Resident stem cells are not required for
exercise-induced fiber-type switching and angiogenesis
but are necessary for activity-dependent muscle
growth
Ping Li, Takayuki Akimoto, Mei Zhang, R. Sanders Williams and Zhen Yan
Am J Physiol Cell Physiol 290:1461-1468, 2006. First published Jan 11, 2006;
doi:10.1152/ajpcell.00532.2005
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Point:Counterpoint: Satellite cell addition is/is not obligatory for skeletal muscle
hypertrophy
R. S. O'Connor and G. K. Pavlath
J Appl Physiol, September 1, 2007; 103 (3): 1099-1100.
[Full Text] [PDF]
Am J Physiol Cell Physiol 290: C1461–C1468, 2006.
First published January 11, 2006; doi:10.1152/ajpcell.00532.2005.
Resident stem cells are not required for exercise-induced fiber-type switching
and angiogenesis but are necessary for activity-dependent muscle growth
Ping Li, Takayuki Akimoto, Mei Zhang, R. Sanders Williams, and Zhen Yan
Department of Medicine, Duke University Medical Center, Durham, North Carolina
Submitted 21 October 2005; accepted in final form 6 January 2006
endurance exercise; adaptation
are terminally differentiated and
no longer have the ability to proliferate, but they contain a pool
of myogenic stem cells, also called satellite cells, that reside
between the basal lamina and sarcolemma of myofibers (19).
Upon stimulation, such as injury and increased work overload,
satellite cells are activated and undergo proliferation and differentiation to participate in muscle regeneration and hypertrophy (1, 28, 34). It is well known that endurance exercise,
defined as repetitive, prolonged exercise of submaximal intensity, induces phenotypic changes in skeletal muscles that lead
to enhanced exercise capacity and improved metabolic homeostasis (6, 32). Recent advances in molecular biology have
significantly improved the understanding of exercise-induced
skeletal muscle adaptation, as reported in previous studies of
the skeletal muscle signaling-transcription network (2, 7, 17,
22, 30, 33). However, little is known about cellular processes
such as satellite cell proliferation and differentiation in skeletal
muscle adaptation induced by endurance exercise.
ADULT SKELETAL MUSCLE FIBERS
Address for reprint requests and other correspondence: Z. Yan, Department
of Medicine, Duke University Medical Center, Independence Park Facility,
4321 Medical Park Dr., Suite 200, Durham, NC 27704 (e-mail: zhen.yan
@duke.edu).
http://www.ajpcell.org
Considering that satellite cell and other types of stem cells
reside in adult skeletal muscle and could migrate from other
peripheral tissues (5, 16, 19, 23), one might expect that these
stem cells participate in skeletal muscle adaptation in response
to exercise training if they undergo proliferation and differentiation. Indeed, increased satellite cell (muscle precursor) proliferation has been reported in various animal models of endurance exercise, including treadmill running, voluntary running, and chronic motor nerve stimulation (12, 14, 20, 25), as
well as under conditions in which satellite cell proliferation
induced by muscle injury and fiber-type transformation evoked
by motor nerve stimulation was enhanced (18, 29). Accordingly, it has been hypothesized that myogenic stem cell proliferation is directly involved in or prerequisite to contractile
activity-induced fiber-type switching. We recently obtained
evidence that voluntary running stimulates cell cycle gene
expression and promotes cell proliferation in mouse skeletal
muscle (8) before the onset of fiber-type switching and angiogenesis (2, 3, 31). These findings prompted us to ascertain the
functional role of cell proliferation in skeletal muscle adaptation in this physiological exercise model.
In this study, we hypothesized that resident stem cell proliferation induced by voluntary running plays an essential role
in fast-to-slow fiber-type switching, enhanced angiogenesis,
and/or increased muscle mass. We first characterized temporal
features of cell proliferation in mouse skeletal muscle during
voluntary running using 5-bromo-2⬘-deoxyuridine (BrdU) labeling in vivo. We then determined the functional fates of the
proliferative cells using long-term BrdU labeling. Finally, we
used X-ray irradiation to ablate resident stem cell activity to
ascertain the functional role of resident stem cell proliferation
in skeletal muscle adaptation. In contrast to our hypothesis, our
results have shown that only changes in muscle mass, not
fiber-type switching or angiogenesis, are dependent on resident
stem cell proliferation.
MATERIALS AND METHODS
Animals. Male C57BL/6J mice (8 wk of age) were obtained from
The Jackson Laboratory (Bar Harbor, ME), housed in temperaturecontrolled (21°C) quarters, maintained on a 12:12-h light-dark cycle,
and provided with water and chow ad libitum. All procedures involving these animals conformed to the Guidelines for the Care and Use
of Laboratory Animals published by the National Institutes of Health
and were approved by the Duke University Animal Use Committee.
Voluntary running model. Mice were randomly assigned to sedentary control and running groups. Mice in the running group were
housed individually in cages equipped with locked running wheels for
3 days for acclimatization, and they were allowed to run by unlocking
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Li, Ping, Takayuki Akimoto, Mei Zhang, R. Sanders Williams, and
Zhen Yan. Resident stem cells are not required for exercise-induced fibertype switching and angiogenesis but are necessary for activity-dependent
muscle growth. Am J Physiol Cell Physiol 290: C1461–C1468, 2006. First
published January 11, 2006; doi:10.1152/ajpcell.00532.2005.—Skeletal
muscle undergoes active remodeling in response to endurance exercise training, and the underlying mechanisms of this remodeling
remain to be defined fully. We have recently obtained evidence that
voluntary running induces cell cycle gene expression and cell proliferation in mouse plantaris muscles that undergo fast-to-slow fibertype switching and angiogenesis after long-term exercise. To ascertain
the functional role of cell proliferation in skeletal muscle adaptation,
we performed in vivo 5-bromo-2⬘-deoxyuridine (BrdU) pulse labeling
(a single intraperitoneal injection), which demonstrated a phasic
increase (5- to 10-fold) in BrdU-positive cells in plantaris muscle
between days 3 and 14 during 4 wk of voluntary running. Daily
intraperitoneal injection of BrdU for 4 wk labeled 2.0% and 15.4% of
the nuclei in plantaris muscle in sedentary and trained mice, respectively, and revealed the myogenic and angiogenic fates of the majority
of proliferative cells. Ablation of resident stem cell activity by X-ray
irradiation did not prevent voluntary running-induced increases of
type IIa myofibers and CD31-positive endothelial cells but completely
blocked the increase in muscle mass. These findings suggest that
resident stem cell proliferation is not required for exercise-induced
type IIb-to-IIa fiber-type switching and angiogenesis but is required
for activity-dependent muscle growth. The origin of the angiogenic
cells in this physiological exercise model remains to be determined.
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chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway,
NJ). The following primary antibodies were used for immunoblot
analysis: rabbit anti-MyoD antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) and rabbit anti-␥-tubulin antibody (Zymed Laboratories, San Francisco, CA). The intensities of the bands were quantified using Scion Image software.
Statistical analysis. Data are presented as means ⫾ SE. For the
time course experiments, one-way ANOVA was used to compare
mean values of each time point with those of the sedentary group. The
Dunnett test was used to determine statistically significant differences.
For comparison between the sedentary and trained mice, a two-tailed
Student’s t-test was performed. Two-way ANOVA, followed by the
Newman-Keuls test, was used to compare results in irradiated and
contralateral nonirradiated plantaris muscles from sedentary and
trained mice. P ⬍ 0.05 was accepted as statistically significant for all
analyses.
RESULTS
Voluntary running induces phasic increase of cell proliferation in mouse skeletal muscle. To define the temporal features
of voluntary running-induced cell proliferation in skeletal muscle, we performed in vivo BrdU pulse labeling in plantaris
muscle. As shown by indirect immunofluorescence analysis
(Fig. 1, A and B), voluntary running resulted in a significant
increase in BrdU-positive cells between days 3 and 14 compared with the sedentary control mice (P ⬍ 0.01). The number
of BrdU-positive cells returned to the basal level after 4 wk of
training. This phasic increase in BrdU labeling occurred concurrently with induced mRNA expression of cell division
control protein 6 Cdc6 (Fig. 1C), a cell cycle gene that is
essential for cell proliferation and DNA replication (35).
Voluntary running induces cell proliferation in skeletal
muscle with different functional fates. To better understand the
functional role of cell proliferation in exercise-induced skeletal
muscle adaptation, we traced the functional fate of the proliferative cells by labeling the proliferating cells and allowing
them to differentiate into mature functional cells. The longterm BrdU labeling was achieved by daily intraperitoneal
injection of BrdU during 4 wk of voluntary running. As shown
in Fig. 2A, significantly more BrdU-positive nuclei were detected in plantaris muscles of sedentary control mice after
long-term BrdU labeling (8.6 ⫾ 0.8/mm2) compared with the
pulse-labeling results (0.9 ⫾ 0.2/mm2) (Fig. 1B). Voluntary
running (4 wk) resulted in a 7.5-fold increase in BrdU-positive
nuclei (102.6 ⫾ 25.2 per mm2; P ⬍ 0.01 vs. sedentary control)
in plantaris muscles (Fig. 2, A and B). The percentages of
BrdU-positive nuclei in plantaris muscles were 2.0 ⫾ 0.4% and
15.4 ⫾ 1.6% in sedentary and exercise-trained mice, respectively. The number of BrdU-positive nuclei in soleus muscle
was significantly higher than that in plantaris muscle (12.7 ⫾
1.8 per mm2; P ⬍ 0.001 vs. plantaris muscle) in sedentary
control mice, and voluntary running resulted in a 12.5-fold
increase in BrdU-positive nuclei (159.1 ⫾ 26.5 per mm2; P ⬍
0.001 vs. sedentary soleus muscles). The percentages of BrdUpositive nuclei in soleus muscles were 1.9 ⫾ 0.4% and 20.0 ⫾
3.7% in sedentary and exercise-trained mice, respectively.
Indirect immunofluorescence was used to ascertain the functional identities of these BrdU-positive cells. The BrdU-positive cells in exercised plantaris muscle were of multiple functional fates (Fig. 2C and Table 1). In general, 32.5 ⫾ 3.8% of
the BrdU-positive nuclei were confirmed to be muscle nuclei
(according to relationship to laminin staining) and 28.4 ⫾ 3.2%
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the wheels at the beginning of a dark cycle for 1, 3, 7, 14, or 28 days,
followed by a 24-h resting period (n ⫽ 7 for each time point) as
previously described (4, 31). Sedentary mice (n ⫽ 7) without access
to a running wheel were used as controls. In the long-term BrdU
labeling experiment, mice were randomly divided into sedentary (Sed;
n ⫽ 7) and exercise groups (Ex; n ⫽ 7), and mice in the exercise
group were allowed to run voluntarily for 4 wk. After single-hindlimb
X-ray irradiation, mice were allowed to recover for 3 days before
voluntary running for 2 or 4 wk (n ⫽ 5 for each time point). A
separate group of irradiated mice with no running wheel access were
used as sedentary controls (n ⫽ 5). X-ray irradiation of a single
hindlimb did not have a significant impact on running activity (data
not shown).
BrdU labeling in vivo. To pulse label proliferative cells in skeletal
muscles, the mice were allowed to run voluntarily for 1, 3, 7, 14, or
28 days. A single dose of BrdU (500 mg/kg body wt) was injected
intraperitoneally immediately after the last bout of running activity,
followed by a 24-h resting period (by locking running wheels) before
samples were harvested. For long-term labeling, the mice were allowed to run voluntarily for 28 days, and BrdU (500 mg/kg body wt)
was injected intraperitoneally daily. Sedentary mice injected with
BrdU were used as controls in both pulse-labeling and long-term
labeling experiments.
Ablation of resident stem cells in hindlimb muscles by X-ray
irradiation. Mice were anesthetized with pentobarbital sodium, and
their right hindlimbs were exposed to a single dose of 2,500-rad
ionizing irradiation for 23 min using an XRAD-320 orthovoltage unit
(Precision X-ray, East Haven, CT). The rest of the animal was
shielded from the radioactive source using a 2-cm-thick lead attenuator.
Immunohistochemistry. Muscles were harvested, saturated in 30%
sucrose-PBS for ⬃2 h, placed into optimal cutting temperature (OCT)
tissue-freezing medium (Miles Pharmaceuticals, West Haven, CT),
and frozen in liquid nitrogen-cooled isopentane. Frozen sections (6
␮m) were cut using a cryostat and placed onto microscope slides
(Superfrost Plus; Fisher Scientific, Pittsburgh, PA). Before being
stained, the sections were fixed in 4% paraformaldehyde-PBS for 10
min at 4°C, permeabilized with 0.3% Triton X-100-PBS for 10 min at
4°C, and then blocked in 5% normal goat serum (NGS)-PBS for 30
min at room temperature, followed by incubation with a different
primary antibody in 5% NGS-PBS at 4°C overnight. Rat anti-CD-31
antibody (1:25 dilution; Serotec, Raleigh, NC) and rat anti-laminin
antibody (1:100 dilution; Chemicon International, Temecula, CA)
were used for endothelial cells and basal lamina, respectively. Three
consecutive washes with PBS for 5 min each were followed by
sequential incubation with secondary antibodies (rhodamine or FITC
conjugated, 1:25 dilution; Jackson ImmunoResearch Laboratories,
West Grove, PA) at room temperature for 30 min. The sections were
then fixed in 4% paraformaldehyde-PBS for 5 min, and we continued
to stain them for BrdU. In brief, each slice was treated in 1 N HCl at
37°C for 1 h, followed by incubation with either unconjugated (1:25
dilution) or Alexa Fluor-conjugated mouse anti-BrdU primary antibody (1:25 dilution; Molecular Probes, Eugene, OR) overnight at 4°C.
If unconjugated primary antibody was used, we proceeded to use
secondary antibody as mentioned above. Immunofluorescence was
also performed as described previously (31) for type IIa plantaris
muscle fibers. Images were captured under a confocal microscope
(Olympus, Melville, NY) and further analyzed using Scion Image
software (Scion, Frederick, MD). The percentage area comprising
type IIa fibers was calculated in ⬎100 fibers for each muscle. The
capillary density was calculated by counting CD31-positive cells and
measuring the total area for ⬎100 fibers for each muscle.
Immunoblot analysis. Samples were processed as previously described (3). Protein concentration was determined using the RC DC
protein assay kit (Bio-Rad Laboratories, Hercules, CA). Total protein
(60 ␮g) was resolved on 10% SDS-PAGE gel, transferred onto a
nitrocellulose membrane, and immunodetected using an advanced
CELL PROLIFERATION IN MUSCLE DURING RUNNING
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Fig. 1. Voluntary running induces a phasic increase of cell proliferation in mouse plantaris muscle. A: indirect immunofluorescence staining of plantaris muscle
sections from sedentary control (Sed) and 2-wk exercise-trained (Ex) mice. A section of the duodenum (Gut) was used as a positive control for cell proliferation.
All sections were stained with anti-5-bromo-2⬘-deoxyuridine (anti-BrdU) antibody, followed by FITC-conjugated secondary antibody (green) and nuclear DNA
with propidium iodide (PI; red). B: quantitative data for BrdU-positive nuclei in plantaris muscle (n ⫽ 7 for each time point). **P ⬍ 0.01, statistically significant
difference vs. sedentary control muscle. C: semiquantitative RT-PCR for Cdc6 mRNA. Representative images (top) of RT-PCR products for Cdc6 mRNA and
reference control of Gapdh mRNA are shown. Quantitative data (bottom) also are presented (n ⫽ 7 for each time point). *P ⬍ 0.05, statistically significant
difference vs. sedentary control (running time ⫽ 0).
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Fig. 2. Identification of functional fates for the proliferative cells using long-term BrdU labeling. A: indirect immunofluorescence staining of muscle sections
from sedentary control and 4-wk exercise-trained plantaris muscles with daily intraperitoneal injection of BrdU. All sections were stained with anti-BrdU
antibody, followed by FITC-conjugated secondary antibody (green) and nuclear DNA with PI (red). B: quantitative data for BrdU-positive nuclei in plantaris
(PL) and soleus (SO) muscles (n ⫽ 7). ***P ⬍ 0.001. C: indirect immunofluorescence staining of muscle sections from 4-wk exercise-trained plantaris muscles.
To identify BrdU-positive muscle nuclei (top), sections were stained with anti-BrdU antibody, followed by FITC-conjugated secondary antibody (green) as well
as anti-laminin antibody followed by rhodamine-conjugated secondary antibody (red). Central (arrowhead) and peripheral (arrow) nuclei could be identified
easily. To determine the angiogenic fate of the proliferative cells (bottom), sections were stained with anti-BrdU antibody followed by rhodamine-conjugated
secondary antibody (red) as well as anti-CD31 antibody followed by FITC-conjugated secondary antibody (green). Arrows indicate BrdU-CD31 double-positive
cells. D: immunoblot analysis for MyoD. Representative images of MyoD and loading control ␥-tubulin (top) and quantitative data (n ⫽ 5 for each time point)
are shown. **P ⬍ 0.01, statistically significant difference vs. sedentary control (running time ⫽ 0).
were angiogenic (i.e., CD31-positive) cells. The rest of the
BrdU-positive nuclei were of unknown origin in the interstitial
space outside the basal lamina sheath. The involvement of
myogenic stem cells was also implicated by transiently induced
expression of myogenic regulatory factor MyoD during longAJP-Cell Physiol • VOL
term voluntary running, with peak expression occurring at 2
wk (Fig. 2D). Altogether, these findings provide direct evidence that the majority of the proliferative cells induced by
voluntary running in skeletal muscle participate in myogenesis
and angiogenesis.
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CELL PROLIFERATION IN MUSCLE DURING RUNNING
Table 1. Indirect immunofluorescence identification of
functional fates of proliferative cells in adult mouse skeletal
muscles in response to voluntary running
Muscle cell nuclei
Central myonuclei
Peripheral myonuclei
Endothelial cell nuclei
Plantaris, n ⫽ 7
Soleus, n ⫽ 7
13.8⫾3.8%
18.7⫾1.7%
28.4⫾3.2%
20.3⫾4.5%
14.1⫾1.1%
20.5⫾2.7%
Values are means ⫾ SE; n ⫽ 7 muscles per group. The myogenic fate of the
proliferative cells in 4-wk exercise-trained plantaris muscles was determined
using indirect immunofluoresence staining on the basis of anatomic relationship to basal lamina (stained for laminin) (see Fig. 2C, top). The angiogenic
fate of the proliferative cells was determined using indirect immunofluoresence
staining of 5-bromo-2⬘-deoxyuridine (BrdU)-positive nuclei and the endothelial marker CD31. BrdU-CD31 double-positive cells are considered to have the
angiogenic fate (see Fig. 2C, bottom).
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running at 2 wk. We observed a trend of muscle mass increase
(P ⫽ 0.06) in nonirradiated plantaris muscles at 4 wk of
running, which was completely abolished by irradiation. Irradiated soleus muscles showed a similar response to voluntary
running, except that there also was decreased muscle mass in
the sedentary mice (Fig. 3G).
DISCUSSION
In the present study, we have defined a robust, phasic
increase of cell proliferation in mouse plantaris muscle during
long-term voluntary running. The increase was associated with
fiber-type transformation, enhanced angiogenesis, and muscle
growth. These findings confirmed and extended our previous
observations in the same exercise model that voluntary running
induces cell cycle gene expression and cell proliferation in the
recruited skeletal muscles. Consistent with these findings,
long-term in vivo BrdU labeling revealed that voluntary running-induced cell proliferation contributed to myogenesis and
angiogenesis in plantaris muscles. Most important, we have
demonstrated that ablation of resident stem cell activity blocks
activity-induced muscle growth but has no impact on exerciseinduced fiber-type switching and angiogenesis.
Endurance exercise training has been characterized by the
adaptive changes in fiber-type composition and microvasculature (6, 32), and less attention has been paid to the effect on
muscle mass. It was recently shown that voluntary running
with resistance induces increases in recruited skeletal muscle
mass in mice and rats (13, 15), supporting the notion that
workload is a potent determinant of muscle mass in endurance
exercise. However, it is worth noting that in these studies,
animals that ran with little or no load also showed a trend or a
significant increase in plantaris muscle mass after long-term
voluntary running (7– 8 wk) compared with sedentary control
mice. These findings raised an issue of activity-dependent
muscle growth, which is also supported by the finding that
treadmill running induces moderate muscle growth in rats (26).
Our findings that voluntary running promotes muscle growth in
plantaris and soleus muscles are in complete agreement with
these previous findings. Importantly, we have extended the
findings to confirm that the muscle growth induced by voluntary running in mice is dependent on resident myogenic stem
cell function. X-ray irradiation not only may elicit DNA
damage and cell cycle checkpoint to block satellite cell proliferation but also may result in damage to the microenvironment
of the muscle, and collectively these effects may impair the
ability of satellite cells to fulfill their normal function in
promoting muscle growth in response to exercise.
Several other features of cell proliferation in skeletal muscle
have been revealed in this study. First, we have found that in
sedentary mice, soleus muscle, a muscle that is frequently
recruited for antigravity postural activity, had significantly
more proliferating cells per unit of cross-sectional area than the
adjacent plantaris muscle (Fig. 2B). Second, voluntary running
resulted in an ⬃10-fold increase of BrdU-positive nuclei
shown by either pulse labeling at 2 wk of running or long-term
labeling during 4 wk of voluntary running, and it induced an
⬃10% increase in muscle mass in the recruited plantaris and
soleus muscles (Fig. 3G). Third, irradiation-mediated ablation
of resident stem cells resulted in decreased muscle mass in
frequently recruited soleus muscle, but not in plantaris muscle,
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Resident stem cell activity is not required for voluntary
running-induced fiber-type switching and angiogenesis. To
determine the functional role of resident stem cell proliferation
in skeletal muscle adaptation, we used X-ray irradiation to
ablate resident stem cells in a single hindlimb before the start
of long-term voluntary running. We chose a dose of 2,500 rad
on the basis of our previous finding that this dose of local
irradiation is sufficient to block myogenic stem cell-mediated
skeletal muscle regeneration in vivo (34). One-hindlimb X-ray
irradiation did not significantly affect daily running distance
compared with the nonirradiated mice that exercised for the
same running distance in the above-described time course
experiment (8.7 ⫾ 0.4 km/day for irradiated mice vs. 9.9 ⫾ 0.7
km/day for nonirradiated mice, n ⫽ 5–7; P ⬎ 0.05). X-ray
irradiation did not alter the number of BrdU-positive cells in
sedentary control mice but did reduce significantly the increase
in BrdU labeling in the trained mice (14.6 ⫾ 2.0 BrdU-positive
cells/mm2 in nonirradiated mice vs. 4.9 ⫾ 0.7 BrdU-positive
cells/mm2 in mice with irradiated plantaris muscle; P ⬍ 0.001)
at 2 wk of running (Fig. 3A). To assess the functional consequence of stem cell ablation, we performed indirect immunofluorescence to characterize skeletal muscle adaptation in irradiated muscles after exercise training and compared the results
with the nonirradiated contralateral muscles. Irradiation had no
significant impact on type IIb-to-IIa fiber transformation (Fig.
3, B and C), which was also confirmed using Western blot
analysis for myosin heavy chain (MHC) type IIa protein (Fig.
3D). Irradiation did not affect the increase of CD31-positive
endothelial cells in plantaris muscle after 4 wk of voluntary
running (Fig. 3, E and F).
Resident stem cell activity is required for voluntary runninginduced muscle growth. It has been reported that voluntary
running with increased workload results in significant skeletal
muscle hypertrophy in soleus and plantaris muscles (13, 15). In
those studies, voluntary running with no or low resistance
induced moderate increases in muscle mass. To determine the
effect of long-term voluntary running on muscle mass in
skeletal muscle in the present study, we measured the muscle
mass for the soleus and plantaris muscles in both irradiated and
nonirradiated hindlimbs at 2 and 4 wk of voluntary running
(normalized to body wt) and compared those data with those
from the sedentary mice. Irradiation did not affect the muscle
mass in plantaris muscles from sedentary mice but completely
blocked the increase in muscle mass induced by voluntary
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even in sedentary mice (Fig. 3G). This treatment blocked
voluntary running-induced increases in muscle mass in both
soleus and plantaris muscles. Collectively, these findings
strongly support the notion that contractile activity promotes
muscle growth through myogenic cell proliferation. We have
also confirmed that voluntary running is associated with muscle injury and regeneration, as evidenced by the detection of
central nuclei (Fig. 2C) and induction of MyoD protein expression (Fig. 2D). MyoD plays an essential role in muscle regeneration (21).
The finding that voluntary running-induced cell proliferation
in plantaris muscle reversed beyond day 14 despite continued
exercise suggests that cell proliferation is associated with a
process as a response to running activity in untrained muscle,
but not in trained muscle, and/or associated with the adaptation
process. It is likely that both muscle injury and muscle adaptation contribute to the increased cell proliferation in this
AJP-Cell Physiol • VOL
voluntary running model. If it were due only to muscle
injury, it would suggest that chronic exercise results in
remodeling of the muscle-rendering resistance to the same
injury stimulus.
Regardless of whether satellite cell proliferation is required
for fast-to-slow fiber-type switching has been an intriguing
question in the field of exercise physiology. Previous findings
in animal models of compensatory hypertrophy (surgical removal of synergistic muscles) show that adaptations in MHC
gene expression are not significantly affected by satellite cell
sterilization (1, 27, 28), which argues against the hypothesis
that satellite cell proliferation plays an essential role in contractile activity-induced fiber-type transformation. However,
before our present study, it remained questionable whether the
results were applicable to endurance exercise. Our findings
have extended these previous findings and provide clear evidence, in a physiological model of endurance exercise, that
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Fig. 3. Resident stem cell proliferation is required for voluntary running-induced muscle growth, but not for fiber-type switching and angiogenesis. A:
quantitative data for indirect immunofluorescence for pulse-labeled BrdU-positive nuclei in nonirradiated (NI) or irradiated (IR) plantaris muscles in sedentary
control or 2-wk exercise trained mice (n ⫽ 5). ***P ⬍ 0.001. B: indirect immunofluorescence staining of type IIa myofibers. An increased percentage of type
IIa myofibers was present in trained plantaris muscles, regardless of X-ray irradiation. C: quantitative data for type IIa myofiber surface area in plantaris muscles
(n ⫽ 5). *P ⬍ 0.05. ***P ⬍ 0.001. D: immunoblot analysis for MHC type IIa protein. Representative images of MHC type IIa and loading control ␥-tubulin
are shown.
CELL PROLIFERATION IN MUSCLE DURING RUNNING
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Fig. 3—Continued. E: indirect immunofluorescence staining of CD31-positive capillary endothelial cells in plantaris muscles that were not irradiated (NI) and
in those that were X-ray-irradiated (IR) from sedentary control or 4-wk exercise trained mice. An increase in CD31-positive capillary endothelial cells in trained
plantaris muscles occurred regardless of X-ray irradiation. F: quantitative data for capillary density in plantaris muscles (n ⫽ 5). ***P ⬍ 0.001. G: comparison
of muscle mass (normalized to body wt) in soleus and plantaris muscles that either were or were not irradiated from sedentary control or exercise-trained mice.
*P ⬍ 0.05. **P ⬍ 0.01. ***P ⬍ 0.001.
fiber-type switching does not require active proliferation of
resident myogenic stem cells.
An unexpected finding in this study is that irradiated muscles
showed significant amount of residual activity of cell proliferation during voluntary running. There are several possibilities
that could explain the residual cell proliferation in the irradiated skeletal muscles. First, it is possible that the radiation dose
used was insufficient to ablate all of the resident stem cells,
although we previously used this radiation dose in the same
strain of mice to block cardiotoxin-induced skeletal muscle
regeneration (34). The fact that irradiation resulted in a complete block of voluntary running-induced muscle growth provides evidence that the X-ray radiation dosage was sufficient to
block myogenic cell function. Second, some of the resident
stem cells may have survived irradiation to promote delayed
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cell proliferation. Recent reports have indicated that there are
stem cell populations within skeletal muscle that appear to be
resistant to radiation-induced damage (11). Third, stem cells
from extramuscular tissues may migrate via the circulation to
the skeletal muscle to compensate for the loss of resident stem
cells. This latter scenario is particularly intriguing and could be
of important functional significance. Although increasing evidence supports the hypothesis that resident satellite cells are
self-sufficient as a source of muscle regeneration (9), we
cannot exclude the possibility that irradiated muscle could
undergo hypertrophy with the participation of stems cells other
than resident myogenic stem cells if the mice were allowed to
run for a longer period.
Irradiation had no significant impact in this study on angiogenesis induced by exercise. This finding raised several inter-
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C1468
CELL PROLIFERATION IN MUSCLE DURING RUNNING
ACKNOWLEDGMENTS
We thank Curtis Gumbs for technical support and Jin Yan for careful
proofreading of the manuscript.
Present address of T. Akimoto: Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda
University, Waseda-Tsurumaki 513, Shinjuku, Tokyo 162-0041, Japan.
GRANTS
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R01 AR-050429-01 and American Heart
Association Grant 0130261N (to Z. Yan).
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esting questions regarding the cellular processes for activityinduced angiogenesis in skeletal muscle. Angiogenesis in skeletal
muscle can occur by intussusception or sprouting (for review, see
Ref. 24), which requires increases in endothelial cells, presumably
through cell proliferation. Our findings suggest that either 1)
endothelial cells are resistant to X-ray irradiation, or 2) other
progenitor cells from extramuscular sources participate in exercise-induced angiogenesis. It should be pointed out that irradiation
at the dose used in this study had previously been shown to block
the proliferation of endothelial cells in vitro (10). It is unlikely that
endothelial cells in skeletal muscle are uniquely resistant to
irradiation. Thus endothelial progenitor cells may be recruited
from the circulation. The possibility that circulating endothelial
progenitor cells plays an essential role in endurance exerciseinduced angiogenesis is worthy of future investigation.
In summary, in the present study, we have defined for the
first time some temporal and functional features of cell proliferation in skeletal muscle in a physiological model of endurance exercise. The results indicate that cell proliferation induced
by voluntary running is required for activity-dependent muscle
growth but not for fiber-type switching and angiogenesis.