Activated satellite cells fail to restore myonuclear number in
spinal cord transected and exercised rats
Esther E.
Dupont-Versteegden1,
René J. L.
Murphy2,
John
D.
Houlé2,
Cathy M.
Gurley1, and
Charlotte A.
Peterson1
Departments of 1 Geriatrics and
2 Anatomy, University of Arkansas
for Medical Sciences, and Central Arkansas Veterans Health Care
System, Little Rock, Arkansas 72205
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ABSTRACT |
In this study, possible mechanisms underlying soleus muscle
atrophy after spinal cord transection and attenuation of atrophy with
cycling exercise were studied. Adult female Sprague-Dawley rats were
divided into three groups; in two groups the spinal cord was transected
by a lesion at T10. One group was
transected and killed 10 days later, and another group was transected
and exercised for 5 days starting 5 days after transection. The third group served as an uninjured control. All animals received a
continuous-release 5'-bromo-2'-deoxyuridine pellet 10 days
before they were killed. Transection alone and transection with
exercise lead to activation of satellite cells, but only the exercise
group showed a trend toward an increase in the number of proliferating
satellite cells. In all cases the number of activated satellite cells
was significantly higher than the number that divided. Although the
number of cells undergoing proliferation increased with exercise, no
increase in fusion of satellite cells into muscle fibers was apparent. Spinal cord transection resulted in a 25% decrease in myonuclear number, and exercise was not associated with a restoration of myonuclear number. The number of apoptotic nuclei was increased after
transection, and exercise attenuated this increase. However, the
decrease in apoptotic nuclei with exercise did not significantly affect
myonuclear number. We conclude that apoptotic nuclear loss likely
contributes to loss of nuclei during muscle atrophy associated with
spinal cord transection and that exercise can maintain muscle mass, at
least in the short term, without restoration of myonuclear number.
muscle atrophy; apoptosis; myogenin; spinal cord injury
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INTRODUCTION |
ADULT SKELETAL MUSCLE is capable of changing its size
depending on the demands placed on it. Several conditions, such as
disuse, denervation, malnutrition, and microgravity, lead to atrophy of muscles. Other stimuli, such as resistance exercise and overload, are
associated with muscle hypertrophy. In a previous study we showed that
spinal cord transection rapidly decreased fiber size in affected
muscles and that this atrophy was ameliorated by cycling exercise (12).
The cellular and molecular mechanisms underlying these changes are
poorly understood, but regulation of protein synthesis and degradation
is likely involved (37). In addition, it has been shown that hindlimb
unweighting and spaceflight decrease muscle mRNA levels (4, 17, 38),
and thus the rate of transcription may be changed by conditions that
lead to muscle atrophy. A change in transcription could be the result
of a decrease in nuclear number and/or a decrease in transcription per nucleus.
Myonuclear number has been shown to decrease after spinal cord
isolation (2), spaceflight (3), hindlimb suspension (10), and
denervation (40). This loss of nuclei was most prevalent in slow-twitch
muscles and in slow-twitch fibers of mixed or predominantly fast-twitch
muscles. The mechanism by which these nuclei disappear is not clear,
although a possibility is that of programmed cell death or apoptosis.
Apoptosis is characterized by a series of morphological changes that
accompany the death of cells in a wide variety of tissues. These
changes include chromatin compaction and segregation, rapid overall
cellular condensation, budding to produce membrane-enclosed apoptotic
bodies, and disposal of the apoptotic bodies without an inflammatory
response (18). It has been shown that heterokaryons can exhibit
apoptotic changes in a subset of nuclei without affecting the survival
of the other nuclei within the same cell (11). Adult skeletal muscle
fibers contain numerous nuclei within one cytoplasmic unit, and these nuclei can independently undergo apoptotic changes under certain conditions. Allen et al. (1) showed that hindlimb unweighting increased
apoptotic myonuclei, which appeared to be randomly distributed within
muscle fibers without signs of degeneration. Apoptotic nuclei also have
been observed in muscles of patients with a variety of neuromuscular
diseases (34, 35), in muscles of dystrophic mice (32), and in muscles
undergoing atrophy due to denervation (36). The effect of exercise on
apoptosis is controversial. Dystrophic and normal mice were shown to
have increased amounts of apoptotic nuclei after running exercise (29),
but in a recent study it was shown that resistance exercise decreased
the frequency of apoptotic myonuclei observed after hindlimb
unweighting (1).
On the other hand, it has been demonstrated that hypertrophying
conditions, such as functional overload (2, 21, 41) and endurance
training (6), increase the number of myonuclei. Satellite cells most
likely serve as the source of new myonuclei. Satellite cells are
undifferentiated myogenic stem cells located between the sarcolemma and
the basal lamina (20) and have been shown to be important during normal
muscle growth (23), regeneration (5, 15), and hypertrophy of skeletal
muscles (28, 30, 31). Satellite cells are activated on muscle damage
and go through cell division, after which they may fuse with existing
fibers to repair the damage or form new fibers if the damage is very extensive (for review see Refs. 5 and 15). Whether satellite cell
proliferation is required for hypertrophy is unclear. Studies have
shown that satellite cells are involved in the hypertrophic response
(26) and that
-irradiation of skeletal muscle, which kills dividing
cells, prevents hypertrophy (28). However, others have shown that
hypertrophy occurs in skeletal muscle even after
-irradiation (19).
Satellite cell activation is characterized by increased expression of
MyoD and myogenin, and therefore these factors have commonly been used
as indicators of satellite cell activation (14, 15). MyoD and myogenin
are members of the group of myogenic regulatory factors that are
involved in inducing muscle-specific gene expression during
embryogenesis. However, their role in adult skeletal muscle is less
clear (24). In a previous study we showed that satellite cells become
activated without overt signs of muscle damage with spinal cord
transection alone or in combination with exercise (12). This indicates
that activation of satellite cells, as measured by MyoD and myogenin expression, occurs independently of muscle damage, and factors other
than damage-induced growth factors may be involved in satellite cell
activation. However, the fate and function of these activated satellite
cells were not explored. A possibility is that satellite cells become
activated but do not progress through the cell cycle. Also, the
question remains whether satellite cells contribute to the decrease in
atrophy with short-term cycling exercise observed after spinal cord
transection (12).
The goal of this study was to investigate factors contributing to
muscle atrophy observed with spinal cord transection and the
attenuation of atrophy with cycling exercise. Specifically, the fate of
satellite cells after spinal cord transection and exercise was
investigated, and the involvement of apoptotic nuclear loss in atrophy
was studied. We focused on early cellular responses of the soleus
muscle, as we showed previously that this muscle was most severely
affected by spinal cord transection and responded to exercise with an
attenuation of atrophy (12).
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MATERIALS AND METHODS |
Animals and experimental protocol.
All procedures were performed in accordance with institutional
guidelines for the care and use of laboratory animals. Adult female
Sprague-Dawley rats (180-220 g) were randomly divided into three
groups (n = 4-5); control rats
did not undergo a spinal cord transection and were not exercised. Rats
in the remaining two groups underwent a complete transection of the
thoracic (T10) spinal cord by
creation of an aspiration lesion 2-3 mm long while under
anesthesia with ketamine (60 mg/kg) and xylazine (10 mg/kg). After
surgery, manual expression of the urinary bladder was carried out twice
daily, and rats received penicillin procaine G and a dextrose saline
injection immediately after surgery. Rats in one group did not exercise
and were killed 10 days after transection (tx10). Rats in the remaining
group were subjected to pedaling exercise on a motor-driven bicycle as
described (12, 16) for 60 min each day beginning 5 days after spinal
cord transection (tx10e5). The tx10e5 rats were killed 5 days after the
first exercise session. To measure nuclei that underwent cell division,
rats received a 21-day continuous-release pellet containing 100 mg of
5'-bromo-2'-deoxyuridine (BrdU; Innovative Research
America, Sarasota, FL), which was constructed to give a dose of 0.022 mg BrdU · g body
wt
1 · day
1
(7). Pellets were implanted subcutaneously in the subscapular region in
tx10 and tx10e5 rats at the time of transection and in the control rats
10 days before they were killed. Animals were killed with an overdose
of pentobarbital sodium. Soleus muscles were carefully dissected,
embedded in freezing medium, snap frozen at resting length in liquid
nitrogen-cooled isopentane, and stored at
70°C.
Immunocytochemistry.
Cross sections of soleus muscles were cut on a cryostat (8 µm), air
dried, and stored at
20°C. Myogenin was detected as
described previously (12). Briefly, sections were rehydrated in PBS and reacted with 0.25%
H2O2
to block endogenous peroxidase activity. Sections then were fixed in
2% paraformaldehyde in PBS and permeabilized using 1% Igepal CA-630
(Sigma Chemical, St. Louis, MO) to allow access of antibody to the
nucleus. All subsequent washes and incubations were performed with
0.1% Igepal in PBS. Myogenin antibody was applied at a concentration
of 2-5 ng/µl, and sections were incubated for 1 h. Myogenin
(F5d) antibody was supplied by W. Wright (University of Texas
Southwestern Medical Center, Dallas, TX) (9). An IgG1 biotin-conjugated
secondary antibody (Zymed, San Francisco, CA) was applied at a dilution
of 1:100. After incubation, streptavidin-horseradish peroxidase (Zymed)
was added, and diaminobenzidine (DAB) peroxidase substrate (Vector
Labs, Burlingame, CA) was applied for color development. Sections were
dehydrated and coverslipped. Positive nuclei were counted in an area
occupied by 70-100 fibers. Number of positive myogenin nuclei was
expressed per 100 fibers.
BrdU incorporation was detected using a BrdU antibody (Boehringer
Mannheim, Indianapolis, IN) according to manufacturer's instructions.
Briefly, soleus muscle sections were rehydrated in PBS and reacted with
0.25%
H2O2;
then they were fixed in absolute methanol. Sections were incubated in 2 N HCl for 60 min at 37°C to denature the DNA, then neutralized in
0.1 M borate buffer at pH 8.5. Muscle sections then were incubated in
PBS containing 1.0% Igepal (Sigma Chemical) to permeabilize the
tissue, and all further washes contained 0.1% Igepal. BrdU antibody
was applied at a concentration of 6-8 ng/µl and incubated for 1 h at room temperature. After the sections were washed, a secondary rat
anti-mouse IgG1 biotin-conjugated antibody (Zymed) was applied at 1:100
dilution for 1 h at room temperature. Streptavidin peroxidase was
applied, and then DAB peroxidase substrate (Vector Labs) was applied
for color development. The number of BrdU-positive nuclei per whole muscle section was determined.
Detection of dystrophin and myogenin on the same section was performed
as described previously (12). Briefly, sections were cut at 8 µm and
reacted with 0.25%
H2O2
to block endogenous peroxidase activity. Dystrophin antibody (NCL-DYS2,
Vector Labs) diluted 1:4 in PBS was applied. An alkaline
phosphatase-conjugated IgG secondary antibody (Zymed) was applied; then
the section was incubated with alkaline phosphatase substrate (Vector
Labs) for color development. Myogenin staining was performed as
described above, except blocking was eliminated.
Detection of BrdU and laminin on the same section was performed as
follows. Sections were first stained for BrdU as described above, and
then laminin staining was performed. Laminin antibody (Sigma Chemical)
was applied at 1:40 dilution; then a rat anti-rabbit IgG alkaline
phosphatase-conjugated secondary antibody (Zymed) was added at a 1:100
dilution. Color development was performed using the alkaline
phosphatase substrate kit.
To detect BrdU and dystrophin on the same section, muscle sections were
rehydrated in PBS and incubated in 0.25%
H2O2
in PBS. After the sections were washed, a dystrophin antibody (mouse
anti-human dystrophin, NCL-DYS2, Vector Labs) was applied at a 1:4
dilution; then a rat anti-mouse IgG1 alkaline phosphatase-conjugated
secondary antibody (Pharmigen, San Diego, CA) was applied. The alkaline phosphatase substrate kit was used to yield a red color for dystrophin staining. Sections were then fixed in methanol, and BrdU staining was
performed as described above, but without the blocking step.
To count myofiber nuclei, a Hoechst dye was applied after dystrophin
staining. Dystrophin staining was performed as described above.
Subsequently, sections were fixed in 2% paraformaldehyde, and
Hoechst-33258 nuclear dye (Molecular Probes, Eugene, OR) was applied at
1.2 ng/ml for 30 min. Sections were viewed with a fluorescent microscope with use of an ultraviolet filter package and photographed. Nuclei within the dystrophin-positive sarcolemma were counted in
70-100 fibers, and the number of nuclei was expressed per 100 fibers. Numbers were not expressed per unit fiber cross-sectional area,
because fiber area changes with the experimental manipulations (12).
Detection of apoptotic nuclei.
Apoptotic nuclei were identified by using a TdT-mediated dUTP nick end
labeling (TUNEL) assay (Boehringer Mannheim). This assay is based on
the fact that apoptotic nuclei display DNA strand breaks, and TdT can
be used to label these DNA strand breaks with fluorescein. Incorporated
fluorescein was then detected by anti-fluorescein antibody conjugated
with horseradish peroxidase. TUNEL detection was performed according to
specifications supplied with the assay. Specifically, sections are
fixed in 4% paraformaldehyde at room temperature, blocked in 0.3%
peroxide in methanol at room temperature, and permeabilized in 0.1%
Triton-X and 0.1% sodium citrate at 4°C. TUNEL mix was then added
to the sections at a 1:7.5 dilution with 30 mM Tris, 140 mM sodium
cacodylate, and 1 mM CoCl2.
Labeling mix was incubated at 37°C for 1 h. Sections were rinsed,
and fluorescein antibody was applied for 30 min at 37°C. Sections
were rinsed, DAB substrate (Vector Labs) was added for color
development, and sections were dehydrated and coverslipped. The number
of positive nuclei of a whole muscle section was counted.
Statistics.
To test for statistically significant differences, ANOVA was used; in
the case of significant differences, Tukey's multiple comparison test
was applied. Statistical significance was assumed at
P < 0.05.
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RESULTS |
Satellite cells are activated after spinal cord transection.
After spinal cord transection and transection with exercise, myogenin
expression in soleus muscles was used as an indicator of satellite cell
activation. Soleus muscle cross sections immunoreacted with myogenin
(A-C) or dystrophin and
myogenin antibodies
(D-F) are shown in Fig.
1; the number of myogenin-positive nuclei
is quantitated in Fig. 2. Rare
myogenin-positive nuclei were observed in control soleus muscles (Fig.
1A). The number of
myogenin-positive nuclei was increased sevenfold in soleus muscles of
tx10 rats (Figs. 1B and 2) and
remained elevated in soleus muscles of tx10e5 rats (Figs.
1C and 2). Although exercise decreased
the atrophy associated with transection (compare myofibers in Fig. 1,
B and C) (12), no difference was observed
in myogenin expression with exercise compared with transection alone
(Fig. 2). In transected rat soleus muscle the average number of
myogenin-positive nuclei was 98 nuclei in an area occupied by 100 fibers (Fig. 2). To distinguish activated satellite cell nuclei from
myofiber nuclei that were also expressing myogenin, the sarcolemma was
stained with dystrophin antibody. Nuclei expressing myogenin located
within the dystrophin-positive sarcolemma are myonuclei, and nuclei
expressing myogenin located outside the sarcolemma reside within
satellite cells. Figure 1, D-F,
shows the result of the double staining. No myogenin-positive nuclei in
satellite cells were found in control soleus; however, some myogenin
was detected in myonuclei (Fig. 1, A
and D). In transected soleus
muscles, myogenin-positive nuclei were found within the sarcolemma and
outside the sarcolemma (Fig. 1, E and F). Approximately 10% of the
myogenin-expressing nuclei reside within satellite cells in muscles
from tx10 and tx10e5 rats. Therefore, spinal cord transection is
associated with increased myogenin expression in myofiber nuclei and
with activation of satellite cells regardless of exercise status.

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Fig. 1.
Satellite cell activation in soleus muscle after spinal cord
transection and exercise. Cross sections of soleus muscle of control
rats (A and
D), rats 10 days after spinal cord
transection (tx10; B, E, and
F), and rats 10 days after spinal
cord transection and 5 days of exercise (tx10e5;
C) were immunoreacted with myogenin
(brown) antibody only (A-C) or
with dystrophin (red) and myogenin (brown) antibodies
(D-F). Nuclear staining for
myogenin was increased in soleus muscle of tx10 and tx10e5 rats
(B and
C). Myogenin-positive nuclei were
detected in myofibers (solid arrows) and satellite cells (open arrows).
For A-E bar in
E, 25 µm; bar in
F, 25 µm.
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Fig. 2.
Increased myogenin expression in soleus muscles from tx10 and tx10e5
rats. Number of myogenin-positive nuclei per area of 100 fibers in
soleus muscle cross sections from control, tx10, and tx10e5 rats is
shown. Bars represent means ± SE . * Significantly different
from control (P < 0.05).
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Activated satellite cells do not contribute to attenuation of
atrophy in response to exercise.
To investigate what happens to activated satellite cells after
transection and exercise, continuous-release BrdU pellets were implanted. BrdU incorporation identifies cells that have undergone cell
division after implantation of the pellets. Representative cross
sections of soleus muscles immunoreacted with BrdU antibody are shown
in Fig. 3, and quantitation of the labeled
nuclei is depicted in Fig. 4. Compared with
control (Fig. 3A), the number of
BrdU-positive nuclei in a whole muscle section increased over twofold
with transection and exercise combined (Figs.
3C and
4A), but not with transection alone
(Figs. 3B and
4A). Soleus cross sections were
immunoreacted sequentially with laminin and BrdU antibodies to estimate
the total number of muscle nuclei (satellite cell nuclei + myofiber
nuclei) that had undergone cell division. Laminin is a protein present
in the basal lamina surrounding muscle fibers, and nuclei within the
basal lamina are satellite cell nuclei or myofiber nuclei. Examples of
this staining are shown in Fig. 3,
D-F, and quantitation is shown in
Fig. 4B. Most of the BrdU-stained
nuclei were located outside the basal lamina and are therefore
nonmuscle nuclei. A small percentage of BrdU-positive nuclei were
muscle nuclei, and Fig. 4B shows that
the number of BrdU-positive muscle nuclei tended to increase with
transection and exercise combined. However, this increase failed to
reach significance (P = 0.15). The
average number of muscle nuclei, i.e., satellite cells and myofiber
nuclei combined, per whole soleus muscle section of tx10 animals was 8 and that of tx10e5 animals was 12. These numbers are significantly
lower than the number of activated satellite cells as measured by
myogenin positivity. This indicates that only a small subset of
activated satellite cells subsequently divide. To determine whether any
activated satellite cells that had divided also fused into myofibers
and whether the frequency differed between experimental groups, soleus cross sections were double stained with dystrophin and BrdU antibodies. Because myofiber nuclei are thought to be postmitotic, BrdU-positive nuclei within the sarcolemma most likely derive from satellite cells
that recently fused into the fiber. BrdU-positive nuclei present in
soleus muscle are shown in Fig. 3,
G-I, and are quantitated in Fig.
4C. The number of BrdU-positive
myofiber nuclei was not different between the three groups (Fig.
4C), indicating that there was no
change in fusion of satellite cells with myofibers after transection
alone or with transection and exercise combined.

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Fig. 3.
Localization of nuclei that had divided, thereby incorporating
5'-bromo-2'-deoxyuridine (BrdU) in soleus muscle. Cross
sections of soleus muscles from control rats (A,
D, and G), tx10 rats
(B, E, and
H), and tx10e5 rats
(C, F, and
I) were immunoreacted with BrdU
antibody (brown) alone (A-C),
laminin (red) and BrdU antibodies
(D-F), or dystrophin (red) and
BrdU antibodies (G-I). More
BrdU-positive nuclei were observed after exercise
(C). Some BrdU-positive nuclei were
observed within basal lamina (inside laminin stain) mainly in exercised
rats (arrow, F). Very few positive
nuclei were observed within sarcolemma (inside dystrophin stain,
G-I). Bar for
A-C in
C, 25 µm. Bar for
D-I in
I, 25 µm.
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Fig. 4.
Quantitation of BrdU incorporation in soleus muscles. Numbers of total
BrdU-positive nuclei (A),
BrdU-positive nuclei within basal lamina
(B), and BrdU-positive nuclei within
sarcolemma (C) were counted per
whole soleus muscle cross section of control, tx10, and tx10e5 rats.
Bars represent means ± SE. * Significantly different from
control; # significantly
different from tx10 (P < 0.05).
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Myofiber nuclei are lost after spinal cord transection and are not
restored after exercise.
The results described above suggest that even though satellite cells
appear to be activated, they do not seem to be directly involved in
restoration of muscle fiber size in response to short-term cycling
exercise. This led us to investigate whether myonuclear number changed
in response to transection alone or transection and exercise. Myofiber
nuclei of soleus muscles from control rats (Fig.
5), tx10 rats (data not shown), and tx10e5
rats (data not shown) were counted after a dystrophin stain combined
with Hoechst dye. Nuclei inside the dystrophin-positive sarcolemma were
considered myofiber nuclei. With spinal cord transection, there was a
decrease of 25% in the number of myofiber nuclei (see Fig.
7A). In control soleus muscle,
nuclei were found in almost every myofiber on a given cross section,
whereas in transected soleus muscles many small fibers did not exhibit
nuclei on a cross section. The myofiber nuclear number did not increase
with exercise compared with transection alone (see Fig.
7A). To investigate a possible
mechanism of myofiber nuclear loss with transection, soleus muscle
cross sections were assayed for TUNEL reactivity, an indicator of
apoptotic nuclei (Fig. 6,
A-C). Control soleus muscles
showed little TUNEL positivity (Fig.
6A), but the number of
TUNEL-positive nuclei was increased >35-fold with spinal cord
transection (Fig. 6B, quantitated in Fig.
7B).
Most of the TUNEL-positive nuclei are not muscle nuclei. The number of
TUNEL-positive nuclei decreased ~60% with exercise compared with
transection alone (Figs. 6C and
7B) but remained significantly
higher than control.

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Fig. 5.
Identification of myonuclei in soleus muscle cross sections. A
representative cross section from control soleus muscle stained with
dystrophin (red) and reacted with Hoechst dye (bright blue) is shown.
Bar, 25 µm.
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Fig. 6.
Increased frequency of apoptotic nuclei with spinal cord transection.
Cross sections of soleus muscles from control
(A), tx10
(B), and tx10e5
(C) rats were assayed for
TdT-mediated dUTP nick end labeling (TUNEL) reactivity (brown). Arrows
indicate examples of TUNEL-positive nuclei. Bar for
A-C in
C, 25 µm.
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Fig. 7.
Quantitation of myonuclear number and apoptotic nuclei after spinal
cord transection and exercise. A:
myofiber nuclei per 100 fibers for soleus muscles of control, tx10, and
tx10e5 rats. B: number of
TUNEL-positive nuclei on a whole cross section of soleus muscles from
control, tx10, and tx10e5 rats. Bars represent means ± SE.
* Significantly different from control;
# significantly different
from tx10 (P < 0.05).
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DISCUSSION |
In this study we investigated myonuclear number and found that there
was a 25% decrease in myonuclei accompanying atrophy after spinal cord
transection. Atrophy in soleus muscle has been shown to be associated
with a loss of myonuclei, independent of the manner in which atrophy
was induced (2, 3), thereby maintaining the myonuclear domain
(DNA-to-cytoplasmic ratio). The mechanism by which these nuclei are
lost is unclear, but it may be through apoptosis. Apoptotic nuclear
loss has been shown to increase with a number of muscle diseases (32,
34, 35) and with denervation (36). Furthermore, a recent study showed that apoptotic nuclear loss was increased with atrophy associated with
hindlimb suspension (1). In the present study we found that the number
of apoptotic nuclei dramatically increased with spinal cord transection
and accompanying atrophy. With exercise, there was a decrease in the
number of apoptotic nuclei compared with transection alone. Because
apoptosis is a rapid process, the number of TUNEL-positive myonuclei at
any given time is likely an underestimate of the actual number of
nuclei that have undergone apoptosis over the 10 days after
transection. Thus these results suggest that apoptotic nuclear loss is
one potential mechanism by which myonuclear number decreases during
muscle atrophy in response to spinal cord transection.
As the number of myonuclei decreased with atrophy due to spinal cord
transection, we determined whether exercise-related reduction of
atrophy was associated with a restoration in myonuclear number and
whether satellite cells were involved in this response. We showed
previously that muscle fiber cross-sectional area of soleus muscles
decreased to ~40% of its original size 10 days after spinal cord
transection. Moreover, we found that cycling exercise prevented this
decrease in muscle fiber size (12, 16), and we hypothesized that
satellite cells are involved in the attenuation of atrophy after
exercise. Surprisingly, satellite cell activation, as measured by
myogenin expression, was observed with spinal cord transection alone
and appeared unaffected by exercise. The signals for satellite cell
activation with transection alone are unclear, because no overt muscle
damage occurs (12). It is known that myogenin expression increases in
myonuclei in response to a decrease in electrical activity caused by
denervation (13) or application of a neurotoxin (42). We also showed
previously that MyoD and myogenin mRNA of whole soleus muscle increased
transiently after spinal cord transection (12), likely because of the
decrease in electrical activity in muscle after transection. It is
possible that satellite cells also respond to a decrease in electrical
activity in muscle. At issue was whether the activated satellite cells
subsequently divide and fuse into fibers. We hypothesized that exercise
might promote this process, allowing satellite cells to replenish
myonuclei lost with atrophy. Double-labeling techniques with the BrdU
antibody in combination with antibodies against components of the basal lamina and sarcolemma demonstrated that in the exercised soleus there
seemed to be a preferential increase in replicating muscle cells,
although most labeled nuclei were from nonmuscle cells. It has been
shown that denervation increases the number of dividing satellite cells
and connective tissue cells (22), but denervation is associated with
nerve damage extending into the muscle, which is not the case with
spinal cord transection. Therefore, spinal cord transection alone
induces expression of myogenic regulatory factors in satellite cells,
and thus activation, but does not appear to generate a strong enough
signal for satellite cell proliferation. By contrast, exercise appears
to provide signals necessary for satellite cells to enter the cell cycle.
Even though the number of BrdU-labeled nuclei was increased with
exercise, no increase in BrdU-labeled nuclei inside the muscle fibers
themselves was observed with exercise, suggesting that satellite cells
that had divided had not fused with the muscle fiber. This finding
correlated with the observation that the number of myonuclei did not
increase with exercise compared with transection alone. Thus, at this
early time point after the onset of exercise, satellite cells are not
involved in exercise-induced maintenance of muscle fiber size but may
participate at later time points. The most likely explanation for the
decrease in atrophy is that exercise changes protein synthesis and
degradation in such a way that protein is accumulated in the muscle.
Indeed, it has been shown that protein synthesis is increased to a
greater extent than protein degradation shortly after resistance
exercise, resulting in a net increase in muscle protein balance (27).
However, if the myonuclear domain is to remain constant, nuclei would
have to be added. It is possible that satellite cells will eventually fuse with fibers to restore myonuclear number. Another possibility is
that addition of nuclei to fibers with exercise is not necessary because of the fiber type switching that occurs with spinal cord transection. It has been shown that spinal cord transection is associated with a loss of slow-twitch fibers and an increase in fast-twitch fibers (16). As fast-twitch fibers have a larger myonuclear
domain (2, 39), the loss of nuclei may reflect the fiber type
transformation, which occurs after spinal cord transection. Thus,
whereas exercise results in a reduction of atrophy, but not in
restoration of slow-twitch fiber types (12, 16), satellite cells may
not be required, and the larger myonuclear domains characteristic of
fast-twitch fibers may be stable under these circumstances. To
investigate whether satellite cells contribute to myonuclei, studies
are under way to look at later time points after initiation of exercise
in this model.
An important finding of this study is that the number of activated
satellite cells as measured by myogenin expression is significantly higher than the number of BrdU-positive muscle nuclei in soleus muscles
from tx10 and tx10e5 rats. This is deduced from the fact that we found
~98 myogenin-positive nuclei per 100 muscle fibers, with 10% of
these within satellite cells. As a soleus muscle in adult female
Sprague-Dawley rats contains 2,500-3,000 muscle fibers (25), it
follows that there is expression of myogenin in ~250 satellite cell
nuclei per whole muscle section in tx10 and tx10e5 rats. In
BrdU-positive muscle, there are only 8-12 nuclei per whole muscle
section. This suggests that not all activated satellite cells
subsequently divide. Indeed, Tatsumi et al. (33) suggested that
activation is an event separate from proliferation, since quiescent and
activated satellite cells do not respond in the same way to growth
signals. Therefore, different signals may be necessary for activation
vs. proliferation of satellite cells. Interestingly, Cornelison and
Wold (8) showed that, in some satellite cells associated with isolated
muscle fibers, myogenin is expressed in the absence of proliferation,
supporting the idea that activation and proliferation may be separable
events. There is also the possibility that satellite cells fuse into
muscle fibers on activation without dividing first; however, there is no experimental evidence to support this possibility.
In summary, myonuclear loss was associated with the muscle atrophy
after spinal cord transection, and an increase in apoptotic nuclei is a
potential mechanism to account for this loss. The attenuation of
atrophy by short-term exercise in soleus muscles of spinal cord
transected rats occurs without satellite cell fusion or an increase in
myonuclear number, even though activation of satellite cells is
prominent. Studies are needed to investigate whether satellite cells
will eventually contribute to the increase in muscle fiber size
observed with cycling exercise after spinal cord transection and to
elucidate the signals responsible for the satellite cell activation vs. proliferation.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Child Health and
Human Development Grant HD-35096. E. E. Dupont-Versteegden is a
recipient of National Institute of Arthritis and Musculoskeletal and
Skin Diseases Fellowship AR-08432, and R. J. L. Murphy is a Natural
Sciences and Engineering Research Council of Canada fellowship recipient.
 |
FOOTNOTES |
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.
Address for reprint requests: C. A. Peterson, Dept. of Geriatrics, VA
Hospital, Research 151, 4300 West 7th St., Little Rock, AR 72205 (E-mail: petersoncharlottea{at}exchange.uams.edu).
Received 9 March 1999; accepted in final form 19 May 1999.
 |
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