1Department of Nutrition, 2Department
of Orthopedic Surgery, The University of Tokushima School
of Medicine, Tokushima, Japan.;3Department of
Neuromuscular Research, National Center of Neurology and
Psychiatry, Tokyo, Japan.;4National Institute
of Neuroscience, National Center of Neurology and Psychiatry,
Tokyo, Japan.;5Department of Physiology and Biophysics,
University of California, Irvine, California 92697, USA.;and
6Research Center for Pathogenic Fungi and Microbial
Toxicoses, Chiba University, Chiba, Japan.
Abstract: We obtained the skeletal muscle of rats exposed
to weightless conditions during a 16-day-spaceflight (STS-90).
By using a differential display technique, we identified
6 up-regulated and 3 down-regulated genes in the gastrocnemius
muscle of the spaceflight rats, as compared to the ground
control. The up-regulated genes included those coding Casitas
B-lineage lymphoma-b, insulin growth factor binding protein-1,
titin and mitochondrial gene 16S rRNA and two novel genes
(function unknown). The down-regulated genes included those
encoding RNA polymerase II elongation factor-like protein,
NADH dehydrogenase and one novel gene (function unknown).
In the present study, we isolated and characterized one
of two novel muscle genes that were remarkably up-regulated
by spaceflight. The deduced amino acid sequence of the spaceflight-induced
gene (sfig) comprises 86 amino acid residues and is well
conserved from Drosophila to Homo sapiens. A putative leucine-zipper
structure located at the N-terminal region of sfig suggests
that this gene may encode a transcription factor. The up-regulated
expression of this gene, confirmed by Northern blot analysis,
was observed not only in the muscles of spaceflight rats
but also in the muscles of tail-suspended rats, especially
in the early stage of tail-suspension when gastrocnemius
muscle atrophy initiated. The gene was predominantly expressed
in the kidney, liver, small intestine and heart. When rat
myoblastic L6 cells were grown to 100% confluence in the
cell culture system, the expression of sfig was detected
regardless of the cell differentiation state. These results
suggest that spaceflight has many genetic effects on rat
skeletal muscle. J. Med. Invest. 50:39-47, 2003
Keywords:spaceflight/differential display approach/skeletal
muscle gene/rats
INTRODUCTION
Skeletal muscles, especially antigravity slow-twitch muscles,
are vulnerable to rapid and marked atrophy under microgravity
or its simulated conditions (1, 2). We previously reported
that spaceflight (STS-90) as well as tail-suspension stimulated
the ubiquitination of various proteins, including myosin
heavy chain (MHC), and the accumulation of MHC degradation
fragments in atrophied rat gastrocnemius muscle (3). In
this case, the spaceflight significantly increased mRNA
levels of cathepsin L, proteasome components (RC2 and RC9),
polyubiquitin and a ubiquitin-conjugating enzyme in gastrocnemius
muscle. Based on these findings, we suggest that skeletal
muscle may adapt to microgravity conditions by changing
its gene expression. However, there are few data concerning
the gene expression in skeletal muscle under microgravity
conditions. There is only one report that the gene expression
in the paraspinal muscles of rats exposed to spaceflight
(STS-58) was analyzed by a differential display approach
(4). They demonstrated that the expressions of 42 genes
changed, including heat shock protein 70, myosin light chain
and myocyte enhance factor 2C (MEF2C), and suggested that
MEF2C is a key transcriptional factor in skeletal muscle
atrophy and regeneration under microgravity.
In this study, we also examined the changes in muscle gene
expression after a space shuttle flight (STS-90), using
a differential display approach. We identified 9 genes which
may play distinct pathological roles in microgravity-induced
muscle atrophy, in gastrocnemius muscle atrophied by spaceflight.
Among these genes, we focused on a novel spaceflight-induced
gene, sfig, to elucidate the distinct adaptation of skeletal
muscle to microgravity conditions. The deduced amino acid
sequence of the sfig gene is very similar to that of the
Drosophila CG6115 gene, which may function as a transcription
factor possessing a leucine zipper structure (5). Tail-suspension
up-regulated its expression in gastrocnemius muscle at the
early stage, when muscle atrophy caused by suspension initiated
(3). Our results suggest that genetic studies on the effects
of microgravity are helpful to understand the mechanism
of microgravity-induced muscle atrophy.
MATERIALS AND METHODS
Spaceflight and tail-suspended rats.
As described in detail in a previous report (3, 6), the
rats were launched into space on April 17, 1998, on the
space shuttle Columbia, when they were 8-days old. The shuttle
rats were housed with their dams in research animal holding
facility (RAHF) cages in the orbiter. The shuttle landed
at Kennedy Space Center on May 3, 1998. Approximately 2
hr elapsed under weight-bearing conditions before the animals
were killed, and isolation of the gastrocnemius muscles
from all animals was completed within 75 min. The asynchronous
ground control rats were housed with their dams in cage
conditions that simulated the shuttle's environment, including
the shuttle's ambient temperature, the facilities and the
timing of events of the flight animals.
Six-week-old male Wistar rats were subjected to tail suspension-induced
hypokinesia, a model simulating microgravity conditions,
for the indicated period by using the apparatus described
previously (3, 7). Their tails were suspended to keep their
hind legs off the ground. Rats were housed in a room maintained
at 23°C on a 12-hr light/dark cycle and were allowed
free access to a 20% casein diet and water.
All of the treatments described here were performed according
to the Guide for the Care and Use of Laboratory Animals
(1985) and were approved by the Animal Care Committee of
the National Aeronautics and Space Administration (NASA)
or the National Space Development Agency of Japan (NASDA)
counterpart.
Cell culture.
Rat myoblastic L6 cells were purchased from Dainippon Pharmaceutical
Co. (Osaka, Japan). L6 cells were maintained in tissue culture
flasks at 37°C with 5% CO2/95% air in DMEM, supplemented
with10% fetal calf serum (FCS), 100 units/ml penicillin
and 0.1 mg/ml streptomycin. When the cells were grown to
100% of confluence, the medium was changed to DMEM containing
2% horse serum and the same antibiotics to stimulate differentiation.
Differential display analysis.
For the differential display analysis, we used a fluorescence
differential display kit (Takara, Kusatsu, Japan) with modifications
(8). DNA-free total RNA was isolated with an acid guanidinium
thiocyanate-phenol-chloroform mixture (Nippon Gene, Tokyo,
Japan). Total RNA (300ng) was used for the reverse transcription
(RT) reaction (final volume, 20 µl) with 7.5 units
of AMV RTase XL, 20 mM dNTP and 2.5 mm downstream primer,
which was oligo-dT primer (5'-T11VV-3', V represented A,
C or G) for 10 min at 30°C. Following initial denaturation,
second-strand synthesis and DNA amplification with 2.5 µM
fluorescence-labeled downstream primer, 0.5 µM non-labeled
upstream primer, 10 units of Taq DNA polymerase (Takara)
and 2 µM dNTP (final volume, 20 µl) were achieved
in a thermal cycler through 40 cycles of the following incubations:30
sec at 90°C, 2 min at 40°C and 30 sec at 72°C.
Samples were run on 12% native acrylamide gels, and the
gels were analyzed with an image analyzer (FMBIOII, Takara).
Differentially expressed bands were cut and each fragment
was eluted by boiling in 100 µl of distilled water
and re-amplified by polymerase chain reaction (PCR) using
the same set of primers. Re-amplified DNA fragments were
directly subjected to DNA sequence analysis with a DNA sequencer,
model 373A (Perkin Elmer, Foster City, CA, USA).
Cloning and sequencing.
A Uni-ZAP cDNA library from the skeletal muscle of a 12-wk-old
Wistar rat was purchased from Stratagen (La Jolla, CA, USA).
Re-amplified DNA fragments were used as a probe for plaque
hybridization screening, which was performed according to
the method of Ishidoh et al. (9) with modifications. In
short, recombinant plaques were transferred onto nylon membranes
(Amersham, Little Chalfont, UK) and fixed with ultraviolet
light. After prehybridization in a quick hybridization buffer
(Amersham), hybridization was performed overnight at 60°C
in the same buffer mixed with an isotope-labeled probe.
The membranes were washed and exposed to Kodak X-ray films
at -80°C for the appropriate time. Positive plaques
hybridizing to probes were subjected to second and third
screenings in the same manner.
Phage DNAs containing cDNAs hybridizing the probe were isolated
from plate lysates and subcloned into pBluescript SK(-)
vectors. The plasmids were introduced into E. coli JM109.
Plasmid DNAs were prepared by the alkaline-SDS method. The
nucleotide sequence of the DNA inserts in the multicloning
site of pBluescript vectors was determined by a bi-directional
dideoxy sequencing method using T7 DNA polymerase.
Northern blot analysis.
Total RNA (20 µg/lane) was separated in a 1% agarose
gel, blotted and ultraviolet crosslinked to a nylon membrane
(Amersham). After prehybridization, hybridization of the
membrane was performed in a quick buffer with an isotope-labeled
cDNA probe, as described above. The membranes were washed
and exposed to Kodak X-ray films at -80°C for the appropriate
time, and then the films were developed. Autoradiograph
signals were quantified by densitometric analysis. Each
mRNA level was standardized to that of 18S rRNA.
In vitro translation.
The transcription and translation of cDNA for the sfig gene
in vitro was performed in a rabbit reticulocyte coupled
transcription/translation system (Promega)(10). Briefly,
1µg of linearized template (pBluescript SK containing
a cDNA for sfig gene) was suspended in 50 µl of reaction
mixture (25 µl of rabbit reticulocyte lysate, 1 µl
of T3 RNA polymerase, 20 µM amino acid mixture, 40
unit of RNase inhibitor and biotinylated lysine-tRNA complex)
and then incubated at 30°C for 90 min. This was mixed
with sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) sample buffer and heated at 95°C for 5 min.
The reaction products were subjected to SDS-15%-PAGE and
blotted on a nylon membrane. In vitro translated protein
was detected with streptoavidin-labeled horse radish peroxidase
(Promega) in an enhanced chemiluminescence system.
Other biological analyses.
Creatine kinase (CK) activity was measured by the method
of Szasz et al. (11). Protein concentration was measured
according to the method of Lowry et al. (12) with bovine
serum albumin as a standard.
Statistical analysis.
Data are expressed as mean±SD and were statistically
evaluated by analysis of variance (ANOVA) with SPSS software
(release 6.1;SPSS Japan Inc., Tokyo, Japan). One-way ANOVA
was used to determine the significant effects of tail-suspension
on the measured variable. Individual differences between
groups were assessed using Duncan's multiple range test.
The differences were considered significant at P<0.05.
RESULTS
Genetic effects of microgravity.
The present study examined the alteration of the muscle
gene expression under microgravity using the differential
display approach, i.e. a method to identify the actual differences
between two well-defined biological situations. We performed
differential display analysis for the gastrocnemius muscles
of 16-day-spaceflight and asynchronous ground control rats,
and obtained 9 PCR fragments whose lengths were 200-500
bp as expected. We then sequenced all of these PCR fragments
and compared their DNA homology with the GenBank and the
EMBL databases using BLAST. As shown in Table 1, the expression
of Casitas B-lineage lymphoma-b (Cbl-b), insulin growth
factor binding protein-1, titin and mitochondrial gene 16S
rRNA was significantly up-regulated by spaceflight. In contrast,
the mRNA levels of RNA polymerase II elongation factor-like
protein and NADH dehydrogenase decreased. The physiological
functions of three genes similar to KIAA0368, AF412300 or
AA275180, are still unknown.
Isolation and sequence of a novel gene, sfig.
In this study, we isolated and fully sequenced the cDNA
of sfig, one of three novel genes described above (See #6
in Table 1). The nucleotide sequence of the cDNA for this
gene comprised 1162 bp containing 94 bp in the 5'-noncoding
region, 261 bp in the coding region and 807 bp in the 3'-noncoding
region (Fig. 1A). The ATG codon numbered as 1 must be the
translation initiation site, since the nucleotide sequence
around it (TGAAAATGG) corresponds to Kozak's rule (13) and
an in-frame termination codon (-9 to -7) is found in the
5'-upstream sequence. The deduced amino acid sequence of
the open reading frame codes for 86 amino acid residues.
Calculating from the amino acid sequence, the molecular
mass of rat Sfig protein is 10405.07. A putative leucine-zipper
structure was located at the N-terminal portion (Fig. 1A
and C). No potential N-glycosylation site was found. In
vitro translation assay revealed that a protein with a molecular
mass of about 11 kDa was produced from plasmid containing
the cDNA for sfig gene, but not from a mock vector (Fig.
1B), suggesting that sfig was not a pseudo gene.
The full cDNA sequence of this gene is similar to those
of mouse testis full-length cDNA (AK015530), mouse RIKEN
cDNA 4930469P12 gene (XM_132933), human gene similar to
RIKEN cDNA 4930469P12 (XM_084843), Macaca fascicularis brain
cDNA clone QnpA-16830 (AB049871) and Drosophila melanogaster
CG6115 gene (AAF53585), besides mouse clone 58 growth hormone-inducible
soluble protein mRNA (AF412300). Fig. 1C shows that the
amino acid sequence of the sfig gene, especially a leucine-repeated
sequence (See asterisks) is highly conserved from Drosophila
to Homo sapience.
Expression of sfig gene up-regulated by spaceflight and
tail-suspension.
To confirm the high expression of the novel gene in the
gastrocnemius muscle of spaceflight or tail-suspended rats,
Northern blot analysis was performed. The 16-day-spaceflight
remarkably increased the mRNA level of sfig (Fig. 2A). Tail-suspension
also up-regulated the expression of sfig transcripts in
the early stage and reached the peak value on Day 5, when
a significant loss of gastrocnemius muscle started (3),
as shown in Fig. 2B. The expression of the transcripts returned
to the basal level once after reaching the peak value and
then increased gradually.
Tissue distribution.
The levels of sfig mRNA were high in the liver, kidney and
small intestine, moderate in the large intestine, heart,
lung and brain, and low in the skeletal muscle and spleen
(Fig. 3). Only small amounts of the sfig transcript existed
in the stomach and thymus. Among muscles and a myoblastic
cell line, the sfig transcript was highly expressed in the
cardiac and anterior tibial muscles, followed by the soleus
and gastrocnemius muscles (Fig. 3). Low levels of the sfig
transcript were expressed in smooth muscle from the small
intestine and proliferating L6 myoblastic cells.
Expression of sfig gene during differentiation of myoblastic
cells.
Besides microgravity conditions, the expression of this
gene was stimulated during the differentiation of L6 cells.
When L6 cells grown to 100% of confluence were treated with
DMEM containing 2% horse serum, CK activity, a muscle differentiation
marker, in the cells significantly increased on Day 4 (Fig.
4A). The level of sfig mRNA remarkably increased on Day
2 prior to the CK activation and then sustained a gradual
increase (Fig. 4B).
DISCUSSION
In the present study, we found that 9 muscle genes changed
during spaceflight by using a differential display technique.
All the genes may have pathological significance for spaceflight-induced
muscle atrophy. For example, the imbalanced expression of
mitochondrial genes, i.e. the up-regulated expression of mitochondrial
gene 16S rRNA and the down-regulated expression of NADH dehydrogenase,
indicates mitochondria dysfunction under microgravity conditions.
We previously reported that tail-suspension caused oxidative
stress in hindlimb skeletal muscles (14). These results support
our hypothesis that superoxide anions and/or iron ions leaking
from mitochondria may contribute to this oxidative stress.
Recently, c-Cbl and Cbl-b, adaptor proteins, have been reported
to act as ubiquitin-protein ligases (E3s) for several growth
factor receptors, including the epidermal growth factor receptor,
and to down-regulate the signaling pathway of growth factors
(15, 16). We confirmed that spaceflight significantly stimulated
Cbl-b expression at the mRNA and protein levels, and Cbl-b
played an important role in microgravity-induced muscle atrophy
by down-regulating insulin-like growth factor signaling (unpublished
observation, manuscript submitted). In general, microgravity
conditions decrease the transcription of general genes (17).
The down-regulated expression of RNA polymerase II elongation
factor-like protein indicates that spaceflight may cause decreased
translation as well as transcription. Thus, genetic studies
on the effects of spaceflight are helpful to elucidate the
mechanisms of microgravity-induced muscle atrophy. We are
further examining the pathological roles of other genes identified
in the present study.
The genes identified as sensitive to the STS-90 spaceflight
are not identical to those sensitive to the STS-58 spaceflight.
In the present study, we used younger rats (8-days old at
launch) than those (8-wks old at launch) in the STS-58 mission.
In addition, we used a fast-twitch muscle (gastrocnemius muscle)
for a differential display analysis, rather than a slow-twitch
muscle (paraspinal muscle). The duration of spaceflight in
the STS-90 mission was 16 days, similar to that (14 days)
in the STS-58 mission. Therefore, the age of rats at launch
and the type of skeletal muscle may be important factors which
affect muscle gene expressions in space.
In this study, we also identified and sequenced a novel skeletal
muscle sfig gene. Spaceflight and tail-suspension induced
the remarkable up-regulated expression of sfig mRNA in rat
gastrocnemius muscles, whereas they contained small amounts
of this transcript under normal conditions (1 g). In addition,
tail-suspension up-regulated its expression in the early stage
(Day 5) when muscle atrophy caused by suspension initiated
(3). Changes in sfig mRNA expression by denervation, another
model simulating microgravity, also showed similar patterns
to that by tail-suspension (data not shown). Our results suggest
that this gene may be useful as a marker to detect early muscle
atrophy caused by microgravity and its simulated conditions.
At present, the pathophysiological roles of this novel gene
for microgravity-induced muscle atrophy are unknown. However,
the deduced amino acid sequence of the sfig gene was conserved
from Drosophila to Homo sapiens. In particular, the Drosophila
melanogaster CG6115 gene was reported to code a novel transcription
factor, since there is a putative leucine zipper structure
at the N-terminal domain (5). During the differentiation of
myoblastic cells, the expression of this transcript was significantly
up-regulated prior to CK activation, indicating that this
sfig gene may function as a transcription factor associated
with the differentiation of myoblastic cells. In rat skeletal
muscles under normal conditions (1 g), this mRNA was dominantly
expressed in the cardiac and anterior tibial muscles, which
are resistant to atrophy caused by microgravity (3, 18). These
findings lead us to consider that the expression of this gene
may be secondarily up-regulated against muscle atrophy caused
by spaceflight. Further examinations are necessary to clarify
this hypothesis.
ACKNOWLEDGEMENTS
This study was supported by Grants-In-Aid of “Ground
Research Announcement for Space Utilization" promoted by NASDA
and Japan Space Forum to T. N.
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Abbreviations:ANOVA, analysis of variance;Cbl, Casitas B-lineage
lymphoma;CK, creatine kinase;E3, ubiquitin ligase;FCS, fetal
calf serum;MEF2C, myocyte enhance factor 2C;NASA, National
Aeronautics and Space Administration;NASDA, National Space
Development Agency of Japan;PCR, polymerase chain reaction,
RT, reverse transcription;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis
Received for publication August 12, 2002;accepted August
21, 2002.
Address correspondence and reprint requests to Takeshi
Nikawa, M. D., Ph. D., Department of Nutrition, The University
of Tokushima School of Medicine, Kuramoto-cho, Tokushima
770-8503, Japan and Fax:+81-88-633-7086.
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