Frank W. Booth, Wei Lou, Marc T. Hamilton and Zhen Yan
J Appl Physiol 81:1941-1945, 1996.
You might find this additional information useful...
This article has been cited by 3 other HighWire hosted articles:
PGAM-M expression is regulated pretranslationally in hindlimb muscles and under altered
loading conditions
R. Kell, H. Pierce and S. J. Swoap
J Appl Physiol, January 1, 1999; 86 (1): 236-242.
[Abstract] [Full Text] [PDF]
Role of local contractile activity and muscle fiber type on LPL regulation during exercise
M. T. Hamilton, J. Etienne, W. C. McClure, B. S. Pavey and A. K. Holloway
Am J Physiol Endocrinol Metab, December 1, 1998; 275 (6): E1016-E1022.
[Abstract] [Full Text] [PDF]
Chronic fatigue syndrome
J B Wright and D W Beverley
Arch. Dis. Child., October 1, 1998; 79 (4): 368-374.
[Full Text]
Updated information and services including high-resolution figures, can be found at:
http://jap.physiology.org/cgi/content/full/81/5/1941
Additional material and information about Journal of Applied Physiology can be found at:
http://www.the-aps.org/publications/jappl
This information is current as of May 19, 2009 .
Journal of Applied Physiology publishes original papers that deal with diverse areas of research in applied physiology, especially
those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American
Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society.
ISSN: 8750-7587, ESSN: 1522-1601. Visit our website at http://www.the-aps.org/.
Downloaded from jap.physiology.org on May 19, 2009
Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl
on the following topics:
Biochemistry .. C-type Cytochromes
Biochemistry .. Muscle Proteins
Physiology .. Exertion
Medicine .. Muscular Atrophy
Medicine .. Exercise
Physiology .. Rats
Cytochrome c mRNA in skeletal
muscles of immobilized limbs
FRANK W. BOOTH, WEI LOU, MARC T. HAMILTON, AND ZHEN YAN
Department of Integrative Biology, University of Texas Medical School, Houston, Texas 77030
atrophy; mitochondria; gene expression
MANY INDIVIDUALS EXPERIENCE the fixation of joints with
plaster casts to allow injured bones, ligaments, and
tendons to heal. A common secondary effect of this
treatment is skeletal muscle atrophy (21), which produces decreased muscle strength. Atrophied muscles
also have a reduction in mitochondrial concentration
(3), which contributes to decreased endurance capacity
for low- and moderate-intensity work (16). Most of our
knowledge about the mitochondrial response to limb
immobilization comes from muscles fixed in a neutral
(i.e., resting) or shortened position (see Refs. 1, 4), as
briefly summarized below. After 10 days of limb immobilization, in both fast- and slow-twitch muscles the
capacity of whole muscle homogenates to oxidize pyruvate, b-hydroxybutyrate, palmitate, or glucose is reduced (22). A final common pathway for these substrates is
their oxidative phosphorylation in mitochondria. The functional consequence of these events is that atrophied muscles
in immobilized limbs had lower ATP levels and an apparent increase in their dependence on anaerobic metabolism,
as reflected by the more extensive depletion of glycogen
and enhanced lactate production during continuous contractile activity (31).
The length of a skeletal muscle in an immobilized
limb determines, in part, whether muscle atrophy
occurs. When muscles are fixed at a neutral or shortened position, they atrophy (5, 12, 28, 29). On the other
hand, lengthening of skeletal muscle beyond its neutral
length during limb fixation either attenuates or prevents muscle atrophy, and in some cases produces
hypertrophy (3, 12, 26–29).
Appell (1) has hypothesized that an early decrease in
mitochondrial concentration could be of etiological importance in the initiation of atrophy of skeletal muscle
in immobilized limbs. Appell’s hypothesis predicts that
mitochondrial concentration would not decrease in
lengthened slow muscles because they do not atrophy. A
hypothesis in our present study is that lengthening
would attenuate muscle atrophy but would not prevent
the loss of cytochrome c mRNA in the soleus muscle of
immobilized limbs [contrary to the hypothesis of Appell
(1)]. The rationale for our hypothesis is that mitochondrial concentration in skeletal muscle has been shown
to be correlated with the daily duration of muscle
contraction (8, 10), and because lengthened immobilized muscles do not undergo isotonic contractions,
cytochrome c mRNA concentration would decrease in
muscles fixed in a lengthened position.
Cytochrome c concentration in skeletal muscle has
been shown to be positively correlated with another
mitochondrial protein marker, citrate synthase (10).
We have used cytochrome c expression as an index for
the loss of mitochondria in skeletal muscle that occurs
during limb immobilization. Cytochrome c protein and
mRNA per whole muscle decrease 40% during the first
week of limb immobilization in the shortened position
in young rats (19). These observations imply that a
change in the pretranslational control of cytochrome c
plays a role in its decrease in immobilized limbs.
However, little further molecular information on the
loss of cytochrome c mRNA is available. In a concurrent
study, Yan et. al (32) noted that muscles with less
cytochrome c mRNA had more RNA-protein interaction
in the 38-untranslated region (38-UTR) of cytochrome c
mRNA. Thus we hypothesized that a decrease in contractile activity by limb immobilization would increase
RNA-protein interaction in the 38-UTR of cytochrome c
mRNA.
MATERIALS AND METHODS
Animals. Pathogen-free Sprague-Dawley female rats were
obtained from Harlan (Houston, TX) and assigned randomly
at 7–8 wk of age to control group and hindlimb immobilization groups with ankles fixed in either plantar flexion or
dorsiflexion. Rats were anesthetized with 1.4 ml/kg of a
mixture of ketamine (54 mg/ml), xylazine (2.2 mg/ml), and
acepromazine (3.5 mg/ml) during the application of immobili-
0161-7567/96 $5.00 Copyright r 1996 the American Physiological Society
1941
Downloaded from jap.physiology.org on May 19, 2009
Booth, Frank W., Wei Lou, Marc T. Hamilton, and
Zhen Yan. Cytochrome c mRNA in skeletal muscles of
immobilized limbs. J. Appl. Physiol. 81(5): 1941–1945, 1996.—
Even though immobilization of a slow skeletal muscle in a
lengthened position prevents muscle atrophy, it is unknown
whether this treatment would prevent a decrease in mitochondrial quantity. We found that, regardless of muscle length in
immobilized limbs, the mRNA of a marker for mitochondrial
quantity, cytochrome c, decreased. Cytochrome c mRNA per
milligram of muscle was 62 and 72% less 1 wk after fixation of
the soleus muscle in shortened and lengthened positions,
respectively, than age-matched controls. Cytochrome c mRNA
per milligram wet weight was 36 and 32% less in the tibialis
anterior muscle fixed for 1 wk in the shortened and lengthened positions, respectively, compared with age-matched
controls. Recently, in the 38-untranslated region of cytochrome c mRNA a novel RNA-protein interaction that
decreases in chronically stimulated rat skeletal muscle was
identified.[Z. Yan, S. Salmons, Y. L. Dang, M. T. Hamilton,
and F. W. Booth. Am. J. Physiol. 271 (Cell Physiol. 40): C1157–
C1166, 1996]. The RNA-protein interaction in the 38-untranslated region of cytochrome c mRNA in soleus and tibialis
anterior muscles was unaffected by fixation in either shortened or lengthened position. We conclude that, whereas
lengthening muscle during limb fixation abates the loss of
total muscle protein, the percentage decrease in cytochrome c
mRNA is proportionally greater than total protein. This
suggests that the design of countermeasures to muscle atrophy should include different exercises to maintain total
protein and mitochondria.
1942
CYTOCHROME
C
IN ATROPHIED MUSCLE
tubes with UV light (0.12 J) for 5 min in a Stratalinker
chamber (Stratagene). After being incubated in Laemmli
sample-loading buffer at 95–100°C for 3 min, the reaction
mixtures were subjected to electrophoresis on 10% sodium
dodecyl sulfate-polyacrylamide gels together with prestained
molecular-weight markers (Sigma Chemical). The gels were
dried and placed in contact with Kodak X-OMAT film to
obtain the autoradiogram.
Statistics. Comparison of the values among different groups
was performed with analysis of variance. If a significant
difference was found, the Tukey-Kramer multiple-range test
was employed. For protein concentration, a two-tailed Student’s t-test was employed. P , 0.05 was designated as
significant. Values are expressed as means 6 SE.
RESULTS
Soleus muscle wet weight and total RNA concentration (µg RNA/mg wet wt) were the same as control
values, and the soleus muscle-to-body weight ratio was
increased by 39% (P , 0.05) at the end of the 7-day
fixation of the muscle in a lengthened position (ankle is
immobilized in dorsiflexion) (Table 1). However, soleus
muscle wet weight, total RNA concentration, and muscleto-body weight ratio were 55, 29, and 44% less (P ,
0.05), respectively, than control after 7 days of fixation
in a shortened position (plantar flexion) (Table 1).
Although Babij and Booth (2) previously reported
that fixation of the soleus muscle in a shortened
position for 7 days did not alter its protein concentration, similar data for a 7-day fixation of rat muscle in a
lengthened position have not been previously published. An additional set of rats (n 5 5) was employed to
obtain this information. Fixation in a lengthened position did not alter soleus muscle protein concentration
(183.1 6 6.3 mg/g wet wt for control; 160.3 6 9.0 mg/g
wet wt for 7-day fixation; P 5 0.07, two-tailed Student’s
t-test) and content (15.0 6 1.1 mg/muscle for control;
12.9 6 0.8 mg/muscle for fixation; P 5 0.17). Previously,
Jokl and Konstadt (17) reported that myofibrillar and
sarcoplasmic protein concentration were unchanged in
cat fast- and slow-twitch skeletal muscle that had been
immobilized in the lengthened position for 4 wk.
Cytochrome c mRNA was less in soleus muscles fixed
in either a shortened or lengthened position. Cytochrome c mRNA per microgram RNA was 47 and 65%
less (P , 0.01) in shortened and lengthened positions,
respectively, after 7 days of immobilization compared
with the control group of the same age (Table 1, Fig. 1).
Cytochrome c mRNA concentration per milligram so-
Table 1. Cytochrome c mRNAs in soleus muscles fixed in lengthened or shortened position for 7 days
Position
Body Wt, g
Wet Wt, mg
Wet
Wt-to-Body
Wt Ratio
Control
Shortened (plantar flexion)
Lengthened (dorsiflexion)
154 6 2
121 6 1*
114 6 4*
62.8 6 5.3
28.3 6 2.1*
64.6 6 4.0‡
0.41 6 0.01
0.23 6 0.01*
0.57 6 0.01*‡
Cytochrome c mRNA
Total
RNA, mg/g
IOD/µg RNA
IOD/mg wet wt
IOD/whole muscle
1.7 6 0.1
1.2 6 0.1*
1.9 6 0.3‡
0.384 6 0.023
0.204 6 0.051*
0.133 6 0.011†
0.648 6 0.046
0.249 6 0.058*
0.179 6 0.035*
40 6 4
7 6 1*
12 6 2*
Values are means 6 SE. Muscle weights for each animal were average of both sides. For cytochrome c mRNA analysis, 2 soleus muscles from
a rat were pooled for an observation (control and lengthened groups), and 4 soleus muscles from 2 rats were pooled for a single observation
(shortened group). For control and lengthened groups, n 5 5; for shortened group, n 5 10 rats. Average body weight and soleus muscle weights
of 2 rats pooled for mRNA determination were used to calculate body and muscle weights. IOD, integrated optical density units. * P , 0.01
from control; † P , 0.05 from shortened group; ‡ P , 0.01 from shortened group.
Downloaded from jap.physiology.org on May 19, 2009
zation to both hindlimbs with plaster of paris (5) and at the
time of death.
Total protein. Total protein was determined with a detergent-compatible protein assay (Bio-Rad).
RNA isolation. RNA for Northern blot analysis was extracted from soleus and tibialis anterior (TA) muscles of three
groups of rats: ankles fixed for 7 days in plantar flexion,
ankles fixed for 7 days in dorsiflexion, or controls who were
killed at the end of immobilization according to a procedure
by Chomczynski and Sacchi (7) with RNAzol B (Biotecx
Laboratories) as described earlier (32).
Northern blot analysis. The coding region of the cytochrome c gene was subcloned into pBluescript SK (1) vector
(Stratagene). In brief, the BamH I-EcoR I fragment (958-base
pair) from pRC4 plasmid (25) was inserted into the polylinker
region of pBluescript SK (1) digested with BamH I and EcoR
I. The resulting plasmid pRC4 (Bluescript) was used for
Northern blot analysis of cytochrome c mRNA as described by
Babij and Booth (2). mRNA was quantified by image analysis
of the autoradiogram (BioImage, Millipore). Total RNA per
milligram wet weight was determined by the alkaline hydrolysis procedure of Munro and Fleck (20).
Cytoplasmic extract. Muscle homogenization was described
in Yan et al. (32). The homogenates were centrifuged at
15,000 g for 15 min at 4°C. The supernatant (S15) was
snap-frozen in liquid nitrogen and stored at 280°C until
analysis. The protein concentrations of S15 were determined
by the Lowry method (18).
Radiolabeled-RNA probe. For the cytochrome c 38-UTR
probe, pRC4CAT38 (9) was digested with Bgl II and BamH I
followed by ligation with T4 ligase into pBluescript SK (1). A
116-base pair fragment (11,337 to 11,452) was produced by
digestion with Dra I and in vitro transcription with T3
polymerase (Promega) and [a-32P]uridine 58-triphosphate
(3,000 Ci/mmol; ICN). After transcription at 37°C for 1 h,
RNA transcripts were digested with 1 unit of ribonucleasefree deoxyribonuclease (Promega) for 15 min at 37°C, extracted with phenol-chloroform, and precipitated in ethanol
with 20 µg Escherichia coli tRNA as carrier. The radiolabeled
RNAs were quantified, and their specific activities
(counts · min21 · µg21 ) were determined by liquid scintillation
counting after trichloroacetic acid precipitation (24).
Ultraviolet (UV) cross-linking. UV cross-linking assays
were performed according to Rondon et al. (23) with slight
modification as described earlier (32). In brief, cytoplasmic
extracts (15 µg protein) were incubated at 30°C for 10 min
with excess 1.5 ng 32P-labeled RNA probe (see above) in a
reaction mixture containing 10% glycerol and (in mM) 12
N-2-hydroxyethylpiperazine-N8-2-ethanesulfonic acid (pH
7.9), 15 KCl, 0.25 EDTA, 0.25 dithiothreitol, and 5 MgCl2, as
well as E. coli tRNA (200 ng/µl) in a total volume of 15 µl. The
reaction mixtures were placed on ice after ribonuclease T1
treatment (0.6 units, 37°C, 20 min) and irradiated in open
CYTOCHROME
C
1943
IN ATROPHIED MUSCLE
leus wet weight was 62 and 72% less (P , 0.01) in
shortened and lengthened positions, respectively, compared with the control group (Table 1). Cytochrome c
mRNA content per whole soleus muscle was 82 and
70% less (P , 0.01) in shortened and lengthened
positions, respectively, than the control group (Table 1).
To determine whether the observed phenomena are
only specific to soleus muscles, we performed the
measurements on the TA muscles. The TA muscle wet
weight was 37% (P , 0.05) and 13% (P , 0.01) less
when it was immobilized in a shortened position (dorsiflexion) and lengthened position (plantar flexion), respectively, compared with control values (Table 2).
However, fixation of the TA muscle in a lengthened
position maintained the muscle-to-body weight ratio.
In a separate set of rats (n 5 5), fixation in a lengthened
position did not alter TA muscle protein concentration
(219.7 6 1.2 mg/g wet wt for control; 211.1 6 4.1 mg/g
wet wt for 7-day fixation; P 5 0.08) but decreased
protein content (76.0 6 2.5 mg/muscle for control; 58.8
6 1.3 mg/muscle for fixation; P , 0.05). Goldspink (12)
found that the protein content of the rat extensor
digitorum longus (EDL) muscle was the same as in an
age-matched control after 2, 4, and 7 days of immobilization in the lengthened position. Lengthening of the
rabbit EDL by immobilization for 3 days did not alter
its protein concentration, which indicates that wet
weight reflects protein content in this animal (13). Total
RNA concentration (µg RNA/mg wet wt) was the same
as the control values at the end of the 7-day fixation
Table 2. Cytochrome c mRNAs in tibialis anterior muscles fixed in lengthened or shortened position for 7 days
Position
Body Wt, g
Wet Wt, mg
Wet
Wt-to-Body
Wt Ratio
Control
Lengthened (plantar flexion)
Shortened (dorsiflexion)
140 6 2
119 6 3*
118 6 4*
587 6 48
509 6 33*
369 6 40†‡
4.2 6 0.1
4.3 6 0.1
3.1 6 0.2†‡
Cytochrome c mRNA
Total
RNA, mg/g
IOD/µg RNA
IOD/mg wet wt
IOD/whole muscle
1.4 6 0.2
1.6 6 0.3
1.3 6 0.1
0.304 6 0.043
0.182 6 0.024†
0.209 6 0.019†
0.435 6 0.056
0.296 6 0.077†
0.278 6 0.022†
254 6 29
152 6 46†
103 6 17†
Values are means 6 SE (n 5 5). Two muscles from a rat were pooled for each observation of muscle weight, RNA, and cytochrome c mRNA.
* P , 0.05 from control; † P , 0.01 from control; ‡ P , 0.05 from lengthened group.
Downloaded from jap.physiology.org on May 19, 2009
Fig. 1. Autoradiogram of Northern blot (A) for soleus muscles from rats with no treatment or with hindlimbs fixed
in either a shortened (plantar flexion) or a lengthened position (dorsiflexion). Total RNA (10 µg) was added to each
lane, electrophoresed on 1% agarose denaturing gels, blot transferred to Hybond N1, and hybridized with a
958-base pair radiolabeled probe from pRC4 rat somatic cytochrome c probe (25). See Table 1 for tabulated data.
Ethidium bromide staining (B) of 28S and 18S on gel after electrophoresis and before transfer shows approximate
equality of total RNA loading.
1944
CYTOCHROME
C
IN ATROPHIED MUSCLE
DISCUSSION
When soleus and TA muscles were immobilized in
lengthened position, cytochrome c mRNA levels per
Fig. 2. RNA-protein interaction in the 38-untranslated region of
cytochrome c mRNA. Equal amounts of protein (15 µg) from whole
soleus muscle homogenates were incubated with 1.5 ng of 32P-labeled
riboprobe. Autoradiogram from ultraviolet cross-linking assay with
radiolabeled riboprobe for 116 bases (11,337 to 11,452) of the
38-untranslated region of cytochrome c mRNA. Molecular masses (in
kDa; left) were estimated from prestained markers (Sigma Chemical). Groups were control, shortened (plantar flexion) for 7 days, and
lengthened (dorsiflexion) for 7 days. FP, free probe.
whole soleus and TA muscles were 30 and 60%, respectively, of control. In contrast, total protein per whole
soleus and TA muscles was 86 and 77%, respectively, of
control. Thus fixation of muscle in a lengthened position not only did not prevent loss of cytochrome c
mRNA, but the decrease in cytochrome c mRNA predicts a decline in cytochrome c protein that is greater
than total protein. Although the attenuation of muscle
atrophy when muscles are lengthened in immobilized
limbs confirms previous reports (3, 12, 28, 29), the
observation that cytochrome c mRNA content is not
maintained in muscles immobilized in a lengthened
position is new. The disassociation of muscle mass from
the loss of cytochrome c mRNA supports the hypothesis
of differential gene regulation of mitochondrial and
contractile protein pools by different cell signals. Examples are that resistance training enlarges muscle
mass whereas mitochondrial concentration remains
constant (15), endurance training increases mitochondrial concentration whereas muscle mass is unaltered
(6), and muscle mass decreases whereas mitochondrial
density increases during chronic stimulation (30). The
hypothesis that mitochondrial and contractile proteins
are modulated by different signaling pathways from
contractile activity deserves further study.
Results of the present study support the idea that
neither passive stretch nor isometric contractions can
maintain the concentration of mitochondria in skeletal
muscle in immobilized limbs. Fournier et al. (11) found
that integrated electromyograph (EMG) was unchanged
when the soleus muscle was fixed in a lengthened
position during immobilization. This EMG activity
must reflect isometric contraction because the joints
were fixed. Hnı́k et al. (14) found similar results for the
soleus muscle and extended these observations to the
TA. The fixation of the ankle in neither plantar flexion
nor dorsiflexion had any appreciable effect on EMG
activity in the TA. Hnı́k et al. (14) concluded that,
because immobilization in the lengthened position maintained, but did not increase, EMG activity in either the
soleus or TA muscle, passive stretch, rather than EMG
activity, appeared to be the factor mainly responsible
for lessening atrophy of muscles fixed in the lengthened
position. Previous reports indicate a direct correlation
exists between duration of low-intensity isotonic contractions and cytochrome c protein concentration in
skeletal muscle (8, 10). Hindlimb unloaded muscles
undergo random isotonic contractions. Because it has
previously been shown that cytochrome c mRNA per
whole soleus muscle was decreased 54, 45, and 61% by
7 days of hindlimb unloading, limb immobilization of
the soleus muscle in a shortened position, and denervation, respectively (2), the isotonic contractions occurring in the hindlimb-unloaded muscle are insufficient
in duration to maintain cytochrome c mRNA.
Because muscles with more contractile activity have
higher concentrations of cytochrome c mRNA and less
RNA-protein interaction in the 38-UTR of cytochrome c
mRNA as determined by gel mobility shift and UV
cross-linking assays (32), we hypothesized that decreasing loaded isotonic contractions of the soleus muscle
Downloaded from jap.physiology.org on May 19, 2009
in both shortened and lengthened TA muscles (Table 2).
Cytochrome c mRNA levels were less than the control
values in the TA muscles immobilized in both the
shortened and lengthened positions as normalized by
three methods. Cytochrome c mRNA (integrated optical
density unit per microgram of RNA) was 31 and 40%
less (P , 0.01) in shortened and lengthened positions,
respectively, after 7 days of immobilization compared
with controls of the same age (Table 2). Cytochrome c
mRNA per milligram wet TA weight was 36 and 32%
less (P , 0.01) in shortened and lengthened positions,
respectively, than in the controls (Table 2). Cytochrome
c mRNA per whole TA muscle was 59 and 40% less (P ,
0.01) in shortened and lengthened positions, respectively, compared with control group values (Table 2).
These data again demonstrate that muscle atrophy during
immobilization can be attenuated by lengthening the
muscle, but cytochrome c mRNA levels decrease as long
as the muscle is immobilized.
No consistent change in cytoplasmic protein interaction with a 116-base riboprobe (11,337 to 11,452) from
the 38-UTR of rat somatic cytochrome c mRNA in
cytoplasmic extracts was noted from soleus muscles
fixed for 7 days in either lengthened or shortened
position (Fig. 2). Similar observations were observed in
TA muscle (data not shown).
CYTOCHROME
C
IN ATROPHIED MUSCLE
would increase this RNA-protein interaction. This hypothesis did not hold up. Protein interaction with the
38-UTR was not changed by fixation of the soleus
muscle in either a shortened or lengthened position.
Thus the decrease in cytochrome c mRNA was not due
to the increase in RNA-protein interaction in the
38-UTR of cytochrome c mRNA. In summary, passive
lengthening of the immobilized soleus muscle does not
prevent the loss of cytochrome c mRNA, even though
loss in its muscle mass was attenuated; and decreased
cytochrome c mRNA levels in the immobilized muscles
are not due to increased RNA-protein interaction in the
38-UTR of cytochrome c mRNA. The greater losses of
cytochrome c mRNA (70%) than of total protein (14%)
in the whole soleus muscle during immobilization in a
lengthened position underscore that countermeasures
to atrophy in limb immobilization must include specific
exercises to signal increases in both contractile and
mitochondrial gene expression.
Received 6 November 1995; accepted in final form 22 May 1996.
REFERENCES
1. Appell, H.-J. Muscular atrophy following immobilization. A
review. Sports Med. 10: 42–58, 1990.
2. Babij, P., and F. W. Booth. a-Actin and cytochrome c mRNAs in
atrophied adult rat skeletal muscle. Am. J. Physiol. 254 (Cell
Physiol. 23): C651-C656, 1988.
3. Booth, F. W. Time course of muscular atrophy during immobilization of hindlimbs in rats. J. Appl. Physiol. 43: 656–661, 1977.
4. Booth, F. W. Effect of limb immobilization on skeletal muscle. J.
Appl. Physiol. 52: 1113–1118, 1982.
5. Booth, F. W., and J. R. Kelso. Production of rat muscle atrophy.
J. Appl. Physiol. 34: 404–406, 1973.
6. Booth, F. W., and D. B. Thomason. Molecular and cellular
adaptation of muscle in response to exercise. Perspectives of
various models. Physiol. Rev. 71: 541–585, 1991.
7. Chomczynski, P., and N. Sacchi. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162: 156–159, 1987.
8. Dudley, G. A., W. M. Abraham, and R. L. Terjung. Influence of
exercise intensity and duration on biochemical adaptations in
skeletal muscle. J. Appl. Physiol. 53: 844–850, 1982.
9. Evans, M. J., and R. C. Scarpulla. Both upstream and intron
sequence elements are required for elevated expression of the rat
somatic cytochrome c gene in COS-1 cells. Mol. Cell. Biol. 8:
35–41, 1988.
10. Fitts, R. H., F. W. Booth, W. W. Winder, and J. O. Holloszy.
Skeletal muscle respiratory capacity, endurance and glycogen
utilization. Am. J. Physiol. 228: 1029–1033, 1975.
11. Fournier, M., R. R. Roy, H. Perham, C. P. Simard, and V. R.
Edgerton. Is limb immobilization a model of disuse? Exp.
Neurol. 80: 147–156, 1983.
12. Goldspink, D. F. The influence of immobilization and stretch on
protein turnover of rat skeletal muscle. J. Physiol. Lond. 264:
267–282, 1977.
13. Goldspink, D. F., V. M. Cox, S. K. Smith, L. A. Eaves, N. J.
Osbaldeston, D. M. Lee, and D. Mantle. Muscle growth in
response to mechanical stimuli. Am. J. Physiol. 268 (Endocrinol.
Metab. 31): E288–E297, 1995.
14. Hnı́k, P., R. Vejsada, D. F. Goldspink, S. Kasicki, and I.
Krekule. Quantitative evaluation of electromyogram activity in
rat extensor and flexor muscles immobilized at different lengths.
Exp. Neurol. 88: 515–528, 1985.
15. Holloszy, J. O., and F. W. Booth. Biochemical adaptations to
exercise in muscle. Annu. Rev. Physiol. 38: 273–291, 1976.
16. Holloszy, J. O., and E. F. Coyle. Adaptations of skeletal muscle
to endurance exercise and their metabolic consequences. J. Appl.
Physiol. 56: 831–839, 1984.
17. Jokl, P., and S. Konstadt. The effect of limb immobilization on
muscle function and protein composition. Clin. Orthop. Clin. Orthop.
Rel. Res. 174: 222–229, 1983.
18. Lowry, O. H., and J. V. Passonneau. A Flexible System of
Enzymatic Analysis. New York: Academic, 1972.
19. Morrison, P. R., J. A. Montgomery, T. S. Wong, and F. W.
Booth. Cytochrome c protein-synthesis rates and mRNA contents during atrophy and recovery in skeletal muscle. Biochem. J.
241: 257–263, 1987.
20. Munro, H. N., and A. Fleck. Mammalian Protein Metabolism.
New York: Academic, 1969, vol. 13, p. 481–483.
21. Paulos, L. E. Knee rehabilitation after knee ligament injury and
surgery. In: The Knee, edited by W. N. Scott. St. Louis, MO:
Mosby, 1994, p. 945.
22. Riffenberick, D. H., and S. R. Max. Substrate utilization in
disused rat skeletal muscles. Am. J. Physiol. 226: 295–297, 1974.
23. Rondon, I. J., L. A. MacMillian, B. S. Beckman, M. A.
Goldberg, T. Schneider, H. F. Bunn, and J. S. Malter.
Hypoxia up-regulates the activity of a novel erythropoietin
mRNA binding protein. J. Biol. Chem. 266: 16594–16598, 1991.
24. Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular
Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor,
NY: Cold Spring Harbor Laboratory Press, 1989.
25. Scarpulla, R. C., K. M. Agne, and R. Wu. Isolation and
structure of a rat cytochrome c gene. J. Biol. Chem. 256:
6480–6486, 1981.
26. Simard, C. P., S. A. Spector, and V. R. Edgerton. Contractile
properties of rat hind limb muscles immobilized at different
lengths. Exp. Neurol. 77: 467–482, 1982.
27. Spector, S. A., C. P. Simard, M. Fournier, E. Sternlicht, and
V. R. Edgerton. Architectural alterations of rat hind-limb
skeletal muscles immobilized at different lengths. Exp. Neurol.
76: 94–110, 1982.
28. Summers, T. B., and H. M. Hines. Effect of immobilization in
various positions upon the weight and strength of skeletal
muscle. Arch. Phys. Med. Rehabil. 32: 142–145, 1951.
29. Thomsen, P., and J. V. Luco. Changes of weight and neuromuscular transmission in muscles of immobilized joints. J. Neurophysiol. 7: 245–251, 1944.
30. Williams, R. S., M. Garcia-Moll, J. Mellor, S. Salmons, and
W. Harlan. Adaptation of skeletal muscle to increased contractile activity. J. Biol. Chem. 262: 2764–2767, 1987.
31. Witzmann, F. A., D. H. Kim, and R. H. Fitts. Effect of hindlimb
immobilization on the fatigability of skeletal muscle. J. Appl.
Physiol. 54: 1242–1248, 1983.
32. Yan, Z., S. Salmons, Y. L. Dang, M. T. Hamilton, and F. W.
Booth. Increased contractile activity decreases RNA-protein
interaction in the 38-UTR of cytochrome c mRNA. Am. J. Physiol.
271 (Cell Physiol. 40): C1157–C1166, 1996.
33. Yan, Z., S. Salmons, J. Jarvis, and F. W. Booth. Increased
muscle carnitine palmitoyltransferase II mRNA after increased
contractile activity. Am. J. Physiol. 268 (Endocrinol. Metab. 31):
E277–E281, 1995.
Downloaded from jap.physiology.org on May 19, 2009
We express our deep appreciation for the help we received from the
late James Pastore, who assisted our research. We thank Brad
Goodwin and his staff for animal care.
This research was supported by National Aeronautics and Space
Administration Grant NAGW 3908.
Address for reprint requests: F. W. Booth, The Univ. of TexasHouston Health Science Center, Medical School, Dept. of Integrative
Biology, 4.100 MSB, 6431 Fannin, Houston, TX 77030 (E-mail:
fbooth@girch1.med.uth.tmc.edu).
1945