highlighted topics
J Appl Physiol 95: 2171–2179, 2003.
First published August 1, 2003; 10.1152/japplphysiol.00500.2003.
Physiology of Aging
Selected Contribution: Identification of differentially expressed
genes between young and old rat soleus muscle during recovery
from immobilization-induced atrophy
J. Scott Pattison,1 Lillian C. Folk,2 Richard W. Madsen,3 and Frank W. Booth1
1
Departments of Biomedical Sciences and of Pharmacology and Physiology, and the Dalton
Cardiovascular Institute University of Missouri at Columbia; 2Department of Veterinary Pathobiology
and 3Department of Statistics, University of Missouri at Columbia, Columbia, Missouri 65211
Submitted 12 May 2003; accepted in final form 24 July 2003
muscle mass and
strength play in overall health and homeostasis has
been somewhat underappreciated. For example, the
loss of skeletal muscle mass and strength is significant
because it is associated with an increased incidence of
death (30). Major modes by which skeletal muscle mass
can be lost include physical inactivity, chronic health
disorders, and aging (termed sarcopenia) (8). The average time course of sarcopenia is that by the age of 50
yr, ⬃10% of peak muscle mass is lost, and another 30%
disappears by 80 yr of age (27). The health implications
of muscle loss in the elderly are further exacerbated by
the fact that aged populations seem to have a limited
capacity to recover skeletal muscle mass after a bout of
atrophy (29, 35). Skeletal muscles from old humans
and animals also do not hypertrophy as well as their
younger counterparts. In younger individuals, the exact time course for muscle fiber hypertrophy is not well
documented but is thought to require at least 6–7 wk of
regular resistive training at reasonably high intensity
before increases in fiber cross-sectional area are
deemed significant (33). Conversely, in humans averaging 87 yr of age, 10 wk of progressive resistance
training increased thigh area by only 3% and knee
strength by 200%, which Fiatarone et al. (16) attributed to an improved neural recruitment of existing, but
underused, skeletal muscle, not actual hypertrophy.
Animal studies further confirm the human observations in that skeletal muscle atrophied by hindlimb
immobilization regrows on remobilization to its preatrophy size in younger (5, 9) but not older rats (8, 39).
Some studies have found that mechanically overloaded
skeletal muscle hypertrophies less in old than young
rats (1, 4, 12). Thus the capacity of skeletal muscle to
grow in response to increasing mechanical loads appears to be diminished in old human and animal subjects.
Inappropriate levels of factors permitting growth
may underlie impaired muscle regrowth from atrophy
at old age. For example, high-resistance exercise enhanced IGF-IEc mRNA (mechanogrowth factor) level
in the vastus lateralis muscle of young, but not of old,
Address for reprint requests and other correspondence: F. W.
Booth, Univ. of Missouri, Dept. of Veterinary Biomedical Sciences,
E102 Vet. Med. Bldg., 1600 E. Rollins, Columbia, MO 65211 (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
aged; mRNA; rehabilitation; growth
THE INHERENT VALUE THAT SKELETAL
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Pattison, J. Scott, Lillian C. Folk, Richard W. Madsen, and Frank W. Booth. Selected Contribution: Identification of differentially expressed genes between young and
old rat soleus muscle during recovery from immobilizationinduced atrophy. J Appl Physiol 95: 2171–2179, 2003. First
published August 1, 2003; 10.1152/japplphysiol.00500.2003.—
After cessation of hindlimb immobilization, which resulted in
a 27–37% loss in soleus mass, the atrophied soleus muscle of
young but not old rats regrows to its mass before treatment.
We hypothesized that during remobilization the mRNA levels of growth potentiating factor(s) would be present in the
soleus muscle of young (3- to 4-mo-old) but absent in old (30to 31-mo-old) Fischer 344 ⫻ Brown Norway rats or that
mRNAs for growth inhibitory factor(s) would be absent in
young but present in old. Gene expression levels of ⬎24,000
transcripts were determined by using Affymetrix RGU34A-C
high-density oligonucleotide microarrays in soleus muscles
at 3, 6, 10, and 30 days of remobilization after cessation of a
10-day period of hindlimb immobilization. Each muscle sample was applied to an independent set of arrays. Recoveryrelated differences were determined by using a three-factor
ANOVA with a false discovery rate-adjustment of P ⫽ 0.01,
which yielded 64 significantly different probe sets. Elfin,
amphiregulin, and clusterin mRNAs were selected for further confirmation by real-time PCR. Elfin mRNA levels were
less in old than in young rats at 6, 10, and 30 days of
remobilization. Amphiregulin expression exhibited a unique
spike on the 10th day of successful regrowth in young rats
but remained unchanged old. Clusterin mRNA was unchanged in young muscles but was elevated on the 3rd, 6th,
and 10th days of recovery in old soleus muscles. The mRNAs
identified as differentially expressed between young and old
recovery may modulate muscle growth that could highlight
new candidate mechanisms to explain the failure of old
soleus muscle to recover lost muscle mass.
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GENE EXPRESSION CHANGES IN ATROPHIED SOLEUS MUSCLE
MATERIALS AND METHODS
Animals. One hundred male Fischer 344 ⫻ Brown Norway
Fl rats were obtained from the National Institute on Aging
(Harlan Labs, Indianapolis, IN) and were killed at the ages of
3–4 mo (young, n ⫽ 50) and 30–31 mo (old, n ⫽ 50). Five rats
were used per group (four groups: young controls, young
immobilized/recovery, old controls, and old immobilized/recovery) at every time point (five time points: 0, 3, 6, 10, and
30 days) for a total of 20 groups. Half of the young and old
rats were immobilized, whereas the other half of the animals
were kept as controls with normal cage activity. The immobilization protocol has been previously published and is reviewed briefly next (5, 8). Before casting, animals were anesthetized with an intraperitoneal injection of a cocktail containing ketamine (49 mg/ml), xylazine (6.2 mg/ml), and
J Appl Physiol • VOL
acepromazine (2.0 mg/ml) at a concentration of 0.10 ml/100
mg body wt. When rats became anesthetized, both hindlimbs
were fixed from the waist down with wire mesh-reinforced
plaster casts in the plantar-flexed position. Animals were
maintained in the hindlimb-immobilized condition for 10
days. Rats were lightly anesthetized for removal of casts. All
rats were housed 2–3 per cage and maintained on a 12: 12-h
light-dark cycle, in which they received regular rat chow and
water ad libitum. Before muscle removal, animals were anesthetized with an intraperitoneal injection of a cocktail containing ketamine (49 mg/ml), xylazine (6.2 mg/ml), and
acepromazine (2.0 mg/ml) at a concentration of 0.123 ml/100
mg body wt. All animal experimental protocols were approved by the University of Missouri Animal Use Committee.
Soleus muscles were excised, weighed, snap-frozen in liquid
nitrogen, and subsequently powdered in a mortar and pestle
cooled by liquid nitrogen. Both soleus muscles from a single
rat formed a single observation, for which muscle RNA from
a single animal was applied to an individual array. Although
the recovery time points have not previously been published,
the control (31) and the atrophy (J. S. Pattison, L. C. Folk,
R. W. Madsen, T. E. Childs, E. E. Spangenburg, and F. W.
Booth, unpublished observations) data have been published;
these data sets found 682 and 739 differentially expressed
probe sets, respectively, which were too large to be incorporated into a single paper.
Sample processing. Total RNA was extracted from an aliquot of muscle powder when put directly into TRIzol (Invitrogen; Carlsbad, CA) and homogenized on ice by using a Polytron homogenizer (Kinematica; Lucerne, Switzerland) on setting 7 for three pulses of 15 s each. RNeasy columns (Qiagen;
Valencia, CA) were employed to further purify the extracted
total RNA. Methods for sample preparation described in
detail in the Affymetrix Expression Analysis Technical Manual (Santa Clara, CA) are briefly described next. cDNA synthesis was done on 10-␮g aliquots of purified total RNA by
using a T7-(dT)24 primer (100 pmol/␮l). cDNA synthesis
reactions were executed by using components of the Superscript Choice kit (Invitrogen; Carlsbad, CA), with all incubations done in a Mastercycler Gradient thermocycler (Eppendorf; Westbury, NY). The resulting double-stranded cDNA
was quantified by use of a PicoGreen kit (Molecular Probes;
Eugene, OR). One microgram of double-stranded cDNA was
used in the in vitro transcription reaction employing biotinylated nucleotides and reagents provided in the BioArray
high yield RNA transcript labeling kits (Enzo Diagnostics;
Farmingdale, NY). The cRNA product was further purified by
use of RNeasy columns (Qiagen). The purified biotinylated
cRNA was then fragmented and subsequently hybridized to
Affymetrix rat genome U34A, B, and C arrays. All arrays
were analyzed by fluorescent intensity scanning according to
Affymetrix protocols (Affymetrix Expression Analysis Technical Manual). The hybridization and scanning of the arrays
were performed in the University of Missouri DNA Core
Facility (Columbia, MO).
GeneChip analysis. The rat genome U34 array set contained 26,388 probe sets. The present experiment interrogated the relative abundance of ⬃24,000 genes and EST
clusters (based on UniGene Build 34). Each probe set contained 16 perfectly matched (complementary) 25-mers, corresponding to different regions along the length of a transcript. Similarly, 16 mismatched pairs (encoding a single
mutated base) that did not perfectly complement a mRNA’s
sequence were used as a measure of nonspecific background
binding. Microarray Suite 5.0 software (Affymetrix) was
used, which employs a one-sided Wilcoxon’s signed-rank test
to calculate a P value reflecting the significance of differences
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human subjects (19). Delivery of exogenous IGF-I has
been shown to rescue muscle from sarcopenia in old
rodents (3, 8). Because many growth factors have been
shown to alter muscle growth (17), the hypothesis was
generated that a deficiency of multiple growth factors
(an unknown “growth factor milieu”) contributes to the
incomplete regrowth of skeletal muscle in old animals.
Another observation supporting a growth factor deficiency in old skeletal muscle is that a nearly complete
or complete muscle regrowth is observed after myotoxin application (7). The effect of myotoxin treatment
is to remove all muscle fiber protein and sarcolemma
(sparing satellite cells, basal lamina, and nerves),
which putatively releases growth factors for a massive
regeneration process (7). Welle (38) contends that, although skeletal muscle has the capacity to regenerate
itself, this process is not activated during the gradual
age-related loss of skeletal muscle. In support of the
aforementioned postulate, data from Fiatarone et al.
(15) suggest that weight lifting did induce regeneration
in the elderly, as evidenced by increased embryonic
myosin heavy chain expression but was not sufficient
to elicit muscle growth. Thus the hypothesis was generated that mRNAs for a subpopulation of growth
factors would be differentially expressed between the
regrowing soleus muscle of young rats and the nonregrowing soleus muscles of old rats. Affymetrix microarrays containing ⬎24,000 genes and expressed sequence tags (ESTs) were employed to provide a global,
unbiased determination of differential mRNA expression. To produce a muscle atrophied by inactivity for
testing regrowth, young and old rats had their hindlimbs immobilized for 10 days, which resulted in a
27–37% atrophy of the soleus muscle. Changes in gene
expression were then determined from soleus muscle
samples at postimmobilization days 3, 6, 10, and 30. By
doing a comprehensive mRNA assay, future hypotheses can be generated and subsequently tested, such as
whether there is a group of genes involved in a common
senescence program that prevent muscle growth that
could potentially be altered, whether there are unidentified growth factors critical to muscle growth that are
lacking in old muscle recovery, or whether there are
potential molecular targets that can be manipulated to
enhance growth of sarcopenic muscle or prevent sarcopenia altogether.
GENE EXPRESSION CHANGES IN ATROPHIED SOLEUS MUSCLE
1
The supplementary materials for this article are available online
at http://jap.physiology.org/cgi/content/full/00500.2003/DC1.
J Appl Physiol • VOL
Statistical methods. A three-factor ANOVA was employed
to compare the signal values of young and old soleus groups
(immobilized/controls) after 0, 3, 6, 10, and 30 days postimmobilization. Furthermore, a false discovery rate (FDR) adjusted P ⫽ 0.01 was applied to correct for the multiple
ANOVAs performed on 13,916 probe sets that had been detected as present. As rats were killed in groups of five over a
40-day period, a one-way ANOVA was performed to determine
whether any one control group differed from the other
groups. Groups of young and old rats were analyzed separately. For real-time PCR analysis, the differences in ⌬⌬CTs
were analyzed with a 2 ⫻ 2 ANOVA at each time point with
P ⬍ 0.05 set as significant for individual comparisons between different experimental groups (young control, young
immobilized/recovery, old control, and old immobilized/recovery).
Database searching. The target sequences for the probe
sets that differed significantly were analyzed with nucleotide
BLAST analysis to identify known genes and to determine
significant gene homologies with other species (http://
www.ncbi.nlm.nih.gov/BLAST/). The target sequence is the
region of a given gene or EST that was probed by the RG U34
arrays. Further information about a given sequence and its
homologs and orthologs was procured from the Locuslink,
Homologene, OMIM, mouse genome, rat genome, NetAffx,
and Proteome databases (http://www.ncbi.nlm.nih.gov/
LocusLink/, http://www.ncbi.nlm.nih.gov/HomoloGene/, http://
www.ncbi.nlm.nih.gov/entrez/, http://www.informatics.jax.org/,
http://rgd.mcw.edu/, https://www.affymetrix.com/analysis,
https://www.incyte.com/proteome/databases). A gene’s biological processes and molecular functions were classified by the
defined gene ontologies given in the aforementioned databases.
RESULTS
After 10 days of hindlimb immobilization, old and
young rats lost comparable percentages of soleus mass:
27 and 37%, respectively (Pattison et al., unpublished
observations). However, 30 days of recovery after the
cessation of immobilization, young animals fully recovered their soleus mass to preatrophy size, whereas old
animals recovered no significant muscle mass. Old
soleus muscle mass did not differ between recovery
days 0 and 30 (P ⫽ 0.948) (Fig. 1).
A FDR adjustment for multiple testing was used,
with the FDR taken to be 0.01. In this set of tests, the
FDR level corresponded to an unadjusted P ⬍ 4.6 ⫻
10⫺5. There were 64 probe sets that showed P ⬍ 4.6 ⫻
10⫺5 in testing the overall model in the three-way
ANOVA. With the use of an FDR adjustment for multiple testing at a level of 0.01, one would predict ⬃1
false positive out of the 64 significant differences.
Three examples of differentially expressed mRNAs
were selected for discussion, and their results are presented next. Elfin mRNA was less in the old rats at the
6th, 10th, and 30th recovery day (Figs. 2). Amphiregulin expression showed a unique spike in expression
(⬃2-fold) in young soleus muscles after 10 days of
reloading but no increase during recovery in the old
soleus muscle (Fig. 3). Clusterin mRNA expression
showed no change at any recovery time point in the
soleus muscle of young rats while displaying increased
expression in old soleus muscle at the 3rd, 6th, and
10th days of recovery (Fig. 4). Changes in mRNA ex-
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between the perfectly matched and mismatched probe pairs,
on the basis of their fluorescence intensities. The resulting P
values were used as a qualitative assessment of the ability to
detect a given transcript, where P values ⬍ 0.04 were called
“Present,” P values between 0.04 and 0.06 were called “Marginal,” and P values ⬎ 0.06 were called “Absent.” Only probe
sets that were Present or Marginal in ⱖ60% of the samples
for an experimental group were analyzed statistically. In
total, 13,916 probe sets were sufficiently detected in at least
one of the experimental groups, i.e., young or old soleus
muscles. Microarray Suite 5.0 software (Affymetrix) utilizes
statistically based algorithms to determine transcript abundance on the basis of fluorescence intensities (termed “signal”). The signal for each probe set was calculated as the
one-step biweight estimate of the combined differences of all
of the probe pairs in a probe set. All array data were normalized by scaling to target signal of 150. The calculated signal
value was the variable utilized for all subsequent statistical
analyses. Fold changes were calculated by dividing the mean
signal intensities of the groups to be compared. In the past,
microarray data analyses have been criticized as being “quite
elusive about measurement reproducibility” (11). However,
Bakay et al. (2) have reported that experimental error among
Affymetrix microarrays is not a significant source of unwanted variability in expression profiling experiments (R2 ⫽
0.979). Duplicate arrays were found to have small variability
(R2 ⫽ 0.981) in the present experiment. Microarray data are
available on the GEO database, in MIAME format (see Supplemental Data Tables 1 and 21).
Real-time PCR. Total RNA samples were purified over an
RNeasy column (Qiagen). Total RNA was reverse transcribed
into double-stranded cDNA during the previous cDNA synthesis for array sample processing with a T7-oligo(dT)24
primer. The resulting double-stranded cDNA was quantified
by use of a PicoGreen kit (Molecular Probes). All 5⬘ nuclease
assays consisted of reactions containing 25 ng of cDNA, 250
nM MGB probe, 900 nM primers and Taqman7 Universal
PCR Master Mix (ABI), in a 25 ␮l volume in duplicate with
an ABI Prism 7000 sequence detection system. If a duplicate
contained a range of ⬎0.3 cycle time (CT), it was reassayed.
All probe/primer combinations were designed using PrimerExpress 2.0 (ABI) (Primer/probe sequences are in Supplementary Table 31). Real-time PCR data were analyzed for
relative changes in expression by use of the ⌬⌬CT method,
according to User Bulletin no. 2 ABI PRISM 7700 sequence
detection system. Relative efficiency plots were run to validate use of the ⌬⌬CT method, where all slopes were ⬍0.1. All
targets were normalized to p38 mRNA expression. The expression of p38 was examined with an ANOVA on the factor
combinations, examining the pairwise combinations of interest (young recovery vs. old recovery on days 3, 6, 10, and 30).
Expression of p38 remained unchanged in the pairwise combinations of interest. Data are expressed as the calculated
fold differences between different experimental groups
(young control, young immobilized, old control, and old immobilized). Because of the limited amount of RNA isolated,
some samples were exhausted before real-time PCR analysis,
whereby the entire old 6-day control group was depleted;
thus old controls from adjacent time points (3- and 10-day old
controls) were used for statistical analyses of the gene expression changes at the 6th day of recovery. All other groups
had n ⫽ 3–5 for real-time analyses.
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GENE EXPRESSION CHANGES IN ATROPHIED SOLEUS MUSCLE
Fig. 3. Amphiregulin mRNA expression is elevated only in young
soleus muscles at 10 days of recovery compared with old. Values are
means ⫾ SE; n ⫽ 5 rats/group. * Actual P ⬍ 4.6 ⫻ 10⫺5 from
age-matched control.
Numerous clinical conditions produce skeletal muscle wasting, each employing various combinations of
physical inactivity, poor nutrition, increased cytokines,
decreased anabolic factors, denervation, and reduced
blood flow. Examples of these clinical conditions are
catastrophic illness (cancer, chemotherapy, AIDS, etc.)
(18), aging anorexia (18), hip fracture (29), and physical inactivity such as limb immobilization (8). Unfortunately, the recovery of muscle mass from these conditions seems to be limited in older humans. For example, the postoperative strength of 77-yr-old patients
was impaired compared with 36-yr-old patients after
surgery, in part because of diminished muscle mass
(37). Significant ipsilateral quadriceps muscle atrophy
(predominantly in type 2B and 2A fibers) occurred in
64-yr-old patients with chronic osteoarthritis of the
hip. After total hip arthroplasty and rehabilitation, the
wasting persisted 5 mo postoperatively judged by muscle ultrasound and strength measurements (35). Dependence on others to perform lower extremity physical activities of daily living tripled from prefracture
level, after hip fracture, in 81-yr-old patients; this
dependence remained doubled 2 yr postfracture (29).
Depending on the population studied and function being assessed, an estimated 25–75% of those who are
independently living before a fracture can neither walk
independently nor achieve their previous level of independent living within 1 yr after their fracture (see Ref.
29 for references). The outcome of muscle wasting is
low muscle strength, which Metter et al. (30) observed
is associated with increased mortality, and they further stated that it was presumably a result of low
muscle mass, which they did not measure. Here, we
sought to examine differences in gene expression of
young growing and old nonregrowing muscles independent of any clinical condition, thereby allowing us to
examine only effects that are age induced on failed
muscle regrowth from atrophy.
Fig. 2. Elfin mRNA values in the soleus muscle are greater in young
than old rats on the 6th, 10th, and 30th days of recovery from 10 days
of hindlimb immobilization. Values are means ⫾ SE; n ⫽ 5 rats/
group. * Actual P ⬍ 4.6 ⫻ 10⫺5 from age-matched control.
Fig. 4. Clusterin expression is elevated in old soleus muscles from
days 3–10 of recovery, whereas no changes were observed in young.
Values are means ⫾ SE; n ⫽ 5 rats/group. * Actual P ⬍ 4.6 ⫻ 10⫺5
from age-matched control.
pression for Elfin, amphiregulin, and clusterin were
confirmed by real-time PCR (Table 1).
DISCUSSION
J Appl Physiol • VOL
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Fig. 1. Failure of soleus muscles from old rats (B) to recover mass
lost during 10 days of hindlimb immobilization (Imm), when the
soleus muscles of young rats (A) show a full recovery to preatrophy
mass from the same treatment. Wet weight of the soleus muscle
(grams) is plotted as a function of recovery days after ending 10 days
of hindlimb immobilization. Values are means ⫾ SE; n ⫽ 5 rats/
group. *P ⬍ 0.05.
GENE EXPRESSION CHANGES IN ATROPHIED SOLEUS MUSCLE
Table 1. Real-time PCR confirmed the microarray
data that Elfin and amphiregulin mRNAs were
significantly greater in the soleus muscles of young
than the old rats whereas clusterin mRNA levels
were significantly less in the young
Array Data
Time Point
Groups
Fold
6 days
10 days
30 days
YI vs. OI
YI vs. OI
YI vs. OI
⫺1.56
⫺1.67
⫺1.99
10 days
YI vs. OI
3 days
6 days
10 days
YI vs. OI
YI vs. OI
YI vs. OI
Real-time Data
P value
Fold
P value
3.88E-08
4.58E-10
3.33E-16
⫺1.44
⫺1.62
⫺1.90
0.033
0.004
⬍0.001
⫺3.41
0.002
2.42
2.30
1.99
⬍0.001
⬍0.001
⬍0.001
Elfin
Amphiregulin
⫺3.61
5.53E-10
Clusterin
2.24
2.33
2.99
5.49E-13
6.39E-08
8.14E-12
In the present study, an animal model of hindlimb
immobilization was employed to produce atrophy of the
soleus muscle for the purposes of testing the hypothesis that mRNAs for various growth factors would be
differentially expressed between the regrowing soleus
muscles of young rats and the nonregrowing soleus
muscles of old rats after the immobilization was removed. The hypothesis was based on the failure of the
soleus muscle of old rats to regrow after atrophy from
hindlimb immobilization (8, 39), whereas young muscles did successfully regrow after immobilization (5, 9,
Fig. 1 of present study). Growth factors were selected
for the hypothesis because two groups have shown
rescue of old skeletal muscle from atrophy by addition
of exogenous IGF-I (3, 8), whereas the upregulation of
endogenous growth factors does not appear to improve
muscle growth (19). It was, therefore, deduced that an
undefined growth factor milieu might be missing
within the old, atrophied skeletal muscle and that
microarrays could be employed as a global screen to
identify potential genes for future study. Microarrays
with ⬃24,000 genes and ESTs were employed for an
unbiased assessment of the hypothesis. The strategy
was that identification of differentially expressed
mRNAs between regrowing young and nonregrowing
old soleus muscles would provide unexpected new candidates for growth-stimulatory factors missing in the
soleus muscle of old rats but present in young or
inhibitory factors present in old that were absent in
young.
Elfin mRNA was higher in the soleus muscle of
young than old rats on recovery days 6, 10, and 30 (Fig.
2). Elfin (previously named CLIM1) is a member of the
J Appl Physiol • VOL
Enigma family of cytoplasmic proteins that contain a
NH2-terminal PDZ domain and a series of COOHterminal LIM domains (25). COOH-terminal LIM domain proteins are associated with the cytoskeleton and
act as modular protein-binding interfaces mediating
protein-protein interactions in the cytoplasm and nucleus by influencing the localization and activity of its
specific protein partners (21, 23). In a blot of 16 human
tissues, Elfin mRNA was expressed highest in the
heart and next highest in skeletal muscle (26). Elfin
colocalizes with ␣-actinin at Z-discs in the human myocardium (24), is expressed throughout the developing
heart at embryonic day 8.5 (25), and colocalizes with
actin stress fibers in C2C12 myoblasts (25). An emerging concept in muscle cell biology is that cytoskeletalassociated proteins may serve as molecular messengers that enable muscle cells to sense load or stretch
and then signal a physiological response (10, 13). Elfin
could be such a molecule. From the above context, we
hypothesize that limited signaling from the Z-disc in
the soleus muscle of old rats recovering from atrophy
may contribute to its insufficient regrowth.
Amphiregulin mRNA was significantly increased in
the soleus muscle at the 10th recovery day in only the
young, but not in the old, rats (Fig. 3). Amphiregulin,
also known as schwannoma-derived growth factor, is
an autocrine growth factor as well as a mitogen for
astrocytes, Schwann cells, and fibroblasts. Amphiregulin is also a mitogen for adult neural stem cells (15).
Nerve terminal disruption, exposed junctional folds,
and postsynaptic areas that contained little or no
postjunctional folds were present at the neuromuscular junction of the soleus muscle on the 5th day of
hindlimb immobilization, suggesting neural plasticity
(14). Neuromuscular remodeling occurs in old skeletal
muscle (6). Therefore, considering the above information, the lack of increase in amphiregulin mRNA in the
old soleus muscle sets up the hypothesis for future
studies that insufficient amphiregulin gene expression
contributes to the failure of old skeletal muscle to
recover from immobilization-induced atrophy because
of insufficient neural neutrophic factors.
Whereas amphiregulin mRNA was significantly
greater in the soleus muscle of young than in old rats at
the 10th recovery day, the age effect was reversed for
clusterin mRNA. Old rats had significantly more clusterin mRNA in their soleus muscles during recovery
than young rats (Fig. 4). Peak expression of clusterin
mRNA (⬃3-fold increase) was observed at 3 days of
recovery and remained significantly elevated through
10 days of recovery in old soleus muscle recovery.
Conversely, young soleus muscles showed no significant changes in clusterin expression from control levels
in any of the time points measured. Clusterin (also
known as complement lysis inhibitor, SP-40, sulfated
glycoprotein 2, testosterone-repressed prostate message 2, and apolipoprotein J) is a heterodimeric-conserved glycoprotein that is expressed in a wide variety
of tissues and found in all human fluids (36). Its precise
function is still uncertain, although it has been implicated in several diverse physiological processes such as
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YI, young immobilized/recovery; OI, old immobilized/recovery. For
fold change and unadjusted P value for the microarray data for the
groups compared, P ⬍ 4.6 ⫻ 10⫺5 was significant. For fold change
and unadjusted P values resulting from real-time PCR analyses for
the groups compared, P ⬍ 0.05 was considered significant. Fold
changes are compared relative to the young-age groups. A negative
fold change indicates that the old value was significantly less than
the young’s respective value, and a positive fold change indicates
that the old was significantly greater than the young.
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GENE EXPRESSION CHANGES IN ATROPHIED SOLEUS MUSCLE
Table 2. Probe sets that had significant differences in expression as determined by a 3-factor ANOVA
Unadjusted
P Values
EST weakly similar to rat plateletderived growth factor receptor-␣
AI171772
4.9E-18
EST similar to mouse glutathione
transferase zeta 1
(maleylacetoacetate isomerase)
Myosin heavy chain IIb
AI169075
4.9E-17
L24897
1.2E-15
EST function unknown
EST similar to human CGI-38, brainspecific protein
Serine protease inhibitor (Spin2b)
EST similar to mouse tropomyosin 2␤
AI227690
AI171466
1.7E-14
7.8E-13
M38566
AA858875
1.3E-12
3.8E-10
EST function unknown
Aldehyde oxidase
AI234712
AA926200
3.8E-10
6.7E-10
EST similar to mouse D-dopachrome
tautomerase
EST function unknown
EST similar to rat carboxypeptidase B1
AA945149
5.1E-09
AA818271
AI230199
7.8E-09
1E-08
EST function unknown
PDZ and LIM domain 1 (elfin)
EST weakly similar to rat cytochrome
P450, subfamily 2A, polypeptide 1
EST function unknown
Metallothionein (Mt1a)
AA858817
U23769
H31125
1.4E-08
1.6E-08
4.1E-08
AI105451
M11794
1.7E-07
1.9E-07
Interleukin 15
U69272
2.4E-07
EST similar to mouse metallothionein 2
Glutamine synthetase (glutamateammonia ligase)
AI176456
M91652
3E-07
5.5E-07
EST function unknown
EST highly similar to mouse
tropomyosin 3␥
Glutamine synthetase (glutamateammonia ligase)
AI137344
AI170847
5.8E-07
7.6E-07
AI232783
1.5E-06
EST function unknown
Clusterin (complement lysis inhibitor,
SP-40,40, sulfated glycoprotein 2,
testosterone-repressed prostate
message 2, apolipoprotein J)
EST similar to mouse pre-B-cell
colony-enhancing factor
EST similar to mouse FXYD domaincontaining ion transport regulator 1
(phospholemman)
Janus kinase 2 (a protein tyrosine
kinase)
AI177033
M64733
1.9E-06
1.9E-06
AI177755
2.2E-06
AA799645
2.5E-06
U13396
2.7E-06
EST function unknown
EST function unknown
Phospholipase A2, group IIA
(platelets, synovial fluid)
AI013978
AI172352
X51529
3.1E-06
3.8E-06
4.3E-06
Metallothionein 3
AA924772
5E-06
Functions as Curated From Gene Ontology Databases
ATP binding, antigen binding, protein amino acid
phosphorylation, transmembrane receptor protein tyrosine
kinase signaling pathway, organogenesis, cell proliferation,
signal transduction, platelet-derived growth factor, ␣-receptor,
cell surface receptor linked signal transduction
Enzyme, isomerase, transferase, tyrosine catabolism, glutathione
transferase, phenylalanine catabolism, maleylacetoacetate
isomerase, glutathione peroxidase, amino acid metabolism
Muscle motor activity, myosin ATPase activity, muscle myosin,
actin binding, striated muscle contraction, cytoskeleton
organization and biogenesis, muscle development
EST function unknown
Gene function unclear/unknown
Serpin
Actin binding, muscle contraction, muscle development, muscle
thin filament tropomyosin, structural protein of muscle
EST function unknown
Aldehyde oxidase, inflammatory response, xanthine
dehydrogenase, oxygen and reactive oxygen species metabolism,
oxidoreductase, electron transport, molybdenum binding
Isomerase, melanin biosynthesis from tyrosine
EST function unknown
Carboxypeptidase A, proteolysis and peptidolysis, protein
degradation
EST function unknown
Response to oxidative stress, intracellular signaling cascade
monooxygenase, electron transport
EST function unknown
Heavy metal binding, heavy metal resistance, response to heavy
metal, heavy metal ion transport, cell stress
Cytokine, immune response, cell proliferation, cell-cell signaling,
signal transduction, positive regulation of cell proliferation
Heavy metal binding, copper homeostasis
Glutamate-ammonia ligase, glutamine biosynthesis, regulation of
neurotransmitter levels, amino acid metabolism, ligase,
nitrogen fixation, nitrogen metabolism
EST function unknown
Actin binding, muscle contraction
Glutamate-ammonia ligase, glutamine biosynthesis, regulation of
neurotransmitter levels, amino acid metabolism, ligase,
nitrogen fixation, nitrogen metabolism
EST function unknown
Lipid metabolism, plasma glycoprotein, complement activation,
fertilization (sensu Animalia), reproduction, anti-pathogen
response, cell death
Cytokine, cell-cell signaling, signal transduction, positive
regulation of cell proliferation
Ion transport, chloride channel, chloride transport, muscle
contraction
ATP binding, protein tyrosine kinase, cell motility, JAK-STAT
cascade, mesoderm development, regulation of cell cycle,
intracellular signaling cascade, protein amino acid
phosphorylation, cell death/apoptosis, control of cell
proliferation, cell differentiation
EST function unknown
EST function unknown
Oncogenesis, phospholipase A2, tumor suppressor, calciumdependent secreted phospholipase A2, hydrolase, lipid
catabolism, calcium ion binding, regulation of cell growth
Heavy metal binding, neurogenesis, cell proliferation, metal ion
homeostasis
Continued
J Appl Physiol • VOL
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Accession
No.
Gene Name
GENE EXPRESSION CHANGES IN ATROPHIED SOLEUS MUSCLE
2177
Table 2.—Continued
Gene Name
Accession
No.
Unadjusted
P values
Functions as Curated From Gene Ontology Databases
EST similar to mouse chemokine
(C-X-C motif) ligand 14
Homer, neuronal immediate early
gene, 2
FXYD domain-containing ion
transport regulator 6
(phosphohippolin)
EST function unknown
EST similar to human cytidine
deaminase
Calmodulin 1 (phosphorylase
kinase ⌬)
EST similar to mouse mitochondrial
ribosomal protein L9
EST similar to human myosin light
chain 1 slow a
Microtubule-associated proteins
1A/1B light chain 3
EST similar to mouse testis expressed
gene 189 (also known as MORFrelated gene 15)
EST similar to rat A kinase (PRKA)
anchor protein (yotiao)
AA874803
5.1E-06
AB007690
5.1E-06
AI179595
8.7E-06
AI228301
AA859352
9.7E-06
1.1E-05
X13933
1.1E-05
AA956460
1.2E-05
AI230220
1.2E-05
U05784
1.3E-05
Structural constituent of muscle, calcium ion binding activity,
muscle development, muscle myosin, myosin
Gene function unclear/unknown
Al137864
1.4E-05
Chromatin modeling, chromatin remodeling complex
X62951
1.5E-05
EST similar to human methylmalonyl
CoA epimerase
Retroviral-like ovarian specific
transcript 30-1 mRNA
EST weakly similar to mouse
ribosomal protein L23
Fumarylacetoacetate hydrolase
AI172417
1.6E-05
Centrosome, cytoskeleton, protein binding, signal transduction,
synaptic transmission, small molecule transport, N-methyl-Daspartate receptor-associated protein, anchor protein, neuronal
transmission
Gene function unclear/unknown
U48828
1.7E-05
Gene function unclear/unknown
AA944741
1.8E-05
Protein biosynthesis, cytosolic ribosome (sensu Eukarya)
M77694
1.8E-05
Collagen, type 1, ␣1
Z78279
1.8E-05
Insulin-like growth factor binding
protein 3
M31837
1.8E-05
Crystallin, ␮
AI233209
1.9E-05
Metallothionein (Mt1a)
AI102562
2E-05
EST similar to mouse tropomyosin 3␥
EST function unknown
Lipopolysaccharide binding protein
AI170775
AI172057
L32132
2.2E-05
2.3E-05
2.7E-05
EST highly similar to mouse myosin,
heavy polypeptide 1, skeletal
muscle, adult (MHC2X/D)
EST weakly similar to human
aldehyde dehydrogenase 1 family,
member B1
EST weakly similar to human
transducin-like enhancer of split 1
[E(sp1) homolog, Drosophila]
EST function unknown
Proteasome (prosome, macropain) 26S
subunit, ATPase 2
S68736
2.7E-05
Metabolism, fumarylacetoacetase, tyrosine catabolism, amino
acid metabolism, phenylalanine catabolism
Collagen, cell adhesion, extracellular matrix, skeletal
development, epidermal differentiation, structural protein of
bone, extracellular matrix component
Insulin-like growth factor binding, signal transduction, cell
migration/motility, growth factor binding, regulation of cell
growth
Sensory organ development, vision, ornithine cyclodeaminase,
photoreception
Heavy metal binding, heavy metal resistance, response to heavy
metal, heavy metal ion transport, cell stress
Actin binding, muscle contraction
EST function unknown
Lipopolysaccharide binding, acute-phase response, cellular
defense response, response to pathogenic bacteria, lipid
binding, lipid transport, xenobiotic metabolism
Cytoskeleton organization and biogenesis, muscle motor, muscle
myosin
AA998174
2.8E-05
Aldehyde dehydrogenase, carbohydrate metabolism
AA875084
2.8E-05
Development, signal transduction, histogenesis and
organogenesis, cell fate specification
rc_AI014132at
D50694
3.1E-05
3.4E-05
Ceruloplasmin
AI010470
3.4E-05
EST similar to mouse laminin ␥1
AI071644
3.5E-05
EST function unknown
Amphiregulin (schwannoma-derived
growth factor)
EST function unknown
AA875032
X55183
3.8E-05
3.9E-05
EST function unknown
ATP binding, nucleotide binding, virulence, 26S proteasome,
adenosinetriphosphatase, proteolysis and peptidolysis, protein
degradation
Ferroxidase, copper binding, iron homeostasis, copper
homeostasis, oxidoreductase
Chemotaxis, cell adhesion, signal transduction, extracellular
matrix, endoderm development, protein complex assembly
EST function unknown
Growth factor, cell proliferation, cell-cell signaling, cytokine
AA925603
4.6E-05
EST function unknown
EST function unknown
Cytidine deaminase, nucleobase, nucleoside, nucleotide and
nucleic acid metabolism
Calcium ion binding, protein binding, G-protein-coupled receptor
protein signaling pathway, cell cycle
Structural constituent of ribosome
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J Appl Physiol • VOL
Cytokine, chemokine, immune response, chemotaxis, cell-cell
signaling, signal transduction, response to injury
Metabotropic glutamate receptor signaling pathway, actin
binding, protein binding
Ion transport
2178
GENE EXPRESSION CHANGES IN ATROPHIED SOLEUS MUSCLE
J Appl Physiol • VOL
We thank Dr. Gary Allen for providing access to the Bioinformatics Consortium at the University of Missouri and Dr. Mark McIntosh
for leadership in establishment of the Affymetrix Core facility at the
University of Missouri. We also thank Dr. Espen Spangenburg for
thoughtful discussions and Aaron Wheeler for assistance in database
searching.
DISCLOSURES
The research was supported by National Institute on Aging Grant
AG-18881.
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developmental remodeling, apoptotic disease states as
well as neurodegeneration in Alzheimer’s disease,
response to injury, and other stresses (22). Rat clusterin is expressed at high levels in dying cells, suggesting an involvement in the process of cell death, with a
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Other mRNAs identified as differentially expressed
fell into a host of functional categories (listed in Table
2). Other known growth factor-related mRNAs identified with three-factor interactions included interleukin-15, IGF-binding protein-3, an EST similar to
mouse pre-B-cell colony-enhancing factor, and an EST
weakly similar to rat PDGF receptor-␣. However, some
of the aforementioned growth factor-related mRNAs
seemed to show more of an atrophy-specific effect than
a true recovery effect. The atrophy-specific effects have
been reported in detail elsewhere (Pattison et al., unpublished observations). The hypothesis of the present
study that a group of “growth factor milieu” mRNAs
would be differentially regulated between young, regrowing and old soleus muscle failing to regrow was
not conclusively supported (Table 2). However, the
failure to find a disproportionate number of growth
factor mRNAs should not be interpreted to mean that
these are unimportant in soleus muscle regrowth, because changes in only a few critical growth factors
could ameliorate the failed muscle regrowth of old
soleus muscles after hindlimb immobilization.
In summary, the present analysis identified 64 new
candidates, including 38 ESTs, whose inappropriate
gene expression could play some role in the failure of
old skeletal muscle to regrow to its preatrophy mass
after ending immobilization. Among these, the growth
factor amphiregulin supports our initial hypothesis
that a deficiency of multiple growth factor mRNAs (an
unknown growth factor milieu) contributes to the incomplete regrowth of skeletal muscle from inactivityinduced atrophy in old animals. Future work could
show roles for Elfin between regrowing young muscle
and nonregrowing old muscle gene expression in maintenance and growth of muscle mass. Finally, the upregulation in old skeletal muscle of clusterin mRNA,
whose protein has been associated with increased cell
death, could contribute to the poor regrowth of old
atrophied muscle.
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