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1Department of Physiology and Biophysics, University of California at Irvine, Irvine 92697; and 2Brain Research Institute and 3Department of Physiological Sciences, University of California at Los Angeles, Los Angeles, California 90095
Submitted 28 March 2003 ; accepted in final form 23 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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50%, with the greatest degree of loss
occurring during the first 8 days. Throughout the SI duration, muscle protein
concentration was maintained at the control level, whereas myofibrillar
protein concentration steadily decreased between 4 and 15 days of SI, and this
was associated with a 50% decrease in myosin heavy chain (MHC) normalized to
total protein. Actin relative to the total protein was maintained at the
control level. Marked reductions occurred in total RNA and DNA content and in
total MHC and actin mRNA expressed relative to 18S ribosomal RNA. These
findings suggest that two key factors contributing to the muscle atrophy in
the SI model are 1) a reduction in ribosomal RNA that is consistent
with a reduction in protein translational capacity, and 2)
insufficient mRNA substrate for translating key sarcomeric proteins comprising
the myofibril fraction, such as MHC and actin. In addition, the marked
selective depletion of MHC protein in the muscles of SI rats suggests that
this protein is more vulnerable to inactivity than actin protein. This
selective MHC loss could be a major contributor for the previously reported
loss in the functional integrity of SI muscles. Collectively, these data are
consistent with the involvement of pretranslational and translational
processes in muscle atrophy due to SI. myosin heavy chain messenger ribonucleic acid; actin messenger ribonucleic acid; poly(A) messenger ribonucleic acid; ribonucleic acid; deoxyribonucleic acid; protein translation
Recently, Grossman and coworkers (16) have used a novel approach to render the muscles virtually electrically silent while maintaining a functionally and anatomically intact neuromuscular connectivity. This procedure is referred to as spinal cord isolation (SI) and was initially described by Tower (34). This intervention (SI) creates a unique model that eliminates all weight-bearing and neural activity-dependent influences on the muscles but maintains neural activity-independent influences, such as trophic interactions between the motoneurons and nerves and muscles. Thus the SI model is suitable for establishing a baseline of zero electrical activity and weight bearing for defining the atrophy process.
Whereas muscles subjected to SI have been characterized in terms of
defining the changes in muscle contractile protein phenotypes and general
histological, morphological, and functional properties
(16,
19,
20,
28,
29), the changes in the basic
molecular makeup of the muscle have not been examined. For example, it is not
clear how the total muscle protein and myofibril concentrations are altered
during the rapid atrophy process. Also, the muscle concentrations of total
RNA, total DNA, and total messenger RNA pool have not been examined, and these
are important parameters impacting homeostatic protein balance of the target
muscle. Therefore, the primary goals of the experiments described herein and
in a companion paper (18) were
to identify the basic molecular events occurring in the rapidly atrophying
soleus during the first 15 days after SI intervention. In this paper, we
tested two working hypotheses. First, the atrophy induced by SI occurs rapidly
due to the inability of the muscle to maintain sufficient ribosomal RNA and
sarcomeric protein mRNA levels necessary for translating and maintaining
sufficient amounts of muscle protein to counteract the ongoing protein
degradation processes. Second, in contrast to previous data from models of
unloading-induced atrophy (9,
26,
31,
33), suggesting that protein
translational events impact the atrophy process very briefly, we propose that
processes impacting protein translation occur throughout the atrophy response,
i.e., until the muscle reaches a new steady state of reduced muscle mass. To
test these hypotheses, we determined the levels of the myofibrillar protein
pools and the total myosin heavy chain (MHC) and actin protein, as well as the
corresponding relative and total mRNA expression. In this way, we could track,
as a function of time, the respective amounts of mRNA and protein pools for
the two major muscle proteins, i.e., actin and myosin, which account for
40% of total muscle protein. The temporal relationships between specific
mRNAs and corresponding proteins enabled us to estimate the role of
pretranslational and translational processes in contributing to the protein
accretion. We also examined changes in both the concentration and content of
DNA, total RNA, total mRNA, and total protein. These variables collectively
are thought to be critical in determining muscle protein homeostasis.
Combined, the results of this study indicate that the muscle wasting induced in the SI model occurs to a significant extent from an inability of the skeletal muscles to maintain a normal level of sarcomeric protein accretion. This decreased sarcomeric protein accretion occurs because of two abnormalities: 1) an early and sustained loss of ribosomal RNA that is consistent with a general deficit in the translational capacity and 2) inadequate availability of key sarcomeric gene mRNAs impacting MHC to a greater extent than actin. In a companion paper (18), we demonstrate that the inadequate availability of MHC and actin mRNAs results from decreased transcriptional activity for these two genes.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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230 ± 5 g were assigned randomly to either a normal
control (NC; n = 35) or a SI (n = 35) group. Animals in each
experimental group subsequently were assigned randomly into subgroups
(n = 7 each) and studied on the day of (time = 0) and 2, 4, 8, and 15
days after SI surgery. The rats in the SI group were anesthetized by using an
intraperitoneal injection of ketamine hydrochloride (70 mg/kg body wt) and
acepromazine maleate (5 mg/kg body wt) and were subjected to complete spinal
cord transections at both a midthoracic and an upper sacral level plus
bilateral deafferentation between the two transection sites, as described
previously (16,
28). The procedures for the
care and maintenance of spinal cord-injured animals have been detailed
previously (16,
27). These procedures were
approved by the Institutional Animal Care and Use Committee at the University
of California-Los Angeles, and they conformed to the animal care procedures
and standards recommended by the American Physiological Society. Tissue processing. At each time point, animals from the appropriate subgroups were weighed, deeply anesthetized with pentobarbital sodium (100 mg/kg), and then decapitated. The soleus muscles (as well as other limb muscles not reported in this study) were removed, quickly dissected free of connective tissue, quick frozen on dry ice, and subsequently stored at -80°C until used for biochemical and molecular analyses. Analyses for all subgroups involved n = 6 or 7, as presented in the data profiles.
Biochemical and molecular analyses. A preweighed portion of each muscle sample was homogenized in 20 volumes of a homogenization buffer, which contained 250 mM sucrose, 100 mM KCl, 5 mM EDTA, and 10 mM Tris base. Myofibrillar proteins were quantitatively extracted from a known volume of the total homogenate by a modification of the original procedure described by Solaro et al. (30) and were suspended into a known volume of 100 mM KCl, 10 mM Tris, and 1 mM EDTA, pH 7.4.
Muscle total DNA concentration calculation was based on total DNA concentration in the total homogenate, as was determined by using a fluorometric assay (21). Muscle total protein and myofibrillar protein concentration was based on protein concentration of the muscle whole homogenate and myofibril suspension, respectively, as determined by the Bio-Rad protein assay. The whole homogenate was diluted to a final protein concentration of 1 mg/ml in a storage buffer containing 50% glycerol, 100 mM Na4P207, 5 mM EDTA, and 2 mM 2-mercaptoethanol (pH 8.8) and stored at -20°C until subsequent analyses for MHC and actin protein content.
MHC and actin protein analysis. Skeletal muscle MHC and actin
proteins were separated on acrylamide gels (10% T, 2.5% C) by using a standard
SDS-PAGE technique (22).
Protein samples were denatured by placing 10 µg of sample in 30 µl of
sample buffer (5% 
-mercaptoethanol, 100 mM Tris base, 5% glycerol, 4%
SDS, and 0.05% bromophenol blue, pH 6.8) and then heating the solution for 2
min at 100°C. Ten microliters of the denatured protein solution
(equivalent to 2.5 µg of total muscle protein) were loaded per lane, and
the gels were run at constant current (30 mA) for 2.5 h at 22°C. In
each gel run, 0.5 µg of purified myosin and 0.5 µg of purified
-skeletal actin (Sigma Chemical) were also denatured and processed on
the same gel to serve as a reference for the migration level of MHC and actin
bands in the total protein samples. The gels were stained with brilliant blue
G 250 (Sigma Chemical), destained, and then scanned by using a Molecular
Dynamics (Sunnyvale, CA) laser scanning personal densitometer (see
Fig. 1A for an
illustration of a sample gel). The MHC and actin bands were identified on the
digitized image, and their intensity was calculated via volume integration of
density within a rectangle containing the entire band with local background
correction. We have validated this method by loading different amounts of
total proteins (0.5- to 4-µg range) on the gel
(Fig. 1A); and MHC and
actin band intensities were directly proportional to the amount of protein
loaded, whereas the actin-to-MHC ratio remained constant over the entire range
(Fig. 1B). With the
use of this method, MHC and actin proteins were expressed as arbitrary units
(AU) per microgram of total protein, and the amount of MHC and actin protein
content was calculated based on muscle total protein content.
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Total RNA isolation. Total RNA was extracted from preweighed
frozen muscle samples by using the TRI reagent (Molecular Research Center,
Cincinnati, OH), according to the company's protocol, which is based on the
method described by Chomczynski
(13). Extracted RNA was
precipitated from the aqueous phase with isopropanol and, after being washed
with ethanol, was dried and suspended in a known volume of nuclease-free
water. The RNA concentration was determined by optical density at 260 nm
(using an optical density 260-unit equivalent to 40 µg/ml). The muscle
total RNA concentration was calculated based on total RNA yield and the weight
of the analyzed sample. The RNA samples were stored frozen at -80°C and
were used subsequently in determining total mRNA [poly(A)], total MHC mRNA,
and -skeletal actin mRNA expression by using slot-blotting
procedures.
RNA slot blotting. One microgram of total RNA was placed in 20
µl of denaturing buffer (10% formaldehyde, 67% formamide, and 0.5 x
MOPS, pH 7) at 60°C for 15 min. Samples were brought up to 100 µl
volume with 6 x SSC and were applied onto a positively charged nylon
membrane (Gene-Screen plus, NEN) by using a slot-blot apparatus (Schleisher
and Schuell). After UV fixation, these membranes were hybridized with three
different probes consecutively as follows. 1) An antisense
-skeletal actin mRNA probe was used to determine -skeletal actin
mRNA expression, or a common antisense MHC mRNA probe was used to determine
the total MHC mRNA expression. The MHC probe is complementary to the coding
region 500 nucleotides upstream from the stop codon of type I MHC mRNA.
This region is 100% identical in all of the MHC isoforms, and the obtained
signal corresponds to the total population of MHC mRNA expressed in the
muscle. 2) An oligo(dT) probe (12- to 18-mer, Life Technology) was
used to detect poly(A) RNA (total mRNA population). 3) An antisense
18S ribosomal RNA probe, whose signal is directly proportional to the amount
of total RNA, was used to normalize for possible variability in the amount of
loaded RNA per slot. Probes were 5' end labeled with P32 by using
-ATP and T4 polynucleotide kinase. Hybridization and washing procedures
were carried out, as described previously
(17). Hybridization signals
were detected and analyzed by using a PhosphorImager and Image Quant analysis
software (Molecular Dynamics). For each sample, the MHC mRNA, actin mRNA, and
dT [poly(A)] signals were normalized to the corresponding 18S signal. The
slot-blot hybridization signal for these probes was strongly correlated with
the amount of loaded total RNA, ranging from 0.25 to 2 µg/slot. The
sequence of oligonucleotides probes used for hybridization is as reported in
Adams et al. (1). Total MHC
mRNA, total -skeletal actin mRNA, and total mRNA were expressed either
as relative to 18S or as content per muscle in AU. This latter value is
obtained based on total RNA content and the specific signal generated relative
to corresponding 18S per 1 µg total RNA.
Statistical analyses. All values are reported as means ± SE. For each time point, treatment effects were determined by ANOVA with Student-Newman-Keuls post hoc testing by using the Prism software package (Graphpad). For certain variables, to assess the time course response to SI, regression analysis (Graphpad, Prism) was performed, whereby x was the SI duration and y the specific variable analyzed. These data were analyzed in terms of the linearity of the curve, the slope of the line, and whether there was a difference between NC and SI for these parameters. For all statistical tests, the 0.05 level of confidence was accepted for statistical significance.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Soleus muscle total protein and myofibrillar protein concentration and content. To analyze the temporal changes in total protein and myofibril concentration, regression lines were generated between the 4- and 15-day time points. The slopes of the regression lines for protein concentration for both the NC and SI groups were not different from zero (NC slope, +1.6 ± 0.8; SI slope, -1.6 ± 0.8) (Fig. 3A), indicating no significant change in protein concentration during the 15-day experimental period. In contrast, the regression lines for myofibril concentration were significantly different between the two groups (Fig. 3B). The slope of the regression line for the SI group was -5.9 ± 0.8 and was significantly different from zero, whereas the slope of the regression line for the NC group was not significantly different from zero. These data demonstrate that the 4- to 15-day interval after SI is associated with a gradual and selective loss of myofibrillar proteins. When the protein data were expressed as the total content per muscle, i.e., mg/g x g/muscle (wt) = mg protein/muscle, there was a progressive decrease in the protein content of the SI relative to the NC group that closely mimicked the pattern of muscle weight loss (compare Fig. 3C with Fig. 2, B and C). These data indicate that the loss in muscle mass resulted from a proportional loss of water and total protein in the muscle. Total protein loss in the soleus muscle of SI rats averaged 60 ± 5% relative to NC at the 15-day time point. When the myofibril data were expressed as the content per muscle, there was a progressive loss in myofibril content in the SI group throughout the experimental period (Fig. 3D). This myofibril loss averaged 65 ± 5% in the SI relative to the NC group at 15 days.
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Total MHC and actin protein concentration and content. Total MHC
protein and actin fractions were separated by gel electrophoresis (see
METHODS). Densitometric analysis of the MHC and actin bands showed
that the SI muscles were associated with a significant decrease in the MHC
protein fraction relative to total protein at the 8- and 15-day time points
(Fig. 4A). In
contrast, the actin portion relative to total protein was maintained constant
throughout the experimental period (Fig.
4B). These changes resulted in a significant increase in
the actin-to-MHC ratio at the longer time points
(Fig. 4C), a parameter
that is relatively constant over a wide range of the amount of protein loaded
in the assay system (Fig.
1B). Both total MHC and actin protein content were
significantly depleted in the SI relative to the NC muscles when expressed on
a permuscle basis at the 4-day time point and thereafter
(Figs. 4, E and
F). MHC protein isoform composition was examined by using
methods described previously
(32) and confirmed previous
findings (19), i.e., the
atrophied muscles of SI rats are transformed to a faster phenotype (data not
shown). For example, after 15 days of SI, the percent type I MHC protein
composition relative to the total MHC pool had decreased by 15%, and the
muscle content was 80% lower in SI relative to NC muscles. In contrast,
the percent type IIx MHC protein composition of the soleus muscle increased
from 1–2% in NC to 18% (P < 0.05) in SI rats. When
corrected to whole muscle, the type IIx MHC content increased from 47 ±
19 to 104 ± 30 AU (P > 0.05). These data suggest that,
whereas there was a net loss in the total MHC protein pool, this loss was
selective to slow MHC isoforms (types I and IIa), whereas other factors
operating at the transcriptional and pretranslational levels most likely
impacted the enhanced expression of the type IIx MHC isoform, which becomes
the major MHC isoform in the soleus muscle after 90 days of SI
(19).
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Actin and total MHC mRNA. The mRNA relative to 18S of both total MHC (Fig. 5A) and actin (Fig. 5B) mRNA markedly decreased during the first 8 days after SI. The total MHC mRNA normalized to 18S returned to the control level between day 8 and day 15. The total MHC and actin mRNA expressed relative to the total RNA per muscle were significantly decreased after 4 days of SI and thereafter (Fig. 5, C and D). Thus there was a critical loss of these two specific sarcomeric mRNAs that could have limited their availability for protein translation in the inactive muscles. The fact that both total MHC mRNA and actin mRNA decreased to a similar extent (Fig. 5, A vs. B), whereas only total MHC protein was decreased relative to total muscle protein (Fig. 4, A vs. B), may indicate that, during the SI-induced atrophy process, the MHC and actin are lost at different rates. The MHC loss is either faster or greater than that of the actin, and this could be the result of the former possessing a higher turnover rate than the latter. Alternatively, these findings could indicate that the SI induces some degradation enzymes (for example, a specific ubiquitin E3 ligase selective for slow MHC) that more specifically target the MHC component of the sarcomeric protein for degradation.
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RNA, mRNA, and DNA concentration and content. In view of the rapid
muscle atrophy in response to SI, we hypothesized that translational processes
were likely contributing to the severe loss in both total and myofibrillar
proteins. We examined the total cellular RNA pool, total mRNA population, and
total DNA in NC and SI muscles as general markers for the status of
translational processes. Total RNA concentration in the SI group rapidly
decreased relative to the NC group during the early time points (from day
2 to day 4, P < 0.05 at day 4) and then gradually
returned to control levels (Fig.
6A). However, the RNA content per muscle progressively
decreased up to 8 days and then remained stable at 55% below control
levels thereafter (Fig.
6B). Combined, these data suggest that there was a
significant reduction in the RNA pool during the timeframe when the muscle was
going through its most rapid state of protein loss. Because 85% of the
total RNA pool is ribosomal, it appears that there is a significant loss of
the key machinery necessary for protein translation in the soleus muscles of
SI rats. These data also indicate that the loss in RNA occurs very early in
the atrophy process.
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The total mRNA [poly(A) mRNA] levels relative to 18S tended to be slightly
higher in the SI relative to NC muscles (P < 0.05 at 8 days)
(Fig. 6C). This
unexpected observation occurred at a time when total MHC and actin mRNA to 18S
were being reduced by 80%. A loss in total mRNA population was observed
only when the total mRNA was expressed as the content per muscle
(Fig. 6D) and simply
reflected the muscle atrophy. These observations suggest that the general
catabolic state in the SI muscles is not associated with a reduction in the
overall availability of the total mRNA pool for translation.
In contrast to total RNA, there was a significant increase in DNA concentration in the SI relative to the NC group after 8 and 15 days of inactivity (Fig. 6E). An increased DNA concentration reflects a greater loss of protein (cytoplasmic mass) than of the number of nuclei, resulting in "cells" becoming smaller in volume and cross-sectional area (16). However, when the DNA was expressed on a per-muscle basis, there actually was a significant net loss of DNA during the atrophy process at all time points, except at 2 days (Fig. 6F). This observation is consistent with the net loss of nuclei in atrophying muscles (4–7).
Responses of markers of cell size and protein synthesis capacity.
Based on the morphology of the muscle fibers in the soleus, as observed
microscopically on transverse and longitudinal cross sections of the muscle,
the majority of whole muscle proteins originates from the muscle-fiber
cytoplasm (unpublished observations). Although whole muscle DNA is
heterogeneous and may be significantly contaminated by nonmuscle cell DNA, it
is largely accepted that whole protein-to-DNA ratio in muscle may be used as a
rough measure of the myonuclear domain, which is also directly proportional to
fiber cross-sectional area
(12,
14). We have used the
protein-to-DNA ratio as an index of muscle-fiber size (cross-sectional area).
Figure 7A shows a
progressive decrease in this ratio in the SI rats, an observation consistent
with the reported decrease in the mean cross-sectional area of the fibers in
the soleus of SI rats (16).
Total RNA is used as a marker of cellular protein translation, because it
consists of 85% ribosomal RNA, which is a major component of the protein
translational machinery. The RNA-to-DNA ratio, commonly used to estimate the
amount of synthetic material per cell (synthetic capacity), is significantly
lower in SI than NC rats at all time points
(Fig. 7B).
Furthermore, the RNA-to-protein ratio, which provides insight into the
synthetic potential of the fiber, is significantly lower in SI than NC rats
during the early period (2 and 4 days) after SI surgery and then gradually
returns to control levels as the muscle mass reaches a new, but lower steady
state (Fig. 7C).
Whereas these variables admittedly are simple ways of demonstrating changes in
cell structure and function, the direction of adaptation is consistent with
the other changes reported herein. Combined, these observations clearly
demonstrate the highly dynamic state of the adaptive events in the muscle,
leading to a significant reduction in the protein balance of the muscle
throughout the period of SI-induced inactivity investigated in the present
study.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Sarcomeric gene expression, myosin, and actin. Myosin and actin
are two of the most abundant proteins expressed in skeletal muscle, combining
to account for 60–65% of the myofibril pool, which in turn accounts
for >50% of the total protein pool. Our findings in this study show that,
between 4 and 15 days of SI, the soleus muscle proteins are progressively
depleted of the myofibril fraction (Fig.
3). The MHC fraction of the total protein decreased by 50% at
8 and 15 days post-SI, whereas the actin fraction did not change
significantly. Interestingly, both MHC and actin mRNAs relative to 18S
ribosomal RNA were lower in the SI than NC group. This response suggests that
the fate of MHC and actin is different, and they may be subjected to
differential processes at the translational and/or posttranslational
levels.
Furthermore, these findings raise two important questions. The first question is: does this MHC-actin imbalance recover to normal levels after longer durations of SI treatment, e.g., after 30, 60, or 90 days? After 60 days of SI, the specific tension, i.e., force per unit cross-sectional area, of the soleus muscle was 33% lower than that for control (29). This finding suggests an intrinsic muscle weakness that cannot be accounted for by the decrease in fiber size alone. Thus we speculate that the imbalance of MHC to actin expression and/or the myofibril deficit relative to total protein observed at these early time points also may apply to longer durations of inactivity.
The second question that these data raise is: do the observed specific losses in myofibril and MHC protein also occur in other models of muscle atrophy, such as hindlimb unloading and denervation? In this context, it has been reported that specific depletion of myosin relative to actin occurs under conditions of acute quadriplegic myopathy, a condition attributed to the administration of nerve-blocking agents and corticosteroids (23, 25).
Previous studies using Northern blots have found varying levels of
reduction in actin and -MHC mRNA in the hindlimb-unloading model during
the early stages (1–7 days) of soleus muscle atrophy, suggesting that
translational processes must dominate the reductions in protein synthesis
because protein synthesis reductions appeared to be greater than the
reductions in mRNA for MHC and actin
(33). Using slot-blotting
hybridization, as well as the RT-PCR approach to determine the levels of total
MHC mRNA, actin mRNA, and the various MHC mRNA isoforms, we found that the
actin and total MHC mRNA concentrations, normalized to the 18S subunit of the
ribosomal system, were markedly reduced in the SI rats
(Fig. 5) and to a much greater
extent in the muscles from the SI rats compared with that reported for the
hindlimb-unloading model. This observation, coupled with the finding that the
ribosomal RNA pool was significantly reduced during the onset of the rapid
phase of atrophy with SI (Fig.
6A), suggests that pretranslational processes could
contribute to the downregulation of these two proteins in the muscles of SI
rats. Importantly, in a companion paper
(18), we provide evidence that
transcriptional activity for both the slow, type I MHC and the actin genes is
reduced by 50% after 7 days of SI. This reduction in transcriptional
activity is consistent with the marked reduction in mRNA expression for these
two genes. These collective observations provide strong evidence that there is
less drive in a variety of key processes that contribute to protein synthesis
relative to protein degradation in the SI model. Furthermore, the effects of
these processes are specific to the protein in question, i.e., fast MHC, slow
MHC, and actin.
Total RNA content. One of the surprising observations in the present study was the rapid loss in the total RNA concentration of the SI muscle. Because the bulk of the RNA in different types of cells is ribosomal, it appears that this key component of protein translation was rapidly downregulated. In the context of this observation, it is interesting that the same phenomenon has been reported in other models of atrophy, such as limb immobilization and hindlimb unloading (33). Furthermore, we have observed that the total RNA is reduced in atrophying human muscle (unilateral limb suspension model; P. A. Tesch, F. Haddad, and K. M. Baldwin, unpublished observations). In contrast, when rodent muscles are functionally overloaded, one of the earliest responses to the overload stimulus is a marked increase in total RNA concentration and content (1). Thus it is becoming increasingly apparent that changes in RNA concentration and content may be key adaptive responses in both atrophic and hypertrophic processes. Clearly, more work is needed on different atrophy and hypertrophy models to determine whether the plasticity of the RNA pool is a common phenomenon for regulating muscle cell mass.
How do the present findings contribute to understanding the process of muscle atrophy? In 1990, Thomason and Booth published a working model of muscle atrophy (33). In that model, it was suggested that protein synthesis rapidly decreases during the first few days of unloading and remains essentially constant until a new atrophied steady state is reached (Fig. 4 in Ref. 33). In contrast, muscle degradation was proposed to be gradually turned on, reach a peak several days after the onset of muscle unloading, and then return to control levels as the muscle mass reaches a new steady state. The findings presented herein, although on a different atrophy model, also suggest that reductions in pretranslational events are likely to be impacting protein synthesis to a much greater extent as the atrophy process initially unfolds (hence protein loss) and reaches its steepest rate of decline (Figs. 2 and 3). This scenario is consistent with the observation that the decline in the actin and MHC mRNA pools (Fig. 5) occurs very early, and these mRNA levels are maintained at a low level throughout the atrophy response (Fig. 5). Combined, these findings suggest that 1) the pretranslational processes impacting protein synthesis contribute to the net loss in muscle protein well into the atrophy process, i.e., beyond the early stages of unloading; and 2) the protein translational mechanisms may be playing a greater role in muscle loss throughout the atrophy process than what is traditionally thought.
Summary. The slow antigravity rat soleus skeletal muscles
atrophied rapidly in response to inactivity induced by SI. The atrophy
response was associated with a severe loss in muscle protein that matched the
loss in muscle mass. Between 4 and 15 days of SI, we observed a gradual loss
in myofibrillar concentration coupled with 50% reduction in the MHC
fraction of the total protein. Muscle atrophy and protein losses were coupled
to the regulation of key mRNA substrate levels along with the machinery to
translate them. These findings indicate that the rapid reduction in protein
and muscle can be attributed to pretranslational, translational, and perhaps
posttranslational processes. Furthermore, the present study provides important
evidence that those atrophy processes that occur in the absence of
weight-bearing activities, such as chronic disuse and spaceflight, are not
solely regulated by protein degradation processes. Rather, they are strongly
influenced by events that negatively impact on the muscle's ability to
generate protein.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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B. Aravamudan, C. B. Mantilla, W.-Z. Zhan, and G. C. Sieck Denervation effects on myonuclear domain size of rat diaphragm fibers J Appl Physiol, May 1, 2006; 100(5): 1617 - 1622. [Abstract] [Full Text] [PDF]
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F. Haddad and G. R. Adams Aging-sensitive cellular and molecular mechanisms associated with skeletal muscle hypertrophy J Appl Physiol, April 1, 2006; 100(4): 1188 - 1203. [Abstract] [Full Text] [PDF]
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M. J. Toth, P. A. Ades, M. M. LeWinter, R. P. Tracy, and A. Tchernof Skeletal muscle myofibrillar mRNA expression in heart failure: relationship to local and circulating hormones J Appl Physiol, January 1, 2006; 100(1): 35 - 41. [Abstract] [Full Text] [PDF]
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H. Zhong, R. R. Roy, B. Siengthai, and V. R. Edgerton Effects of inactivity on fiber size and myonuclear number in rat soleus muscle J Appl Physiol, October 1, 2005; 99(4): 1494 - 1499. [Abstract] [Full Text] [PDF]
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 M. J. Toth, D. E. Matthews, R. P. Tracy, and M. J. Previs Age-related differences in skeletal muscle protein synthesis: relation to markers of immune activation Am J Physiol Endocrinol Metab, May 1, 2005; 288(5): E883 - E891. [Abstract] [Full Text] [PDF]
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 O. Schakman, H. Gilson, V. de Coninck, P. Lause, J. Verniers, X. Havaux, J. M. Ketelslegers, and J. P. Thissen Insulin-Like Growth Factor-I Gene Transfer by Electroporation Prevents Skeletal Muscle Atrophy in Glucocorticoid-Treated Rats Endocrinology, April 1, 2005; 146(4): 1789 - 1797. [Abstract] [Full Text] [PDF]
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M. J. Toth, D. E. Matthews, P. A. Ades, M. D. Tischler, P. Van Buren, M. Previs, and M. M. LeWinter Skeletal muscle myofibrillar protein metabolism in heart failure: relationship to immune activation and functional capacity Am J Physiol Endocrinol Metab, April 1, 2005; 288(4): E685 - E692. [Abstract] [Full Text] [PDF]
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F. Haddad, K. M. Baldwin, and P. A. Tesch Pretranslational markers of contractile protein expression in human skeletal muscle: effect of limb unloading plus resistance exercise J Appl Physiol, January 1, 2005; 98(1): 46 - 52. [Abstract] [Full Text] [PDF]
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J. M. McClung, R. W. Thompson, L. L. Lowe, and J. A. Carson RhoA expression during recovery from skeletal muscle disuse J Appl Physiol, April 1, 2004; 96(4): 1341 - 1348. [Abstract] [Full Text] [PDF]
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F. Haddad, R. R. Roy, H. Zhong, V. R. Edgerton, and K. M. Baldwin Atrophy responses to muscle inactivity. II. Molecular markers of protein deficits J Appl Physiol, August 1, 2003; 95(2): 791 - 802. [Abstract] [Full Text] [PDF]
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