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1 Mental Health Research Institute and 2 Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Myogenin is a muscle-specific transcription
factor participating in denervation-induced increases in nicotinic
ACh receptor (nAChR) gene expression. Although myogenin RNA
expression in denervated muscle is well documented, surprisingly little
is known about myogenin protein expression. Therefore, we assayed
myogenin protein and RNA in innervated and denervated muscles from
young (4 mo) and old (24-32 mo) rats and compared this expression
to that of the nAChR 
-subunit RNA. These assays revealed increased
myogenin protein expression within 1 day of denervation, preceding
detectable increases in nAChR RNA. By 3 days of denervation, myogenin
and nAChR -subunit RNA were increased 500- and 130-fold,
respectively, whereas myogenin protein increased 14-fold.
Interestingly, old rats (32 mo) had 6-fold higher myogenin protein and
~80-fold higher mRNA levels than young rats. However, after
denervation, expression levels were similar for young and old animals.
The increased myogenin expression during aging, which tends to localize
to small fibers, likely reflects spontaneous denervation and/or
regeneration. Our results show that increased myogenin protein in
denervated muscles correlates with the upregulation of its mRNA.
nicotinic acetylcholine receptor; aging; gene expression
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SKELETAL MUSCLE IS A DYNAMIC system that responds quickly and adapts to changes in intrinsic and extrinsic stimuli. A whole repertoire of genes can be activated in response to these stimuli. The family of myogenic basic helix-loop-helix (bHLH) transcription factors (MyoD, myogenin, MRF4, Myf-5, Id) plays a major role in coordinating the muscle developmental program, as well as the process of adaptation (reviewed in Ref. 27). The mechanism by which myogenic bHLH family members regulate gene expression involves homodimerization or heterodimerization with ubiquitously expressed bHLH proteins (E12, E47) and binding to an E-box sequence in the promoter region of the genes (18, 23). Formation of heterodimers with Id, however, prevents binding to the E-box and leads to downregulation of gene expression (2). Expression levels of different myogenic bHLH proteins vary in different muscle types and might regulate and reflect phenotypic and physiological differences among muscles (16, 35).
One of the most studied members of the family of myogenic bHLH factors is myogenin. Myogenin knockout mice have a severe deficiency of skeletal muscle and neonatal lethality (14, 25). In contrast, mice lacking MyoD (31), Myf-5 (3), or MRF4 (37) are viable and do not have skeletal muscle defects. Myogenin participates in the muscle response to a wide variety of conditions. Expression of myogenin mRNA changes in muscle during development (9), passive stretch (20), regeneration (11), denervation (1, 4, 9, 35), disease (29), and aging (10, 22, 24), etc.
One of the hallmarks of skeletal muscle development is innervation,
which is ultimately required for cell survival. When mature innervated
muscle is denervated, an apparent recapitulation of at least portions
of the developmental process takes place (9). After
denervation, an increase in expression of many of the myogenic transcription factors occurs, but the increase in myogenin mRNA appears
to be the most robust (1, 35). Myogenin
message gradually increases during the first few days of muscle
denervation and is followed by an increase in transcription of
activity-dependent genes such as those encoding the nicotinic ACh
receptor (nAChR) subunits (9). This finding is
complemented by the observation that overexpression of myogenin in
adult muscles of transgenic mice leads to an increase in expression of
nAChR transcripts (12). Furthermore, direct injection of a
myogenin antisense expression vector into denervated muscle causes a
significant decrease in the transcriptional activation of the nAChR
-subunit promoter (26). Finally, muscle denervation
extending beyond 1 mo results in diminished myogenin and nAChR subunit
RNA levels over the ensuing months of denervation (1).
These observations suggest that myogenin plays a key role in regulating
the expression of the nAChR subunits after denervation.
Despite evidence suggesting that after denervation, an increased expression of myogenin mRNA leads to activation of other denervation responsive genes, very little is known about the actual myogenin protein expression profile after denervation. Expression of myogenin protein is not only regulated at the level of transcription, but also by mRNA stability, translation efficiency, protein stability, and protein phosphorylation (reviewed in Ref. 27). For example, 8.5 days postcoitus (dpc) mouse embryos accumulate a significant pool of unprocessed myogenin RNA in their developing somites, but do not have detectable levels of myogenin protein until 10 dpc (6, 32, 33). In addition, immunostaining has shown that myogenin protein accumulates in rat muscle fiber myonuclei 3 days after denervation (36). Unfortunately, immunostaining does not allow for a quantitative estimation of myogenin protein expression. Therefore, it is difficult to tell if accumulation of myogenin protein after denervation is a result of increased protein expression or a posttranslational modification that leads to the accumulation of the protein in the nuclei.
To determine if myogenin RNA levels reflect protein levels, we assayed
myogenin RNA and protein expression in innervated and denervated young
(4 mo) and old (24-32 mo) rats. We related these changes to nAChR
-subunit mRNA expression, which is regulated by myogenin
(12, 26). In the present study, gastrocnemius muscles from young rats were denervated over a period of 4 mo and
myogenin and nAChR -subunit RNAs and myogenin protein expression were characterized. Experiments on old animals were also included in
this study on the basis of the observation that the levels of myogenin
and nAChR 
- and 
-subunit mRNAs were significantly higher in
muscles from 31-mo-old rats than from either 3- or 18-mo-old rats
(10, 22). Our data showed that after
experimental denervation of muscles from young and old rats and during
the normal aging process, an increase in myogenin mRNA expression
correlated with an increase in myogenin protein that was localized to
muscle nuclei. Compared with myogenin mRNA expression, accumulation of
nAChR -subunit mRNA after experimental denervation was delayed for ~1 day but, in general, followed the pattern of myogenin expression both after denervation and during aging. In innervated muscles from old
rats, increased basal levels of myogenin expression were consistent
with the presence of a population of denervated and/or regenerating
muscle fibers.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and muscle denervation. Experiments were performed on 4- to 32-mo-old Wistar rats of the WI/HicksCar strain maintained at the University of Michigan. Two to five animals were used for each time point (n = 54). Rats were anesthetized with ether during the induction of experimental denervation and before euthanasia. For denervation experiments, the right legs of 4-mo-old rats were denervated by a high sciatic nerve section in the hip region of the hindlimb. The proximal stump of the sciatic nerve was ligated and then implanted and sutured into the hip musculature. The distal stump was implanted as far from the proximal stump as possible. This procedure results in permanent denervation of the lower hindleg (34). Denervation experiments were carried out over a period of 1 to 120 days. Gastrocnemius and tibialis anterior muscles were then excised for analysis. Usually muscles from the contralateral legs served as a control. In agreement with previously published observations (15), we could not detect differences between muscles from unoperated animals and from contralateral legs of operated animals (data not shown). All operations and subsequent animal care were carried out in accordance with the guidelines of the Unit for Laboratory Animal Medicine at the University of Michigan and National Institute of Health Guide for the Care and Use of Laboratory Animals.
RNA isolation and RNase protection assay.
Total RNA was isolated by homogenizing the muscles in Trizol (GIBCO
BRL, Grand Island, NY) followed by the single-step purification method
as described by the manufacture protocol. Antisense probes used to
detect muscle creatine kinase (MCK), myogenin, and the nAChR
-subunit were the same as those described by Adams et al. (1). RNase protection assays were carried out as
previously described (1). The probe for MCK was included
in each experiment and served to normalize for differences in the
amount of RNA in each of the samples as has been reported previously
(1). The RNA for MCK was not regulated by any of the
conditions employed in this report. The myogenin probe was made from
mouse DNA and consistently protects two bands when hybridized to rat
RNA. RNase resistant hybrids were analyzed on 6% polyacrylamide-8 M
urea gels. After electrophoresis, gels were dried and exposed to the X-ray film. Probe signals were quantified by scanning densitometry, and
values were normalized to the RNA signal obtained for MCK. The
specificity of the protected bands was confirmed by hybridizing probes
to tRNA, which resulted in no protected fragments on the gel. Probe
integrity was monitored for each experiment by running an aliquot of
nonhybridized probe on each gel.
Western blotting. Rat muscles were dissected, frozen in liquid nitrogen, pulverized, and homogenized in solution containing 20 mM Tris · HCl (pH 6.8), 4% (wt/vol) SDS, 1 mM of phenylmethylsulfonyl fluoride (PMSF), and 1 µm each of leupeptin and pepstatin A. Protein concentrations were determined using the Bio-Rad detergent compatible protein assay (Hercules, CA). Protein samples were mixed with loading buffer, subjected to SDS-PAGE (10%), and transferred electrophoretically to Immobilon-P membranes (Millipore, Bedford, MA). Gels with identical samples were stained with Coomassie brilliant blue and used as an additional control of equilibration of protein loading. After transfer Immobilon-P membranes were blocked in Blotto buffer containing 5% dry milk in PBS-0.05% Tween 20 (PBST) and then incubated overnight at 4°C with mouse monoclonal antibody against myogenin (clone F5D, obtained from the Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA). Immunodetection was done using peroxidase-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Lab, West Grove, PA) with subsequent chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Band intensity was quantified by scanning densitometry.
Immunohistochemistry. Dissected muscles were fixed in 2% (wt/vol) paraformaldehyde, washed in PBS, embedded in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC), and sectioned on a cryostat (10 µm). Sections were incubated in blocking buffer (20% goat serum in PBST) for 1 h and then in the solution of anti-myogenin antibody (50% F5D hybridoma supernatant-50% PBST) overnight at 4°C. Cy3-conjugated goat anti-mouse secondary antibody was used for visualization. Nuclei were stained with a bis-benzimide solution (Sigma, St. Louis, MO) in PBST.
Statistics.
Means and SE were determined for samples from different animals. To
determine differences in mean values of expression of myogenin and
nAChR -subunit RNA and myogenin protein, one-way analyses of
variance were performed. If the F statistic of the analysis
of variance showed significance, differences among means were detected
by using the Tukey-Kramer multiple comparisons post hoc test. The level
of significance was set a priori at P < 0.05. Values
are expressed as means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Myogenin and nAChR mRNA expression in aged rodent muscle suggest
the existence of denervated fibers.
Muscle denervation results in increased myogenin and nAChR mRNA levels
(1). After 10 days of denervation in young (4 mo old)
rats, myogenin mRNA increased 300-fold (P < 0.05; Fig.
1). Similarly, nAChR -subunit mRNA was
increased ~100-fold (P < 0.05) after 10 days of
denervation (Fig. 1). Thus myogenin and nAChR RNA levels appeared to
reflect whether the muscle was innervated or denervated.
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-subunit mRNA (Fig. 1). On the
basis of the pattern of gene expression during short-term denervation,
the 80- to 90-fold increase in the expression of myogenin and nAChR -subunit in muscles from 32-mo-old rats suggested that a significant number of muscle fibers in old animals were denervated.
Expression of myogenin in denervated young muscle.
RNase protection and Western blot assays were used to determine if
myogenin protein reflected myogenin RNA levels after denervation of
skeletal muscle in young rats. In addition, because myogenin appears to
mediate denervation-induced increases in nAChR gene transcription
(12, 27, 28), we also assayed
nAChR -subunit RNA levels after muscle denervation. From 1 to 120 days of denervation, the overall pattern of expression of the myogenin
protein paralleled that of the myogenin RNA (Fig.
2). Myogenin and nAChR -subunit RNA
levels increased after muscle denervation, reaching their maximum level
by 30 days (Fig. 2A). However, these levels of myogenin and
nAChR RNAs were not maintained and decreased significantly (P < 0.05) in muscles that remained denervated for
>30 days (Fig. 2A). Therefore, it appears that the level of
transcription of myogenin-responsive genes, such as the nAChR
-subunit, follows the level of myogenin RNA expressed in the muscle.
These results suggest that myogenin RNA will reflect myogenin protein
levels.
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-subunit and
myogenin RNAs increased as myogenin protein increased (Fig. 2). After
10 days of denervation there was a modest, but discernible, decrease in
the expression of both myogenin mRNA and protein (P < 0.05). In contrast, after 10 days of denervation, the level of
expression of the nAChR -subunit was still increasing (Fig.
2A). Around 1 mo of denervation, myogenin RNA and protein and the nAChR -subunit RNA had already reached their highest level
of expression (~600-, 14-, and 600-fold, respectively) and then their
mRNAs began to decline (Fig. 2). By 120 days postdenervation, the
levels of myogenin and nAChR RNA declined to ~20 and 10%
(P < 0.05), respectively, of the levels observed at 30 days postdenervation. Myogenin protein level at 120 days
postdenervation was ~70% (P < 0.05) of the 30-day
postdenervation value. The smaller decrease in myogenin protein between
30 and 120 days of denervation may be a consequence of muscle atrophy,
resulting in a substantial shift in the nuclear protein to total
protein ratio (see DISCUSSION).
Myogenin is a nuclear protein that mediates gene activation. Therefore,
we examined when we could first observe myogenin accumulation within
myonuclei using immunohistochemistry. Immunostaining with antibody
against myogenin showed no staining in innervated (Fig. 3A) and 1-day denervated
muscle (not shown). However, by 2-3 days of muscle denervation
there was a significant accumulation of myogenin protein in myonuclei
(Fig. 3). In experimentally denervated muscle, practically all
myonuclei were stained for myogenin (Fig. 3, C and
D). After 120 days of denervation, muscle fiber
cross-sectional area was decreased significantly, but the size of the
nuclei appeared unchanged (Fig. 3E), and the majority of the
myonuclei was positive for myogenin (Fig. 3, E and
F).
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Expression of myogenin in aging muscle.
The accumulation of myogenin protein in muscle as animals age
is shown in Figs. 4 and
5. From 4 to 17 mo of age, myogenin protein levels did not change significantly (P > 0.05). However, by 24-32 mo of age, myogenin levels increased
(Fig. 4). All myonuclei were negative for myogenin immunostaining in
muscles from 4-mo-old rats (Fig. 5A). In muscles from
17-mo-old rats, a very small number of normal-sized fibers exhibited
myonuclear staining for myogenin (Fig. 5C). The number of
myogenin-positive fibers increased with age. There was a significant
population of small fibers whose nuclei showed detectable myogenin
accumulation in muscles from 26-mo-old rats (Fig. 5E).
Within the 26-mo-old muscle there were also some small regenerating
fibers, recognized by centrally located nuclei, that were myogenin
positive (data not shown). Some larger fibers had centrally located
nuclei and were myogenin negative (Fig. 5, E and
F), suggesting that these fibers represent
regenerated and innervated fibers. However, the majority of the fibers
from 26-mo-old muscles was normal in size and had myogenin-negative nuclei (Fig. 5E).
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Comparison of young and old denervated muscle.
In muscles from 24- to 32-mo-old rats, myogenin and nAChR - and
-subunits mRNA increased >10-fold compared with 4-mo-old controls
(Refs. 10, 22, and 24 and Fig. 1). These changes in gene expression
during aging are presumed to reflect the presence of a population of
denervated muscle fibers. To assess the ability of old animals to
respond to denervation, we compared levels of myogenin and nAChR
-subunit mRNA expression in muscles from young (4 mo) and old (24 mo) rats after denervation. After 10 days of denervation, the magnitude
of the increase in myogenin and nAChR -subunit mRNA was greater for
young than for old animals, but the absolute level of expression in
denervated muscles was similar (Fig.
6A). Western blotting
experiments suggested a similar pattern of expression for the myogenin
protein (Fig. 6B). Finally, immunostaining of innervated
muscles showed little to no detectable myogenin-positive myonuclei in
fibers from young rats and the majority of fibers from old rats (Fig.
7, A and B and
E and F). After 30 days of denervation, a
significant accumulation of myogenin was observed in practically all
myonuclei in muscles from both young and old rats (Fig. 7, C
and D and G and H).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Several studies have implicated myogenin as an important regulator
of gene expression during muscle development, denervation, and aging
(4, 9, 22, 24,
35). Myogenin is expressed at high levels during the late
stages of myoblast differentiation and during myotube formation. After
myotube innervation and maturation, myogenin returns to a low level of
expression. In contrast to the innervated state, after denervation of
mature muscle, myogenin expression increases dramatically and appears
to induce the expression of activity-dependent genes such as those
encoding the nAChR subunits (9). Although changes in
myogenin mRNA after denervation and during aging have been reported
previously (1, 9, 22, 24), we are unaware of any data that compare the level of
myogenin protein expression with its mRNA profile. Our experiments
showed that after denervation, the pattern of myogenin protein
expression was similar to that of the myogenin mRNA. However, despite
this similarity, there were considerable differences in the magnitude of the changes observed for these two molecules. For example, at 3 days
postdenervation of young rats, myogenin RNA increased ~500-fold,
whereas myogenin protein only increased 14-fold above control. This
difference in the magnitude of the response to denervation may simply
reflect differences in transcriptional and translational controls that
ultimately influence myogenin protein levels. Indeed, it was previously
reported that protein synthesis is slower in unweighted muscles then in
control (17). In addition, RNA and protein stability also
likely contribute to the steady-state levels reported here. Regardless
of the reason for these differences, it is clear that the modest
increase in myogenin protein, relative to its RNA, is sufficient for
mediating the muscle's response to denervation as demonstrated by the
increase in nAChR -subunit mRNA.
Although, in general, myogenin protein levels reflected their RNA level
in short-term denervated muscle, this was not the case for long-term
denervated muscle (Fig. 2). The level of myogenin mRNA expression
between 30 and 120 days of denervation decreased ~80%, whereas
myogenin protein expression decreased only ~30% during this same
time. This is an intriguing observation because nAChR -subunit RNA
levels seem to follow myogenin RNA, but not protein (Fig. 2). There are
several possible explanations for the apparent discrepancy between the
maintained level of myogenin protein expression after long-term
denervation and the significant decrease in nAChR -subunit mRNA
expression. First, myogenin activity can be suppressed by
phosphorylation (reviewed in Ref. 27). In our assays we only measured
total myogenin protein levels, and the amount of active
(nonphosphorylated) myogenin in muscle might be significantly smaller.
Second, other regulatory factors, such as Id (1,
2) or the recently described MyoR (21) and
Mist 1 (19), could be inhibiting nAChR -subunit mRNA
expression after prolonged denervation by forming inactive complexes
with myogenin. Consistent with this idea, we have previously shown an
increase in Id expression after 1 mo of muscle denervation (1). Third, one must consider muscle atrophy and decreased muscle volume after long-term denervation. After 2-4 mo of
denervation, muscle mass decreases by >70% (5) and
muscle fiber volume decreases by ~90% compared with innervated
control muscle (34). In contrast, the relative volume of
the nuclei per fiber increases (34). In our experiments,
we compared the level of myogenin expression to the total amount of
protein in muscle. Consequently, relatively large decreases in
cytoplasmic proteins may have obscured decreases in the level of
myogenin protein after long-term denervation.
Our results showing that 2-3 days after denervation practically all myonuclei stain for myogenin are consistent with the observation that after short-term denervation most of the increase in myogenin expression is localized to the existing myofibers (35, 36). In innervated and 2- and 3-day denervated myofibers, the number of activated satellite cells is very small, representing ~4% of total number of nuclei (30, 34). Between 1 and 2 mo of denervation, the number of satellite cell nuclei increases to 8-12% and then decreases gradually to ~2% after 7 mo of denervation (30, 34). Consequently, the positive myogenin staining that we observed after 120 days of denervation likely reflects a mix between existing and newly formed fibers.
The pattern of myogenin and nAChR -subunit mRNA expression reported
in the present study is similar to previously reported observations
(1, 4, 9, 35).
Using Northern blotting experiments, Eftimie et al. (9)
reported a 40-fold increase in myogenin mRNA in muscles from mice 2 days after denervation. The accumulation of nAChR -subunit mRNA was
delayed and reached a maximal level (70-fold) 7 days after denervation.
Buonanno et al. (4) reported that the maximal level of
myogenin transcripts was found in gastrocnemius muscle 4 days after
denervation. In the experiments of Voytik et al. (35),
myogenin mRNA reached a maximal level of 150- to 200-fold above the
contralateral innervated muscle within the first 10 days after
denervation and then gradually decreased to ~50 times higher than
control at 28 days of denervation. Adams et al. (1)
reported an increase in both myogenin and nAChR subunit mRNA expression
in short-term denervated muscle and a decline in these RNAs as
denervation continued beyond 1 mo. The minor differences between
studies in the temporal and quantitative increases of myogenin and
nAChR subunit RNAs after denervation undoubtedly reflect variability
between species, muscles evaluated, and the methods used to evaluate
and quantify changes in gene expression. Nonetheless, after
denervation, the rapid and dramatic increase in myogenin expression
followed by nAChR subunit mRNA expression and then the subsequent
decline in expression of both molecules remain a consistent observation
across studies.
Several lines of evidence suggest that old muscles have a population of
denervated muscle fibers due to loss of motoneurons (reviewed in Ref.
7). Consistent with this observation, 31-mo-old rats express ~10-fold
higher levels of myogenin and nAChR -subunit mRNA than 2-mo-old rats
(10, 22). Complementing these data, Musaro et
al. (24) reported a significant increase in mRNA
expression for myogenin, MyoD, and AChR -subunit message in 2-yr-old
mice. In agreement with previously published results (10,
22, 24), our data showed a gradual increase
in myogenin and nAChR -subunit mRNA expression in aging animals. The
present study, for the first time, showed that the increase in myogenin
mRNA expression in old rats correlated with an increase in myogenin
protein expression. Muscles from 32-mo-old rats had ~80 times more
myogenin mRNA and 6 times more myogenin protein than muscles from
4-mo-old rats. Furthermore, myogenin immunostaining showed that the
majority of the myogenin-positive fibers in 24- to 32-mo-old rats is
small-diameter atrophic fibers that are located in close proximity.
This proximity likely results from spontaneous denervation during aging
accompanied by collateral sprouting from nearby axons that leads to
fiber type grouping (reviewed in Ref. 8). On denervation of these fibers, one would then observe clustering of myogenin-positive fibers
as we described. Indeed, myogenin-positive fibers likely reflect the
diminished capacity of motoneurons in old animals to sprout and/or
maintain collateral sprouting. Some myogenin-positive fibers from old
rats are also positive for the embryonic isoform of myosin and for
neural cell adhesion molecule (A. Borisov and E. Dedkov,
personal communication), which suggests the presence of a
regenerative process. Whether these fibers are refractory to
reinnervation is not known.
The results described above indicate that muscles from old animals have
a population of presumably denervated fibers that express elevated
levels of myogenin and nAChRs. To determine if muscles in old animals
maintain their ability to respond to experimental denervation, we
compared the response of muscles from young and old animals to
short-term denervation. Despite initial differences in myogenin mRNA
and protein and nAChR -subunit mRNA in innervated muscles from young
and old rats, the level of expression was similar in young and old
animals after denervation. Marsh et al. (22) reported
similar results for young and old rats in response to injections of the
myotoxin bupivacaine. In their experiments, old rats had much higher
initial levels of myogenin than young rats. After bupivacaine
injection, myogenin increased in young and old muscles to similar
levels, but muscles from old animals were characterized by incomplete
recovery of mass and a prolonged elevation of myogenin mRNA expression.
These data suggest that innervated muscle fibers in old animals
maintain their ability to activate the process of adaptation to
experimental denervation but, once denervated, are less likely to
become reinnervated.
In summary, our data indicate that myogenin protein levels reflect RNA levels and either can be used as an indicator of a muscle's response to denervation. These results suggest that the increased myogenin expression detected in old animals is a result of spontaneous muscle denervation. In addition, myogenin levels correlate with the activation of target genes such as those encoding nAChR subunits. Finally, young and old muscle exhibit similar changes in myogenin and nAChR RNAs and myogenin protein after denervation, suggesting that aged muscle maintains signaling cascades involved in activation of these genes and presumably participating in adaptation to muscle denervation.
Perspectives
Aging is accompanied by muscle fiber denervation. In addition, muscles that become denervated for extended periods of time (beyond a couple of months) are poorly reinnervated. The limiting factors for these phenomena are not known. Therefore, an important issue for aging research is to identify mechanisms mediating muscle denervation and reinnervation and to identify ways to facilitate reinnervation after partial denervation. We show here that young and old muscle respond to denervation by upregulating nAChR and myogenin gene expression in a similar fashion. Because myogenin is an important regulator of muscle-specific gene expression, it appears that fibers in muscles of old animals are capable of responding appropriately to a denervation event. These results suggest that factors extrinsic to muscle are playing an important role in reinnervation of denervated fibers in old animals. However, there may be subpopulations of fibers in muscles of old animals that do not respond to denervation adequately. For example, the population of small myogenin-positive fibers we identified in the old animals may differ in their ability or capacity to attract axonal sprouts and express factors necessary for reinnervation. Therefore, future experiments must focus on this population of denervated fibers to determine if they are intrinsically different from their young counterparts.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Eduard Dedkov for helpful discussions.
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FOOTNOTES |
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* T. Y. Kostrominova and P. C. D. Macpherson contributed equally to this work.
This work was supported by National Institute of Health Grants NS-25153 (to D. Goldman) and AG-10821 (to D. Goldman and B. M. Carlson).
Address for reprint requests and other correspondence: T. Y. Kostrominova, Dept. of Cell and Developmental Biology, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0616 (E-mail: kostromi{at}umich.edu).
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. §1734 solely to indicate this fact.
Received 20 October 1999; accepted in final form 17 February 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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