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(The FASEB Journal. 2003;17:1812-1819.)
© 2003 FASEB

Serial analysis of gene expression in the skeletal muscle of endurance athletes compared to sedentary men

MAYUMI YOSHIOKA1, HIROAKI TANAKA*, NAOKO SHONO{dagger}, ERIC E. SNYDER{ddagger}, MUNEHIRO SHINDO* and JONNY ST-AMAND

Molecular Endocrinology and Oncology Research Center, Laval University Medical Center (CHUL) and Laval University, Ste-Foy, Quebec, G1V 4G2, Canada;
* Faculty of Sport and Health Science, Fukuoka University, Jonan-ku, Fukuoka, Fukuoka, 814-0184, Japan;
{dagger} Department of Preventive Medicine, Saga Medical School, Saga, Saga 849-8501, Japan; and
{ddagger} Pennington Biomedical Research Center, Baton Rouge, Louisiana, 70808, USA

1Correspondence: Molecular Endocrinology and Oncology Research Center, Laval University Medical Center (CHUL), 2705 Boul. Laurier, Ste-Foy (Québec) G1V 4G2, Canada. E-mail: Mayumi.Yoshioka{at}crchul.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Physical exercise produces several adaptive changes in skeletal muscle. However, the molecular mechanisms of these effects are poorly understood. We performed serial analysis of gene expression (SAGE) to quantify the global gene expression profile in sedentary and endurance-trained muscle. A total of 10869 SAGE tags was sequenced and represented 4727 genes. The genes most expressed in muscle are mainly involved in contraction and energy metabolism. Thirty-three genes were differentially expressed between endurance athletes and sedentary individuals. Four genes such as myosin binding protein C fast-type, glycogen phosphorylase, and pyruvate kinase were expressed less in endurance athletes, whereas eight genes coding for expressed sequence tag similar to (EST) crystallin alpha B, EST myosin light chain 2, EST surfactant pulmonary-associated protein A1, EST thrombospondin, EST fructose-bisphosphate aldolase A, EST cytochrome oxidase 1, NADH dehydrogenase 3, and G8 protein were up-regulated. Most of the up-regulated tags corresponded to novel genes. On the other hand, different isoforms of fructose-bisphosphate aldolase A were also differentially expressed. The current study underlying the most highly expressed genes allows a better understanding of global muscle characteristics in normal and endurance-trained individuals. Moreover, the current data suggest novel candidate genes that may be responsible for enhanced endurance performance.—Yoshioka, M., Tanaka, H., Shono, N., Snyder, E. E., Shindo, M., St-Amand, J. Serial analysis of gene expression in the skeletal muscle of endurance athletes compared to sedentary men.


Key Words: athletes • endurance exercise • human mRNA • SAGE method and gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EXERCISE TRAINING produces a number of adaptive changes in skeletal muscle in order to enhance endurance performance, which results in improved metabolic efficiency such as an increase in the capacity for clearance, utilization and storage of plasma glucose, free fatty acids, and triglycerides by skeletal muscle (1 , 2) . Excessive exposure to these plasma metabolites has a negative influence on the health of a variety of tissues. Thus, discoveries of exercise training effects on skeletal muscle will likely play a large role in preventing some of the most common diseases currently plaguing sedentary societies. Although many studies have investigated the effects of endurance exercise training on skeletal muscle, some molecular mechanisms mediating the cellular adaptations to exercise training in human skeletal muscle are still poorly understood.

Genetic engineering has developed many tools to investigate the molecular mechanisms of experimental conditions and diseases. With the application of techniques such as differential display (DD) (3) and cDNA microarrays (4) , gene expression patterns can be determined more efficiently. However, the big drawback with DD is the nonquantitative results and a high frequency of false leads (3) . Microarrays have some technical limitations especially when used for quantification (5) . The variable quality of the different DNA sequences spotted on microarrays makes standardization of a single hybridization procedure for all the different transcripts difficult. Moreover, the correct strand of cDNA must be placed on a microarray to permit transcript detection. Finally, microarrays can only measure the expression levels of genes with known sequences that have been used to design the probes on the microarrays. On the other hand, the serial analysis of gene expression (SAGE) method is a powerful strategy to analyze quantitatively, simultaneously, and differentially the expression of all the mRNAs of the cells (6 7 8 9 10 11 12) . Moreover, novel genes can be discovered by this method. For example, Velculescu et al. (8) have characterized the entire yeast transcriptome and discovered numerous novel genes. We have also shown the ability of the SAGE method to characterize the molecular mechanisms responsible for exercise restriction in the rat (12) . Therefore, we used this powerful strategy to investigate, for the first time, the molecular mechanisms responsible for enhanced endurance performance in the human skeletal muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Seven endurance-trained Japanese athletes were volunteers for this study: three 5000 meter runners, two marathon runners, one triathlon competitor, and one cyclist. They had participated in competitive endurance sports and trained (1–3 h/day and 5–6 days/wk) since age of 13. Eleven Japanese sedentary volunteers were selected according to their age, height, and weight to match with the endurance-trained athletes. At the time of the study, all the sedentary subjects had not participated regularly in any sports activities more than for 1 year. However, all the sedentary subjects had participated in competitive sports such as track-and-field events, including middle and long distance running, swimming, ski, badminton, basketball, baseball, soccer, rugby, and American football, when they were junior high school and high school students; five continued the competitive sports at university. Before the study, each subject was fully informed about all experimental procedures which received the approval of the Fukuoka University Medical Ethics Committee. The written consent of each subject was obtained before admission in the study.

Body fat measurement
Body density was measured using the hydrostatic weighing technique (13) . Percentage body fat was estimated from the body density with a correction for the residual lung volume (13) .

Physical fitness evaluation
Since measurement of maximal oxygen consumption is commonly performed to assess cardiorespiratory fitness, maximal oxygen consumption was measured with a progressive ramp exercise test (10 W increases every 1 min until exhaustion at a pedaling frequency of 50 cycles/min) on an electrically braked cycle ergometer (Load Excalibur, Groningen, The Netherlands). Oxygen uptake was measured using a Douglas bag method (Fukuda Irika CR-20, Tokyo, Japan); O2 and CO2 fractions were analyzed by a mass spectrophotometer (Arco system RL-600B-RA, Tokyo, Japan). Leveling off and/or >8 mmol of blood lactate concentration were used as criteria for maximal oxygen consumption.

Muscle biopsies
After subjects were fasted >12 h, muscle biopsies were obtained between 8:00 to 10:00 AM under local anesthesia with lidocaine from the middle portion of the left vastus lateralis using the needle biopsy technique with suction (14) . In the endurance-trained athletes, muscle biopsies were performed at least 2 days after the last bout of endurance training (1–2 h of running or cycling). The muscle sample (~50 mg) was immediately frozen with liquid nitrogen and stored at –80ºC until analysis.

Global gene expression profile
The SAGE method was performed to quantify the expressed transcripts according to the strategy described by Velculescu et al. (7 , 8) , the modification of Kenzelmann and Muhlemann (15) , and other changes we have optimized (12 , 16) . In each group, polyadenylated RNA was purified with the mRNA direct kit (Dynal, Oslo, Norway) from pooled samples to eliminate any individual variation and extract sufficient quantities of mRNA. After the annealing of biotin-5'T18-3' primer, the mRNA was converted to cDNA and cleaved with NlaIII. The 3' restriction fragment was isolated with streptavidin-coated magnetic beads (Dynal, Oslo, Norway) and ligated to one of two annealed linker pairs (12) . Adjacent tags were released by cleavage with the type II restriction endonuclease BsmFI. The blunting kit of Takara shuzo Co. (Otsu, Japan) was used for blunting and ligation of tags. The produced ditags was preliminary amplified by PCR and analyzed by polyacrylamide gel electrophoresis. This additional step has been reported to not influence the profiles (17) . Large-scale PCR amplification and gel purification were performed and the ditags were digested with NlaIII. The band containing the ditags was excised and self-ligated to produce long concatamers (15) . The concatamers between 500 and 1500 bp were isolated by agarose gel and extracted with GeneClean Spin (BIO 101, Vista, CA, USA). These products were cloned into SphI site of pUC19. White colonies were screened by PCR to select inserts for automated sequencing. With our optimizations, we had an average of 33 or more tags per cloned concatemer with >90% of long inserts. The sequence and occurrence of each of the transcript tags were determined using the software SAGEparser.pl developed by Drs. Snyder and St-Amand (18) . This software is freely available to academia (ftp://ftp.pbrc.edu/public/eesnyder/SAGE/). To identify the corresponding transcript, the sequences of 15 bp SAGE tags (NlaIII site plus the adjacent 11 bp) were matched with public database (http://www.ncbi.nlm.nih.gov/SAGE/).

Statistical analysis
Subject’s descriptive characteristics were analyzed by the Student’s t test. The differential expression identified by the SAGE method was statistically evaluated as previously described (19) . The difference in two samples is statistically significant if (N1-kN11/2 - N2-kN21/2) > 0, where N1 and N2 are the tag numbers of one transcript from two samples and N1 >= N2, k = Z1-{alpha}, {alpha} is the significance level. Statistical significance was set at P <= 0.01. Yamamoto et al. (20) examined the reproducibility in tag appearance with respect to final abundance of each tag sequence. Two smaller profiles were randomly made from a single original library with 80,000 tags. They concluded that the sequences appearing >fivefold showed similar values in a comparison of smaller profiles with 4000 tags each. Since we analyzed ~5400 tags in each sample, we focus on transcripts with >fivefold difference with a statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subject’s characteristics
Descriptive characteristics of subjects are shown in Table 1 . Subjects of both groups had similar age, height, body weight, and % body fat. The exhaustion time of the maximal oxygen consumption test in the endurance athletes was not statistically different from sedentary men (24.0±4.8 and 23.1±7.5, mean±SD). As expected, maximal oxygen consumption in the endurance-trained group was significantly higher than in the control group.


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  		Table 1. Descriptive characteristics of the subjectsa

Most abundant genes in human skeletal muscle
One distinct advantage of the SAGE method is its ability to quantify the expression frequency of all genes, known and unknown. Table 2 presents all the genes expressed >0.5% of the total mRNA population in human skeletal muscle. The relative frequency of a given tag was calculated by dividing the observed tag count by the total count of 5435 tags sequenced in the control muscle and 5434 tags in the endurance-trained muscle multiplied by 100. A total of 24 distinct tags was expressed above the 0.5% threshold. Genes that expressed >0.5% in the control muscle were involved in two main functions: contraction and energy metabolism. The highly expressed genes involved in contraction are actin alpha, troponin C, I, and T, myosin light chain 2 (MLC-2), and myosin binding protein C (MyBP-C) slow-type. Genes involved in energy metabolism are ATPase 6, NADH dehydrogenase 1, 2, and 4, myoglobin, cytochrome c oxidase 1-3, creatine kinase, fructose-bisphosphate aldolase A, glycogen phosphorylase, an expressed sequence tag (EST) similar to enolase 3 beta, and phosphoglycerate mutase 2. The tag CATGTCCCTATAAGC did not match with any known genes in the public databases. Another highly expressed tag has a match with the polyadenylated 16 S rRNA coded by mitochondrial DNA. Among the highly expressed genes, only glycogen phosphorylase and fructose-bisphosphate aldolase A were differentially expressed with a ratio greater than 5 between the control and endurance groups.


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  		Table 2. The most abundant genes in human skeletal muscle of control group and their corresponding level of expression of endurance athletesa

Difference in gene expression between the muscle of control and endurance-trained athletes
A total of 10869 SAGE tags was sequenced and derived from 4727 transcript species. Among 33 genes differentially expressed (P<=0.01 and ratio >5), 4 genes were down-regulated and 29 were up-regulated in the endurance athletes compared with the control group. The components of the cell structure and motility, extracellular matrix, energy metabolism, and immunity differentially expressed in the control and endurance-trained muscles are presented in Table 3 . In the cytoskeletal and contractile apparatus, MyBP-C fast-type was down-regulated in the endurance group. On the other hand, EST crystallin alpha B and EST MLC-2 cardiac slow were up-regulated. In the extracellular matrix, EST surfactant pulmonary-associated protein A1 and EST thrombospondin were also expressed more in the endurance group than in the control group. In the glycogenolysis and glycolysis pathway, glycogen phosphorylase and pyruvate kinase were down-regulated. Fructose-bisphosphate aldolase A (UniGene cluster Hs.273415) was down-regulated whereas another isoform, EST fructose-bisphosphate aldolase A (UniGene cluster Hs.128873), was up-regulated. In the oxidative pathway, EST cytochrome oxidase 1 and NADH dehydrogenase 3 were up-regulated. G8 gene located in the class III region of the major histocompatibility complex (MHC) was also up-regulated in the endurance athletes.


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  		Table 3. Components of cell structure and motility, extracellular matrix, energy metabolism, and immunity differentially expressed in control and endurance-trained groupsa

The sequences of SAGE tags matching with uncharacterized and novel genes up-regulated in endurance athletes are presented in Table 4 . No uncharacterized gene was down-regulated >fivefold, whereas an EST was up-regulated in the endurance athletes. Several SAGE tag sequences did not match with any known human gene or EST. Since these novel genes are up-regulated in endurance athletes, we have named them exercise genes 1 to 20. Exercise genes 1 to 12 were up-regulated >10-fold in the endurance athletes. Remarkably, exercise gene 1 was up-regulated >65-fold and exercise gene 2 was up-regulated 32-fold in endurance-trained muscle.


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  		Table 4. Uncharacterized and novel genes up- and down-regulated in endurance athletesa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study provides a better understanding of global skeletal muscle characteristics, the genomic expression profile observed in sedentary individuals and endurance athletes, and the molecular mechanisms responsible for enhanced endurance performance in the human skeletal muscle. Main findings of the present study were the following. 1) Molecular characteristics of human skeletal muscle show that regardless physical activity levels, mainly genes coding for contractile apparatus and energy metabolism were highly expressed. 2) Endurance-trained individuals had higher expression of genes involved in the molecular chaperone, skeletal muscle repairing, and oxidative pathway but a lower expression of genes involved glycogenolysis and glycolysis pathways. 3) Twenty novel genes with elevated expression in the endurance-trained muscle were identified.

Most abundant genes in human skeletal muscle
Physical fitness levels influence skeletal muscle characteristics (21 , 22) , which plays an important role in whole body metabolism (1 , 2) and therefore can influence the risk of chronic disease occurrence (23 , 24) . The recommendation of the Japanese Ministry of Health and Welfare is to maintain >41 mL · kg-1 · min-1 of maximal oxygen consumption for an age group of 20- to 29-year-old men in order to reduce or almost eliminate the risk factors of chronic diseases (25) . In the present study, maximal oxygen consumption of the control subjects was 41.2 mL · kg-1 · min-1, which is in the range of recommended fitness level. Their skeletal muscle’s characteristics showed that genes with a level of expression >0.5% in the control muscle were mainly involved in contraction and energy metabolism. Welle et al. (11) investigated the abundance of mRNA in vastus lateralis from healthy young men using the SAGE method and drew similar conclusions. Almost all the genes expressed >0.5% in the control muscle of the present study were also expressed >0.5% in their study, with exceptions of cytochrome c oxidase 1, MyBP-C slow-type and phosphoglycerate mutase 2. In their study, these genes were also relatively highly expressed (0.32%, 0.21%, and 0.35%, respectively). On the other hand, only two genes among the most abundant genes in the present study were differentially expressed between the control muscle and the endurance-trained muscle. Moreover, the most highly expressed genes such as actin alpha 1 and 2, ATPase 6, NAHD dehydrogenase 1, 2, and 4/4L, 16S rRNA, myoglobin, and myosin light chain 2 were constantly highly expressed (>0.5%) in the control and endurance-trained groups of the present study and the study of Welle et al. (11) . Therefore, it suggests that the most abundant genes in the skeletal muscle are highly expressed regardless of the subject’s physical fitness levels.

Components of energy metabolism differentially expressed
It is well known that endurance exercise training increases muscle mitochondrial density (26) and oxidative enzyme activity (27) . In the present study, genes involved in oxidative pathway (EST cytochrome oxidase 1 and NADH dehydrogenase 3) were up-regulated in the endurance-trained muscle, as reported earlier in other studies (28) . On the other hand, genes involving in the glycogenolysis pathway (glycogen phosphorylase) and glycolysis pathway (fructose-bisphosphate aldolase A and pyruvate kinase) were down-regulated in the present study. Moreover, different isoforms such as fructose-bisphosphate aldolase A (UniGene cluster Hs.273415, Locus ID 226) and an EST similar to fructose-bisphosphate aldolase A (UniGene cluster Hs.128873) were differentially expressed. The EST similar to fructose-bisphosphate aldolase A (UniGene cluster Hs.128873) had 99% identity with rat fructose-bisphosphate aldolase A mRNA sequences (UniGene cluster Rn.1774, GenBank accession no. NM 012495, Locus ID 24189) and 96% identity with mouse (UniGene cluster Mm.16763, GenBank accession no. Y00516, Locus ID 11674) whereas only 61% of identity with human fructose-bisphosphate aldolase A (UniGene cluster Hs.273415, GenBank accession no. BC016800, Locus ID 226) was found. Endurance exercise training has been reported to have a minimum effect on glycolytic enzymes (29) . However, these different expression levels of the isoforms might play a role in glycolytic enzyme responses to the endurance exercise training.

Components of contractile apparatus differentially expressed
MyBP-C is a component of thick filaments located within the A bands of vertebrate striated muscles. The carboxyl-terminal domain of MyBP-C contains myosin binding sites and binds to the rod portion of myosin. MyBP-C also binds to actin and native thin filaments in a Ca2+-dependent manner. However, its function is still uncertain. In addition to a structural role in the thick filaments (30) , a potential role in the regulation of contraction has been suggested since MyBP-C inhibits actin-activated skeletal muscle myosin ATPase (31) . No study has investigated the gene regulation of MyBP-C by endurance training. However, we previously demonstrated that adaptation to immobilization caused an increase in gene expression of the fast isoform MyBP-C (12) . In the present study, MyBP-C fast-type was down-regulated in the endurance-trained muscle, suggesting that endurance-trained muscle might have a facility to contract by decreased inhibition of actomyosin ATPase. Further studies are needed to investigate the role of MyBP-C in the structures and contractile kinetics of skeletal muscle after endurance exercise training and immobilization.

MLCs stabilize the long alpha helical neck of the myosin head and consist of essential light chains (MLC-1 and 3) and a regulatory light chain (MLC-2). MLC-2 is a member of the superfamily of Ca2+ binding protein. The MLC-2 of the heart and skeletal muscle can be reversibly phosphorylated by a specific enzyme system, including a MLC phosphatase and Ca2+-calmodulin-dependent kinase (32) . Based on the introduction of negative charges by phosphorylation, it has been hypothesized that the cross-bridges move away from the filament backbone, thus increasing the probability of attachment and force generation (33) . In fact, extraction of MLC-2 from demembrated skeletal muscle fiber decreases Vmax (34) and actin filament sliding velocity (35) . In the present study, expression of EST MLC-2 cardiac slow was higher in the endurance-trained muscle than in sedentary muscle. MLC-2 ventricular is known to be the major isoform in slow-twitch skeletal muscle fibers. Moreover, Buck et al. (36) have suggested that the MLC-2 ventricular isoform may contribute to the greater power-generating capabilities of the ventricle compared with the atrium. Therefore, higher expression of EST MLC-2 cardiac slow may also contribute to the greater power-generating capabilities of the slow-twitch skeletal muscle in the endurance-trained subjects.

Components of molecular chaperone and muscle repair differentially expressed
Alpha B-crystallin, localizes at Z bands in skeletal muscle (37) , where many cytoskeleton-relating proteins assemble and mechanical stress is transferred; it is a small heat-shock protein (HSP) (38) and can function as a molecular chaperone (39) . In the present study, EST alpha B-crystallin was expressed more in the endurance-trained group and the percentage of type I fiber (slow-twitch fiber) in the endurance-trained muscle was significantly higher than in sedentary muscle (64.3±18.0 and 37.2±12.6, respectively; P<0.01). It is known that alpha B-crystallin is expressed at higher levels in slow-twitch muscles than in fast-twitch muscles (40 , 41) , and overexpression of small HSPs, including alpha B-crystallin, can prevent cells from dying by reactive oxygen species (42) . On the other hand, homozygous knockout of alpha B-crystallin gene and the adjacent gene HSPB2 (an ancient duplication of alpha B-crystallin) show postural defects and other health problems that appear to stem from progressive myopathy (43) . Alpha B-crystallin specifically decreases in atrophied soleus muscle, but the expression of alpha B-crystallin can be sustained if the muscle is passively stretched (40 , 44) . Moreover, expression of alpha B-crystallin is related to maintaining the stability of the cytoskeleton (45) . Since alpha B-crystallin binds actin and intermediate filament proteins such as desmin, vimentin, and peripherin, Koh (38) suggested that small HSP stabilizes actin and intermediate filaments against stress. From these observations and from the fact that slow-twitch muscle is subjected to much more stress such as oxidant injury and higher rate of protein turnover (46) , its seems reasonable to assume that higher expression of EST alpha B-crystallin in the endurance-trained muscle is provided for future stress.

Thrombospondin-1 is located in the extracellular matrix of the anatomical sites where firm bindings are needed, i.e., between muscle fibers and fiber bundles, between the collagen fibers of a tendon, and at myotendinous junctions, osteotendinous junctions and articular cartilage (47) . In the present study, EST thrombospondin in the endurance-trained subjects was significantly higher than in the sedentary subjects. In a previous study (48) , no fibrinogen and minimal thrombospondin antigen were reported in undamaged control muscle. Moreover, after crushing injury, fibrin networks appear immediately, followed by a gradual ordered accumulation of thrombospondin (within a few hours) in the vicinity of the vascular bed and adjacent endomysial connective tissue (48) . Later, thrombospondin becomes associated with connective tissue and basal laminae around muscle fibers throughout the damaged muscle, maximal labeling occurring 3–6 days postinjury (48) . Therefore, the current results suggest that reparation of injured muscle induced by regular endurance training might be needed in the endurance athletes.

Other genes differentially expressed
The SAGE method has many advantages when identifying the genes most expressed 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 physiological responses to a stimulus as well as the less commonly studied, uncharacterized, and even unknown genes. Indeed, the present study has quantified the tag TCCCTATAAGC among the most-expressed genes in the human skeletal muscle as well as MyBP-C which was down-regulated in the endurance-trained muscle. On the other hand, EST MLC-2 cardiac slow, EST thrombospondin, G8 protein MHC class III gene, and EST surfactant pulmonary-associated protein A1 were up-regulated. The function of G8 protein, a novel major histocompatibility complex (MHC) class III gene, is unknown. Pulmonary surfactant is a phospholipid–protein complex that serves to lower the surface tension at the air–liquid interface in the alveoli of the lung and is essential for normal respiration. Thus, it can be suggested that the up-regulation of EST similar to surfactant pulmonary-associated protein A1 might be responsible in part of the higher maximal oxygen consumption observed in trained subjects. Surfactant protein-A, which plays a role in innate host defense of the lung, is also expressed in the eustachian tube. However, no study of surfactant pulmonary-associated protein A1 in the skeletal muscle and its regulation by endurance exercise has been reported previously. Although all the genes had the potential to be included in the present study, the limit of sensitivity in determining transcript abundance was 0.018%. There is also the possibility that transcripts will be missed if they do not contain NlaIII restriction site, especially the short transcripts.

Uncharacterized and novel genes differentially expressed
Many tags did not match with any gene in the human nonredundant division of GenBank. In the present study, one EST was up-regulated in the endurance athletes. Moreover, 20 SAGE tag sequences did not match with any known human gene or EST. Since these novel genes are differentially expressed in the endurance athletes, we have named them exercise genes 1 to 20. Exercise genes 1 to 12 were up-regulated >10-fold in endurance athletes. Remarkably, exercise gene 1 and 2 were >65- and 32-fold up-regulated in endurance-trained muscle. Cloning of the full-length cDNA and further characterization of these genes are needed. The current study has characterized the global gene expression in endurance athletes and highlighted the possible molecular mechanisms responsible for enhanced endurance performance.


   ACKNOWLEDGMENTS
 
This study was supported by the Sanix Sports Foundation (Mr. Munemasa), Uehara Memorial Foundation, and Japanese Ministry of Education, Culture, Sports, Science and Technology.

Received for publication January 9, 2003. Accepted for publication June 9, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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