiber type 1, 2A, 2B, and 2X, as well as smooth and cardiac muscle or other cells (25) . Troponin I, C, and T are coded by different genes (25) . Troponin T is coded by three different genes named according to their expression in cardiac, slow, or fast skeletal muscles (25) . In the current study, no SAGE tag sequence matched with cardiac or slow skeletal muscle genes. Two SAGE tag sequences matched with the fast skeletal gene, which is recognized to be differently spliced to produce different troponin T isoforms (25 , 33) . Different genes code for {alpha} and ß tropomyosins, and they are known to be expressed in fast muscle (25 , 35) . The same gene codes for MLC1-f and MLC3-f, whereas MLC2 is coded by another gene (25 , 34) .

Energy metabolism
The other main category of genes expressed >0.5% in skeletal muscle is the metabolic enzymes involved in energy production. According to the role of the gastrocnemius muscle, which is solicited to maintain continuously the posture and to accomplish aerobic exercise as well as to perform high-intensity activity, enzymes for both oxidative phosphorylation (cytochrome c oxidase I and III, cytochrome b and ATPase subunit 6) and glycolysis (GAPDH, phosphoglycerate mutase, and aldolase A) are highly expressed in gastrocnemius muscle. The gene coding for creatine kinase, which provides a readily available energy supply, was also highly expressed in gastrocnemius muscle. Thus, the skeletal muscle devotes a large part of its functions to producing the enzymes involved in energy metabolism. This production of energy is effectively needed because the muscle consumes most of the energy in the body, even at resting condition (36) .

The mitochondrially encoded genes are very important for the muscle and exercise restriction. Indeed, muscle tissue has numerous mitochondria, and the mitochondrial genes encode for the enzymes essential for energy metabolism. The transcription of the mitochondrially encoded genes has been extensively studied for many years (37 38 39 40) . It is well known that the tRNA serve as punctuation between the mitochondrially encoded genes to produce the mRNA. Moreover, the accession no. X14848 of GenBank describes well the delimitation of the genes in all the mitochondrial genome of rat. As described in Materials and Methods, the SAGE tags are located at the last NlaIII site of the 3' extremity. However, the mitochondrially encoded transcripts are polycistronic and are sometimes prematurely terminated or spliced and polyadenylated at unpredictable locations (26) . The mitochondrial SAGE tags presented in the current study are located at the site predicted from the accession no. X14848 of GenBank.

The only highly expressed gene not involved in the contractile apparatus or energy metabolism was coding for lens epithelial protein (accession no. U20525, unpublished results), which has been reported to be expressed in muscle (accession no. 801010). The comparison (blast) of lens epithelial protein sequence has matched with 21 kd polypeptide under translational control in mouse (accession no. X06407), and tumor protein translationally controlled one in human (accession no. NM 003295.1). The lack of common nomenclature between the different databases is responsible for these different names in different organisms (41) . UniGene also identifies the rat lens epithelial protein as the translationally controlled tumor protein in human (UniGene accession no. SP:P13939), which has cell-housekeeping function with calcium-binding propriety and heat stability and is expressed in almost all the tissues (42) .

Components of protein metabolism differentially expressed after immobilization
None of the constitutive components expressed >0.5% in muscle were differentially expressed after immobilization; however, 40 tags showed more than threefold differential expression between immobilized and control muscle. This small number of differentially expressed genes is in agreement with the conclusion of Lee et al. (5) that sarcopenia and the aging process are unlikely to be a result of large, widespread alterations in gene expression. The change in the expression of few genes can seem to contrast with the large decrease in muscle mass. However, a specific molecular mechanism, such as the ubiquitin-proteasome pathway, may explain the massive protein breakdown (4) . Indeed, the current study has suggested the important role of ubiquitination because the polyubiquitin tag was increased fivefold in immobilized muscle. The increase in polyubiquitin transcripts has been shown to be specifically responsible for the accelerated proteolysis, especially in myofibrillar proteins (43) . Moreover, ubiquitin-mediated proteolysis controls the levels of cyclins and other cell cycle regulators (44) . Another key factor in the protein metabolism, the EF2, was regulated by immobilization. The gene expression of EF2, responsible for the translocation on the ribosome, was decreased more than threefold in immobilized leg. Thus, the current results can explain the previous observations that both proteolysis and protein synthesis are regulated to increase protein breakdown and decrease protein production (6) . To our knowledge, no previous study has investigated the effect of immobilization on the expression of ribosomal proteins. In the muscle-losing mass, the need of ribosomal apparatus for protein synthesis may decrease as shown by the down-regulation of ribosomal protein L22. On the other hand, the tremendous changes and the regeneration of tissue caused by the immobilization stimulus may be associated with induction of some specific ribosomal proteins and synthesis of new types of proteins for the adaptation to the new physiological conditions (45) . Because it is unclear why some mRNAs coding for ribosomal proteins have increased, further studies are needed to investigate the role of some ribosomal proteins in mechanisms such as the inhibition of protein synthesis after immobilization.

Components of contractile apparatus differentially expressed
Adaptations to immobilization in skeletal muscle can induce a shift to fast-twitch isoforms (46) . Other studies have also reported that MHC-2B isoform increased after immobilization (47 , 48) . On the other hand, a decrease in the proportion of slow MHC I isoform has been reported in slow-type muscle such as soleus (49) . In the current study, the gastrocnemius muscle, which is predominantly composed of fiber 2B (27) , was examined and showed an increase in MHC 2B transcript, whereas the other MHC isoforms were not affected by immobilization. Indeed, fast-type muscles are known to be less sensitive to the effects of unloading (46) . Because the expression of slow isoform is already low in gastrocnemius muscle, a further down-regulation may not be necessary (48 , 49) . In contrast, an increase of fast-twitch MHC 2B expression is more advantageous under the conditions of the current experiment with casting that did not allow endurance exercise. Furthermore, adaptation to immobilization has also caused an increase in gene expression of the fast isoform myosin-binding protein C. However, the down-regulation of nebulin, a structural protein, can be associated with a decrease in contractile apparatus assembling and stability. Indeed, nebulin promotes actin nucleation and stabilizes actin filament (50) . Thus, the muscle protein lost after immobilization does not appear to be caused by the direct decrease of contractile protein mRNA, but rather by change in mRNA levels of specific regulators of protein synthesis, proteolysis, and contractile apparatus assembling, such as polyubiquitin, EF2, and nebulin.

Components of energy metabolism differentially expressed
Loss of mitochondrial function is known to occur after immobilization (6 , 51) . However, no previous study has investigated simultaneously the changes in the numerous subunits of the different complexes. In this study, immobilization has produced a decrease in the gene expression of enzymes involved in high-energy ATP production, especially ATP synthase subunit ß, {delta}, and {gamma}; cytochrome c oxidase subunit VIII-h, as well as NADH subunit 2 and rat mitochondrial phosphoprotein MIPP65, the homologue of human NADH-ubiquinone oxidoredutase 9 kb subunit precursor. In reaction to the restriction of endurance exercise and to the decrease in energy supply consecutive to the down-regulation of these enzymes involved in the efficient oxidative system, glycogenolysis may be relatively promoted by the decrease of protein phosphatase 1, which inactivates phosphorylase a, the enzyme responsible for the degradation of glycogen to glucose. Indeed, it has been suggested that there may be a shift in substrate preference in response to states of unloading whereby carbohydrates are preferentially used (46) .

The ATP synthase subunit {alpha} is coded by two genes located on chromosomes 9 and 18 (52) . Thus, increase in the subunit {alpha} seems to reflect a switch to another isoform more appropriate for the energy requirement during immobilization. Similarly, cytochrome c oxidase subunit VIa has two different nuclear-encoded genes (53) . Subunit VIa of cytochrome c oxidase was up-regulated, whereas the muscle-specific subunit VIII-h was down-regulated. Indeed, the subunit VIa and VIII-h genes have different proximal promoter regions and are recognized to be regulated by different factors (54) .

Components of oxidative stress differentially expressed
Oxidative stress is known to be increased by immobilization (3) . Selenoprotein W has been suggested to be involved in oxidative stress and reported to bind to gluthatione (55 , 56) . Only 11 proteins are known to contain selenocysteine, and most of the selenoproteins, such as glutathione peroxidase, are involved in oxidative stress (55) . Selenoprotein W has been cloned recently (57) , and few studies have investigated its role in physiological adaptation. Further studies are needed to determine the role of selenoprotein W in oxidative stress and immobilization because the current study has shown a decrease of gene expression. The gene expression of uroporphyrinogen decarxoylase, which is the last cytosolic enzyme in the biosynthesis of metalloporphyrins, such as peroxidase, magnesium-containing porphyrin, heme, and cytochromes (7) , was increased in immobilized leg. The increase in microsomal iron content after immobilization is associated with the production of hydroxyl radicals (3) . The degradation of iron-containing proteins, such as cytochromes and myoglobin, can increase the content of free cytoplasmic iron. In a previous study, cytoplasmic Cu-Zn-containing superoxide dismutase (SOD) was increased, whereas mitochondrial Mn-SOD was decreased (3) . Further studies are needed to investigate the role of uroporphyrinogen decarxoylase to switch the production of metalloporphyrins from mitochondria to cytoplasm to protect against oxidative stress by synthesis of peroxydase and/or by neutralization of free metals.

Other genes differentially expressed
The SAGE method has many advantages, such as the integration of the tremendous information contained in the genome of 3 billion bp and ~80,000 genes, to identify the most-expressed genes in a cell as well as the genes differentially expressed after an experimental intervention. The SAGE strategy also quantifies the genes that are not candidates for a pathology or a physiological response to a stimulus as well as the less commonly studied, uncharacterized, and even unknown genes. Indeed, the present study has quantified the gene expression of parvalbumin and lens epithelial protein among the most-expressed genes in skeletal muscle as well as myosin-binding-protein C, uroporphyrinogen decarxoylase, and p35srj, which were up-regulated after immmobilization. On the other hand, the gene expression of nebulin, MIPP65, selenoprotein W, neuroendocrine-specific protein-like 1, cyclin G, HDL-binding protein (which is a candidate to be the HDL receptor), and unr have been down-regulated. Neuroendocrine-specific protein-like 1 may be involved in the neuronal injury and reinnervation observed in sarcopenia (5) . The decreased food intake during immobilization can be related to the decreased gene expression of HDL-binding protein (58) . Cyclin G, unr, and p35srj have been reported to play roles in cell cycle and growth (59 60 61) . Although all the genes had the potential to be included in the current study, the limit of sensitivity in determining transcript abundance was 0.04%. There is also the possibility that transcripts will be missed if they do not contain the appropriate NlaIII restriction site, especially the short transcripts.

Uncharacterized and novel genes differentially expressed
Many tags did not match with any gene in rat and mouse nr data bank of GenBank. The matching of many of these tags with the EST data bank confirms that these tags are transcripted from uncharacterized genes. Furthermore, these EST had no homology with any gene in rat, mouse, and human data banks. Three tags differentially expressed after immobilization did not match with any known gene or EST. These tags may represent novel genes not previously characterized. We have named them Immou 1–8 and Immod 1–4 according to their up- and down-regulation by immobilization.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
In conclusion, mainly genes coding for contractile apparatus and energy metabolism are highly expressed in skeletal muscle. Following immobilization, proteolysis can be increased by the induction of polyubiquitin gene expression, whereas protein synthesis and contractile apparatus assembling can be decreased by down-regulation of EF2 and nebulin, respectively. A switch to different isoforms and an increase in specific ribosomal proteins can facilitate the adaptation and regeneration of muscle to immobilization. Changes in the expression of genes involved in energy and cholesterol metabolisms, oxidative stress, growth regulation, and other functions were also observed. Moreover, the current study has targeted novel genes differentially regulated by immobilization. Thus, the current study allows an overview of the most important constituents of muscle cell. These molecular mechanisms associated to muscle atrophy might be also observed in sarcopenia and aging, as well as in common diseases related to sedentarity such as obesity, coronary artery disease, diabetes, hypertension, and syndrome X.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. A. Shima for his technical advice and discussion. We would like to thank B. Heist, Y. Tajiri, and M. Yoshioka for their critical reading of the manuscript, as well as the members of our laboratory for discussion and support.


   FOOTNOTES
 
2 Current address: Osaka University of Health and Sport Sciences, 1558–1 Noda, Kumatori, Sennan Osaka 590-0496 Japan. E-mail: okamura@ouhs.ac.jp

Received for publication May 9, 2000. Revision received August 29, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 

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