J Appl Physiol
90: 1407–1414, 2001.
Exercise-enhanced satellite cell proliferation and new
myonuclear accretion in rat skeletal muscle
HEATHER K. SMITH,1 LINDA MAXWELL,2 CAROL D. RODGERS,3
NANCY H. MCKEE,4 AND MICHAEL J. PLYLEY3
Departments of 1Sport and Exercise Science and 2Pathology, University of Auckland, Auckland,
New Zealand; and 3Faculty of Physical Education and Health and 4Department of Surgery,
University of Toronto, Toronto, Ontario, Canada M5S 1A1
Received 13 September 2000; accepted in final form 6 November 2000
Smith, Heather K., Linda Maxwell, Carol D. Rodgers,
Nancy H. McKee, and Michael J. Plyley. Exercise-en-
hanced satellite cell proliferation and new myonuclear accre-
tion in rat skeletal muscle. J Appl Physiol 90: 1407–1414,
2001.—The effects of increased functional loading on early
cellular regenerative events after exercise-induced injury in
adult skeletal muscle were examined with the use of in vivo
labeling of replicating myofiber nuclei and immunocyto- and
histochemical techniques. Satellite cell proliferation in the
soleus (Sol) of nonexercised rats (0.4 ⫾ 0.2% of fibers) was
unchanged after an initial bout of declined treadmill exercise
but was elevated after two (1.0 ⫾ 0.2%, P ⱕ 0.01), but not
four or seven, daily bouts of the same task. Myonuclei pro-
duced over the 7-day period comprised 0.9–1.9% of myonuclei
in isolated fibers of Sol, tibialis anterior, and vastus interme-
dius of nonexercised rats. The accretion of new myonuclei
was enhanced (P ⱕ 0.05) in Sol and vastus intermedius by
the initial exercise followed by normal activity (to 3.1–3.4%
of myonuclei) and more so by continued daily exercise (4.2–
5.3%). Observed coincident with a lower incidence of histo-
logical fiber injury and unchanged fiber diameter and myo-
nuclei per millimeter, the greater new myonuclear accretion
induced by continued muscle loading may contribute to
an enhanced fiber repair and regeneration after exercise-
induced injury.
eccentrically biased exercise; muscle injury; immunocyto-
chemistry; fiber type; bromodeoxyuridine
A SINGLE BOUT OF STRENUOUS or unaccustomed exercise
induces histological evidence of segmental injury and
subsequent regeneration in a small percentage of fibers
(2, 33), and a latent increase in the activation and
proliferation of satellite cells within both slow- and
fast-twitch muscles of adult rats (8, 15). The satellite
cells are mononucleated myogenic precursors that aid
in the repair or replacement of injured or necrotic
muscle fibers (1, 5) and can also function as a source of
additional myonuclei during adult muscle hypertrophy
(27, 35).
Evidence also suggests that repetition of the same
exercise task results in an attenuated injury response
in muscle (25, 33) and that increased muscle use can
Address for reprint requests and other correspondence: H. K. Smith,
Dept. of Sport and Exercise Science, Univ. of Auckland, Private Bag
92019, Auckland, New Zealand (E-mail: h.smith@auckland.ac.nz).
http://www.jap.org 8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (115.186.033.248) on May 17, 2018.
Copyright © 2001 American Physiological Society. All rights reserved.
enhance subsequent tissue regeneration (4, 16, 38).
Exercise training by progressive treadmill running re-
sults in an elevated satellite cell number (37) and
increased mitotic activity (21), in conjunction with
morphological changes indicative of ongoing muscle
fiber injury and repair. Running training before muscle
autotransplantation results in earlier postsurgical sat-
ellite cell activation than in untrained muscles (26),
and the early regenerative growth (muscle mass and
protein content) of orthotopic (nerve-intact) muscle
grafts after surgery is enhanced by increased mechan-
ical loading (10). In contrast, the withdrawal of normal
contractile loading during early muscle regeneration
does not alter the mitotic activity of satellite cells but
does inhibit subsequent maturational increases in
mass, fiber size, protein concentration, and isomyosin
expression (10, 24). Thus a minimum loading, such as
would be achieved by normal weight-bearing activity,
appears necessary for unimpaired muscle fiber repair
and/or regeneration and is particularly important to
the maturation of newly formed myofibers. Because
satellite cell progeny fuse with existing fibers, the
cumulative increase or accretion of such newly pro-
duced myonuclei would augment the DNA available to
aid in fiber regeneration and maturation and possibly
other adaptive processes. However, the effects of func-
tional loading, beyond that of normal activity levels, on
satellite cell proliferation and the accretion of newly
produced myonuclei during the early regeneration of
adult muscle after exercise-induced injury has not, to
our knowledge, been determined.
We hypothesized that continued overloading by daily
treadmill exercise, compared with normal activity
alone, would enhance satellite cell activation and new
myonuclear accretion in skeletal muscle after exercise-
induced injury.
The aim of the present study was to determine the
effects of either one or daily bouts of locomotory exer-
cise on satellite cell proliferation and new myonuclear
accretion in hindlimb muscles of previously untrained,
adult rats. Our results demonstrated that continuation
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
1407
1408 EXERCISE-ENHANCED ACCRETION OF NEW MYONUCLEI
of the exercise bouts that initially induced focal muscle
injury, compared with normal weight-bearing activity
alone, resulted in an increase in satellite cell prolifer-
ative activity and a greater accretion of new myonuclei
during the early phase of muscle fiber repair and re-
generation. Observed coincident with a lower incidence
of histological fiber injury and unchanged fiber size and
myonuclear number, the new myonuclei likely contrib-
uted to an exercise-enhanced repair, regeneration, or
regrowth of fibers during this time.
METHODS
A total of 64 female Wistar rats (10–12 wk old, 246–306 g)
were used in this study. Rats were housed in a temperature-
controlled room maintained on a 12:12-h light-dark cycle
with food pellets and water available ad libitum. All proce-
dures for animal care and treatment were in accordance with
guidelines of the Canadian Council on Animal Care and
approved by the University of Toronto Animal Care Commit-
tee or by the University of Auckland Animal Ethics Commit-
tee.
All animals were accommodated to human contact and
temporary restraint by daily lifting, moving, holding, and
placement in the treadmill enclosure for 3 days before any
exercise or surgical procedure. Each exercise bout consisted
of 30 min of declined treadmill running (⫺16° grade, 15
m/min). Previous studies have shown that a single bout of
exercise of this nature will induce focal muscle fiber injury in
the soleus (Sol) and gastrocnemius and deep portions of the
quadriceps femoris muscles, whereas the ankle plantar flex-
ors remain unaffected (2, 18, 33).
Experiment 1: In vivo labeling of proliferating satellite
cells. Eight groups of rats were used to examine the effects of
acute and daily exercise on satellite cell proliferation in the
Sol muscle. Rats were killed either as nonexercised (0Ex)
controls (n ⫽ 6); 1 (n ⫽ 10), 2, 4, or 7 (n ⫽ 5/group) days after
a single exercise bout (1Ex); or after two, four, or seven daily
exercise bouts (DailyEx; n ⫽ 5/group) that were each of the
same intensity and duration as 1Ex. Rats in the DailyEx
groups were killed 24 h after the final bout of exercise. This
ensured that the two-, four, and seven-bout DailyEx were
comparable to 1Ex groups with 2, 4, and 7 days of recovery,
with regard to the time elapsed after the initial exercise bout.
A single injection of 5-bromo-2⬘-deoxyuridine (BrdU;
Sigma Chemical, St. Louis, MO) in 0.9% sterile saline (50
mg/kg, 10 mg/ml ip) was administered 1 h before anesthetic
and surgical procedures. Proliferating cell nuclei, including
satellite cells, in the S phase of the cell cycle during this
interval would incorporate the BrdU during DNA replication.
Nonreplicating cell nuclei, including the postmitotic mature
myofiber nuclei and quiescent satellite cells, would remain
unlabeled.
Experiment 2: In vivo cumulative labeling of newly pro-
duced myonuclei. Continuous infusion of BrdU was used for
the identification of myonuclei produced in the Sol, vastus
intermedius (VI), and tibialis anterior (TA) muscles over a
7-day period. Miniosmotic pumps (ALZET 2ML1, Alza Scien-
tific) filled with 2 ml of BrdU solution (25 mg/ml) were
implanted in 0Ex rats (n ⫽ 6) and in rats directly after one
exercise bout (1Ex; n ⫽ 6) or after the first of seven, once-
daily exercise bouts (DailyEx; n ⫽ 6). Rats were first anes-
thetized with methoxyfluorane, and, under aseptic condi-
tions, a preprimed (4 h in sterile saline at 37°C) pump was
placed subcutaneously via a small incision made in the skin
above the scapulae. The pumps, designed to deliver 10 ␮l
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (115.186.033.248) on May 17, 2018.
Copyright © 2001 American Physiological Society. All rights reserved.
solution/h over the implantation period, were left in place
until after the death of the animals 7 days later. The volume
of the remaining contents of each pump was then aspirated
and measured to confirm adequate delivery of the solution
(all contained ⱕ0.35 ml). The concentration of BrdU admin-
istered results in a circulating level of the chemical that far
exceeds that of the endogenous thymidine (9), and thus all
replicating satellite cell nuclei, and daughter nuclei that
subsequently fused to become mature, postmitotic myonu-
clei, should have been labeled with BrdU during the exposure
period. Despite some caution regarding the effects of BrdU on
nuclear activity (3, 36), this same concentration of BrdU
administered for 1–2 wk does not appear to inhibit in vivo
satellite cell proliferation or fusion (6, 20, 29).
Tissue collection and processing. At death, rats were
deeply anesthetized by an intraperitoneal injection of pento-
barbital sodium (Somnotol, MTC Pharmaceuticals), and the
Sol, VI, and TA muscles were excised. Animals were main-
tained under deep anesthesia until all procedures were com-
pleted and were then killed by anesthetic overdose. In exper-
iment 1, samples from consistent midmuscle regions were
coated in embedding medium (Tissue-Tek OCT, Miles, Na-
perville, IL), immersed in isopentane cooled by liquid nitro-
gen, and stored at ⫺80°C until use. Transverse sections (10
␮m thick) were later cut from the stored samples at ⫺20°C,
mounted serially onto adherent-coated slides, and stained for
the identification of proliferating cell nuclei, nonproliferating
myonuclei, fiber type, and general morphology. In experiment
2, muscle samples were fixed overnight (⬇18 h) in Carnoy’s
solution and processed to obtain individual fiber segments
free of interstitial cells and adherent fibroblasts with the use
of procedures described previously (19, 28, 30). Muscle sam-
ples (n ⫽ 6 Sol, 3 TA, 3 VI per group) were rehydrated,
washed in 0.1 M PBS, and incubated in collagenase (141
U/mg, 2.5 mg/ml PBS; type 3, Worthington Biochemical
Lakewood, NJ) for 18 h at 37°C, with intermittent agitation.
The individual fibers were then washed and stained for
newly produced BrdU-labeled, and mature non-BrdU la-
beled, myonuclei.
Immunocyto- and histochemistry. A commercially avail-
able (Boehringer Mannheim Biochemica; Roche Diagnostics)
mouse monoclonal IgG1 primary antibody directed against
BrdU was used to detect the nuclei of cells that had incorpo-
rated the BrdU into their DNA in both muscle sections and
individual fiber segments. Muscle samples were incubated
sequentially with 0.03% hydrogen peroxide in PBS for 30
min, 10% normal horse serum (NHS) in PBS for 20 min, and
the primary antibody, diluted 1:10 in the antibody kit-pro-
vided Tris buffer solution, for 35 min at 37°C. After reincu-
bation with NHS, samples were incubated with biotinylated
anti-mouse IgG (Vector Laboratories, Burlingame, CA), di-
luted in 2% NHS in PBS for 35 min and then avidin-biotin
peroxidase (Vectastain ABC Kit; Vector) for 30 min, and
developed with an aminoethylcarbazole (30 min) or diamino-
benzidine-nickel (5 min) substrate solution (Vector). Fibers
and sections were counterstained with Mayer’s hematoxylin
for the identification of non-BrdU-labeled nuclei. Technical
controls were processed by using the replacement of each
primary and/or its secondary antibody with the diluent only.
One set of BrdU-labeled sections was sequentially labeled
for the identification of the fiber basal laminae by using a
rabbit polyclonal anti-laminin (1:100; Dako) and a biotinyl-
ated, anti-rabbit secondary antibody (1:500; Amersham), fol-
lowed by the avidin-biotin method as above but with a con-
trasting color substrate.
Sections from Sol after 2 days of DailyEx (n ⫽ 5) were also
labeled for BrdU and/or the myogenic regulatory factor MyoD
 EXERCISE-ENHANCED ACCRETION OF NEW MYONUCLEI
(MoAb 5.8A, 1:40; BD Biosciences, San Jose, CA). These
sections were intended to assess the number of BrdU-labeled
nuclei situated within the periphery of muscle fibers that
could be confirmed to be of myogenic origin. The muscle
samples were selected based on the time since the initial
exercise bout, when it was expected that they would exhibit
a larger proportion of BrdU-labeled nuclei of nonmuscle ori-
gin, such as inflammatory or phagocytic cells, than nonexer-
cised muscles, or after a longer recovery period (2, 33).
Sets of sections were also processed for slow (type I) and
fast (type IIa, IIb, IId/x) adult myosin heavy chain (MHC)
isoforms with the use of commercially available monoclonal
mouse IgG1 antibodies (Novocastra Laboratories, Newcastle
upon Tyne, UK) with standard indirect immunohistochemi-
cal methods, as previously described (34). Last, one set of
sections was stained with routine hematoxylin and eosin for
the identification of fibers with degenerative or regenerative
characteristics indicative of prior injury (33). These charac-
teristics included the infiltration by inflammatory or phago-
cytic cells, a disrupted cytoplasm, an atrophic and sharply
angular appearance, an apparent “splitting,” a very small
size and extrafascicular location, and/or the presence of in-
ternalized or centrally located myonuclei (33).
Image analysis and quantification. Muscle samples were
examined by using one of two image analysis systems, each
consisting of a light photomicroscope (Olympus America,
Lake Success, NY; Nikon), color charge-coupled device cam-
era (COHU, San Diego, CA; SPOT, Diagnostic Instruments,
Sterling Heights, MI), personal computer, and image pro-
cessing software (Mocha Image Analysis Software, Jandel
Scientific, San Rafael, CA; ImagePro Plus, Media Cybernet-
ics, Silver Spring, MD). Pixel-to-real-size conversions for
each magnification were made by using images of a stage
slide micrometer with 0.01-mm divisions.
In experiment 1, the number of BrdU-labeled nuclei within
fibers was counted from entire sections of the Sol muscle.
Criteria for the inclusion of BrdU-labeled nuclei as prolifer-
ating satellite cells included a peripheral location within and
abutting the cell boundary and a distinct staining intensity
and size. Later analyses showed no significant difference
between the number of MyoD-positive nuclei per muscle
section and the number of proliferating satellite cells as
determined by the above criteria (P ⫽ 0.24). Double-labeling
of sections with BrdU and MyoD using the current methods
proved difficult because of the nuclear colocalization of both
antigens and the particular difficulty of the detection of
MyoD. Thus, although some of the sublaminal BrdU-labeled
nuclei could be confirmed to be of myofiber origin, the possi-
bility that some of these nuclei were of other cell types cannot
be entirely excluded.
The number of fibers containing BrdU-labeled nuclei and
the number of BrdU-labeled nuclei and hematoxylin-stained
nuclei within each fiber were also tabulated from image fields
(total area ⫽ 2.8 mm2) of each muscle section [582 ⫾ 78 (SD)
total fibers]. Satellite cell proliferation was described by the
number of fibers containing BrdU-labeled nuclei and the
number of BrdU-labeled nuclei calculated as a percentage
of the total number of myonuclei (BrdU-labeled and non-
BrdU-labeled nuclei within fibers). Individual fibers with
BrdU-labeled nuclei were also type classified from fast MHC-
stained serial sections. The percentage of fibers with degener-
ative or regenerative characteristics indicative of prior injury
(33) was determined from hematoxylin-and-eosin-stained
sections (627 ⫾ 100 total fibers) of each Sol muscle. The
overall fiber-type composition of the Sol, VI, and TA was
determined from images (397 ⫾ 114 fibers/muscle) of the
muscles of the 0Ex rats (n ⫽ 6).
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (115.186.033.248) on May 17, 2018.
Copyright © 2001 American Physiological Society. All rights reserved.
1409
In experiment 2, mean fiber segment diameter and the
number of BrdU-labeled and non-BrdU-labeled myonuclei
were determined from images of one focal plane of 16 (Sol and
VI) or 20 (TA) individual fiber segments from each identity-
coded muscle. The fiber segments selected for analysis were
intact, free of surface cells and connective tissue, and pre-
sented an undistorted view for imaging. A minimum of 1,000
myonuclei of each TA (1,030 ⫾ 47) and 1,400 myonuclei of
each Sol (2,474 ⫾ 512) and VI (1,825 ⫾ 371) muscle was
tabulated.
Statistical analyses. One-way ANOVA or, where data did
not follow a normal distribution, nonparametric analyses
(Kruskal-Wallis) were used to compare satellite cell prolifer-
ation over time after 1Ex or DailyEx. A t-test was then used
for the comparison of proliferation between 1Ex and DailyEx
at each time point. One-way ANOVAs were also used to
compare the percentage of newly produced myonuclei, total
myonuclear number, and mean fiber diameter for each mus-
cle among the different exercise groups (0Ex, 1Ex, DailyEx)
or among TA, Sol, and VI under each exercise condition. For
all tests, statistical significance was established at P ⱕ 0.05.
RESULTS
All rats examined over the entire 7-day period in the
two experiments increased in body mass (1.7 ⫾ 2.4%,
P ⫽ 0.001, n ⫽ 28). However, there was no significant
difference (P ⫽ 0.4) between the weight gain of 0Ex
rats (2.6 ⫾ 2.6%) and that of all rats exercised either
once (1.8 ⫾ 2.7%) or seven times (1.0 ⫾ 1.6%). In
experiment 2, Sol wet mass relative to body mass did
not differ among 0Ex (0.51 ⫾ 0.05 mg/g), 1Ex (0.48 ⫾
0.05 mg/g), and DailyEx (0.50 ⫾ 0.05 mg/g) groups.
Experiment 1: Satellite cell proliferation. The num-
ber of BrdU-labeled proliferating satellite cell nuclei
(Fig. 1) observed in whole Sol muscle sections was not
different between 0Ex and any of the time points after
1Ex. However, in DailyEx rats, there was a significant
increase in the number of proliferating nuclei after 2
days of exercise (Fig. 2).
The percentage of fibers with proliferating nuclei,
calculated from image fields in muscle sections, was
0.4 ⫾ 0.2% (1.3 ⫾ 0.7 per 1,000 myonuclei) in 0Ex. A
wide range in the incidence of such fibers (P ⫽ 0.36)
was observed 1 day after 1Ex (0.8 ⫾ 0.9%, 2.8 ⫾ 3.2 per
1,000 myonuclei), with a consistent, low level of cell
replication (0.1–0.4%, 0.8–1.4 per 1,000 myonuclei)
observed 2, 4, and 7 days after 1Ex. The percentage of
fibers with proliferating nuclei was, however, signifi-
cantly greater in DailyEx than 0Ex rats (P ⫽ 0.04),
being highest after two bouts of exercise (1.0 ⫾ 0.2%,
3.4 ⫾ 0.7 per 1,000 myonuclei).
The percentage of fibers demonstrating histological
characteristics of prior injury was significantly greater
7 days after 1Ex (7.4 ⫾ 1.7%) than in 0Ex (4.2 ⫾ 1.7%,
P ⫽ 0.03) or after DailyEx for 7 days (4.7 ⫾ 0.8%, P ⫽
0.01). A 5.9 ⫾ 2.9% (P ⫽ 0.25) incidence of fibers with
degenerative and regenerative characteristics was
found 4 days after 1Ex, yet all other groups showed
consistent mean levels (4.6–5.1%) of affected fibers.
Of the total number of fibers with BrdU-labeled
nuclei, 12.4% reacted positively with the anti-fast
MHC antibody. The overall fiber-type composition of
1410 EXERCISE-ENHANCED ACCRETION OF NEW MYONUCLEI
Fig. 1. A: proliferating satellite cells identified as bromodeoxyuri-
dine (BrdU)-labeled nuclei within the basal laminae of soleus muscle
fibers. Transverse sections were immunolabeled with anti-BrdU
(black) and anti-laminin (red) and counterstained with hematoxylin
(blue). B: the no. of proliferating myonuclei identified by MyoD-
positive staining (red) in soleus muscle sections after 2 days of daily
exercise (n ⫽ 5) was not significantly different from that determined
in the same muscles by the above criteria. Calibration bars ⫽ 50 ␮m.
the Sol was 78% slow MHC, 16% fast MHC, and 6%
slow and fast MHC reactive. Fiber-type composition of
the VI was 42% slow MHC, 55% fast MHC, and 3%
slow and fast; the TA was 5% slow MHC with the
balance reacting for fast MHC.
Experiment 2: Myonuclear accretion. The continuous
infusion of BrdU provided a cumulative, permanent
identification of dividing satellite cells and their prog-
eny, allowing the determination of newly produced
myonuclei accrued within the muscle (Fig. 3).
The number of newly produced nuclei observed after
the 7-day labeling period, expressed as a percentage of
all myonuclei, for the TA, Sol, and VI of 0Ex, 1Ex, and
DailyEx rats, is shown in Fig. 4. In the Sol and VI,
DailyEx resulted in a greater percentage of new nuclei
than did 1Ex and over double the percentage of new
nuclei produced in 0Ex over the same 7-day period (all
P ⬍ 0.05). Exercise had no significant effect on the
accretion of new myonuclei in the TA (P ⫽ 0.09). The
total number of myonuclei counted per millimeter of
fiber segment length did not differ among 0Ex, 1Ex,
and DailyEx groups for any of the muscles examined
(Fig. 5). However, there were more (P ⬍ 0.05) myonu-
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (115.186.033.248) on May 17, 2018.
Copyright © 2001 American Physiological Society. All rights reserved.
clei per millimeter in Sol (126 ⫾ 21) than in VI (96 ⫾
15) and more in Sol and VI than in TA (43 ⫾ 2).
Mean muscle fiber segment diameters were not sig-
nificantly different among 0Ex, 1Ex, and DailyEx
groups for any muscle (Fig. 6).
DISCUSSION
Experiment 1: Satellite cell proliferation. The in-
creased satellite cell proliferation seen in the Sol of rats
that exercised daily for 2 days, compared with 0Ex or
1Ex, falls within the range calculated from prior data
based on transverse sections of the muscle 12 h to 7
days after 2 h of level treadmill exercise (0.3 to 1.8% of
fibers; Ref. 15), yet is lower than that observed 1 day
after 90 min of downhill exercise (8). The exercise in
the present study was shorter in duration and meta-
bolically less demanding than that used in these prior
studies, yet it has previously been shown to elicit fiber
injury and regeneration in the Sol muscle (33, 34). We,
therefore, anticipated that it would be sufficient to
increase satellite cell proliferation, even after a single
bout. However, the exercise only appeared to induce
significant increases in fiber degenerative and regen-
erative characteristics indicative of prior injury at 7
days after 1Ex. Earlier or additional structural injury
and sequelae not visible at the light microscopic level
may have occurred and provided the stimulus for the
increased activation and proliferation of satellite cells
seen in the DailyEx rats. Alternatively, the prolifera-
tion of satellite cells may have been affected by stimuli
associated with the exercise but independent of the
collective histological indexes of injury of the muscle.
Fig. 2. Satellite cell proliferation in the soleus muscle of nonexer-
cised (0Ex) rats (n ⫽ 6); 1 (n ⫽ 10), 2, 4, or 7 (n ⫽ 5/group) days after
1 exercise bout (1Ex); or 1 day after 2, 4, or 7 once-daily exercise
bouts (DailyEx; n ⫽ 5/group). Cell proliferation was determined by
the no. of BrdU-labeled nuclei within fibers of transverse muscle
sections after 1-h exposure to the marker of newly synthesized DNA.
Bars indicate the 25th, 50th, and 75th percentiles; whiskers the 10th
and 90th percentiles. E, Data beyond the 10th or 90th percentiles.
There were no significant differences between 0Ex and 1Ex groups.
* Different from 0Ex and other DailyEx groups, P ⫽ 0.02. ** Different
from 1Ex rats killed at the same time point, P ⫽ 0.01.
 EXERCISE-ENHANCED ACCRETION OF NEW MYONUCLEI
Fig. 3. Myonuclei newly produced over a 7-day period (red) and
mature myonuclei (blue) in isolated muscle fiber segments. Fibers
are from the tibialis anterior (TA; A) and soleus (Sol; B) of a nonex-
ercised rat and from the vastus intermedius (VI; C) and Sol (D)
muscles of a rat 1 day after 7, once-daily exercise bouts. Calibration
bars ⫽ 50 ␮m.
The timing of the increased satellite cell prolifer-
ation after the initial exercise bout meets the mini-
mum duration required for fusion of satellite cells in
growing rats (22, 32) and corresponds to the first
Fig. 4. Accretion of newly produced myonuclei in the TA (n ⫽
3/group), Sol (n ⫽ 6/group), and VI (n ⫽ 3/group) muscles of 0Ex, 1Ex,
and DailyEx rats. The new nuclei are expressed as the mean (⫾SD)
percentage of all myonuclei observed in isolated fiber segments from
each muscle. a Greater than corresponding muscle in 0Ex; b greater
than corresponding muscle in 1Ex, P ⬍ 0.05.
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (115.186.033.248) on May 17, 2018.
Copyright © 2001 American Physiological Society. All rights reserved.
1411
Fig. 5. Mean (⫾SD) no. of myonuclei per millimeter fiber length in
the TA (n ⫽ 3/group), Sol (n ⫽ 6/group), and VI (n ⫽ 3/group) muscles
of 0Ex, 1Ex, and DailyEx rats.
detectable increases in replicating satellite cells af-
ter focal fiber injury (8, 31). The elevation of prolif-
eration seen in rats subjected to a second day of
exercise indicates that new satellite cells were re-
cruited, because any cells already activated after the
first bout of exercise would not yet have completed a
full cell cycle (duration ⬇32 h; Ref. 29). The greater
proliferation seen after two vs. one bout of exercise
also implicates the second bout as an additional
stimulus for the recruitment of available precursor
cells. The subsequent decline in proliferation sug-
gests that, thereafter, the exercise provided no fur-
ther stimulus for the activation or replication of
satellite cells, and/or that the responsiveness of sat-
ellite cells to the exercise stimulus was reduced.
It is apparent from the low level of proliferation in
the 0Ex animals and at 2, 4, and 7 days after 1Ex that,
in the adult rat Sol, some satellite cells are within the
replicative cell cycle, even in the absence of an exter-
nally applied stimulus such as exercise. Thus an in-
creased proliferation may rely on the recruitment of
quiescent satellite cells (8, 15) and be related specifi-
cally to the exercise stimulus and/or the release of
growth factors within the muscle. In this study, the
Fig. 6. Mean (⫾SD) fiber diameter in the TA (n ⫽ 3/group), Sol (n ⫽
6/group), and VI (n ⫽ 3/group) muscles of 0Ex, 1Ex, and DailyEx
rats.
1412 EXERCISE-ENHANCED ACCRETION OF NEW MYONUCLEI
second daily bout of exercise may have provided the
appropriate stimuli to maintain the entry and progres-
sion of these satellite cells through their replicative
cycle.
The proliferation of satellite cells and morphological
indexes of ongoing muscle-fiber degeneration and re-
generation in the Sol muscle of 0Ex rats seen here and
in prior studies (8, 15, 33) support the belief that
satellite cells are recruited at low levels to contribute to
a continual, possibly age-related (21), fiber turnover in
this postural, high-use muscle.
The relative incidence of fibers containing proliferat-
ing satellite cell nuclei and expressing fast MHC com-
pared with the overall percentage of such fibers in the
Sol muscle is consistent with a lower availability
and/or activation of satellite cells in these fiber types
(12, 17). However, the data demonstrate that satellite
cells in fibers expressing fast MHC within the predom-
inantly slow Sol muscle proliferate in response to the
use of the muscle during unaccustomed locomotory
exercise. These fibers are recruited (11) and are sus-
ceptible to injury (33) during downhill exercise yet may
not have the same regenerative capacity as the slow
fibers.
Experiment 2: Myonuclear accretion. In the Sol and
VI, daily exercise enhanced the accretion of new myo-
nuclei compared with normal caged recovery after the
initial bout of unaccustomed exercise.
The greater proportion of new myonuclei found after
DailyEx compared with the 0Ex and 1Ex groups we
accept as resulting from a correspondingly greater in-
crease in the production of myonuclei. Although a con-
current loss of myonuclei by apoptotic or necrotic myo-
cellular or myonuclear elimination may have occurred,
this could not have accounted for the observed relative
difference in the percentage of new nuclei between 0Ex
and DailyEx muscles. The greater than twofold in-
crease in new nuclei seen in Sol and VI with DailyEx
would have required an extreme loss of myonuclei (e.g.,
to less than one-half in the DailyEx rats) that would
have been detected by our myonuclear counts; these
showed no significant change in any muscle over the
7-day period.
Myonuclear production is a function of the number of
satellite cells dividing and the number of divisions of
each particular cell. The latter is dependent on the
duration of the cell cycle and the maintenance of each
cell in an active replicative state. Hence, the newly
produced myonuclei resulted either from the activation
of a corresponding percentage of satellite cells each
replicating once or from a smaller proportion of the
available mitotic cells (and their progeny) completing
more than one replicative cycle.
How could the continued exercise contribute to an
enhanced accretion of new nuclei? The initiation, pro-
gression, and continuation (or termination) of myo-
genic cell proliferation are controlled by a number of
positive and negative regulatory factors (13). The daily
exercise may have increased blood or lymphatic flow
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (115.186.033.248) on May 17, 2018.
Copyright © 2001 American Physiological Society. All rights reserved.
and/or altered the release or distribution of such fac-
tors to and within the working muscles. The nature of
the exercise and the extent of the injury incurred are
also important to the regeneration of the tissue. In this
study, the exercise was of moderate intensity and dura-
tion; involved submaximal, asynchronous, and graded
recruitment of motor units within muscles; and could
be tolerated by the rats on a daily basis. Based on the
moderate metabolic demands and duration of the ex-
ercise, we neither expected nor intended to induce
either an endurance training effect (i.e., increases in
oxidative capacity) or hypertrophy of the muscle fibers.
The fiber injury and regeneration after the exercise
were focal in nature, segmental along fibers, and mod-
erate in extent (e.g., 7.5% of fibers in Sol). The recovery
of the muscle would not have been compromised by a
disruption of the blood supply or innervation or the
connective tissue scarring that can occur with severe
injury. In addition, data from experiment 1 and our
previous study (33) showed that morphological indexes
of prior injury in affected muscles were less extensive
after daily exercise than at the same time point after a
single bout of exercise with only normal caged activity
on subsequent days. Hence, in the present study, some
fiber injury in Sol and VI probably resulted from the
first bout of exercise, and the myonuclei produced
thereafter were contributing to the repair, regenera-
tion, or regrowth of fibers in the exercised muscles. The
continued functional overloading of the muscles may
have enhanced or accelerated these processes. The lack
of change in mean fiber diameter and wet mass of the
muscles with 7 days of exercise or passive recovery
supports the idea that the accretion of new myonuclei
was related to fiber injury and repair, rather than
mature fiber hypertrophy, during this time. However,
the influence of newly produced nuclei on subsequent
fiber adaptation with continued exercise training is not
known.
The observed degree of satellite cell proliferation and
accretion in each of the different leg muscles may
depend on the extent of the initial injury and the
nature of the loading subsequently imposed. Motor
units of the Sol are heavily recruited during locomotory
activities (14), and the muscle is susceptible to tread-
mill exercise-induced injury (2, 33) and has a higher
number of satellite cells (e.g., 5–9% of myofiber nuclei)
than less oxidative muscles (12). Hence, the greater
accretion of new myofiber nuclei after exercise in this
muscle may be a function of these characteristics. The
accretion of new nuclei in VI to the same extent as in
the Sol was not expected, based on the faster fiber-type
composition of the muscle and the probable lower avail-
ability of satellite cells (e.g., 3–4%; Ref. 37). However,
the muscle would be well recruited and can be injured
during treadmill exercise (18). In contrast, the TA is
most active during non-weight-bearing phases of the
rat’s gait, comprises predominantly fast-twitch motor
units that are infrequently recruited during normal
activities, and incurs little injury from downhill tread-
mill exercise (33).
 EXERCISE-ENHANCED ACCRETION OF NEW MYONUCLEI
The cumulative satellite cell mitotic activity in
injured or exercised adult rat muscles has not, to our
knowledge, been reported previously. However, the
number of myofiber nuclei accrued in all muscles
over the 7-day period was lower than that found
recently in the Sol (14%; Ref. 23) and plantaris (11%;
Ref. 7) of nonexercised, young adult rats after 14
days of BrdU infusion. This difference was likely a
function of our shorter infusion period (the delivery
rates of the BrdU from the pumps were the same)
and perhaps the different strain and sex of the rats.
However, because our labeling after 7 days was less
than one-half of that previously reported, the early
dividing satellite cells would have had to remain
mitotically active, going through more than one rep-
licative cycle before fusion, to accelerate myonuclear
production over the 14-day period. The consistent
proliferation indexes we observed in experiment 1 do
not support this possibility, and this discrepancy
remains to be explained.
The use of intact, isolated fibers may result in an
underestimation of the number of new myonuclei by
the exclusion of those associated with very small or
necrotic fibers. However, the exercise was sufficient
to enhance myonuclear accretion even in the mature,
intact fibers that were examined over the 7-day pe-
riod.
Our findings suggest that daily exercise evokes
further satellite cell cycle entry and/or maintenance
of proliferating cells within the cell cycle above that
after the initial bout of exercise alone. In addition,
over a 7-day period, daily exercise results in a
greater net accretion of newly produced myonuclei in
the Sol and VI muscles compared with that after a
single bout of exercise with a subsequent passive
recovery. The increased proportion of new myonuclei
may be due to their greater production to augment
the available transcriptional regulation of proteins
required for fiber repair, regeneration, or regrowth
during this time. The implications of greater new
myonuclear accrual on satellite cell availability and
fiber adaptability with continued exercise training
have yet to be determined.
Funding for this work was provided through the Natural Sciences
and Engineering Research Council of Canada (to M. J. Plyley) and
the University of Auckland Research Fund (to H. K. Smith).
REFERENCES
1. Allbrook D. Skeletal muscle regeneration. Muscle Nerve 4:
234–245, 1981.
2. Armstrong RB, Ogilvie RW, and Schwane JA. Eccentric
exercise-induced injury to rat skeletal muscle. J Appl Physiol 54:
80–93, 1983.
3. Bischoff R and Holtzer H. Inhibition of myoblast fusion after
one round of DNA synthesis in 5-bromodeoxyuridine. J Cell Biol
44: 134–150, 1970.
4. Buckwalter JA and Grodzinsky AJ. Loading of healing bone,
fibrous tissue, and muscle: implications for orthopaedic practice.
J Am Acad Orthop Surg 7: 291–299, 1999.
5. Carlson BM and Faulkner JA. The regeneration of skeletal
muscle fibers following injury: a review. Med Sci Sports Exerc 15:
187–198, 1983.
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (115.186.033.248) on May 17, 2018.
Copyright © 2001 American Physiological Society. All rights reserved.
1413
6. Carson JA and Alway SE. Stretch overload-induced satellite
cell activation in slow tonic muscle from adult and aged Japa-
nese quail. Am J Physiol Cell Physiol 270: C578–C584, 1996.
7. Dangott B, Schultz E, and Mozdziak PE. Dietary creatine
monohydrate supplementation increases satellite cell mitotic
activity during compensatory hypertrophy. Int J Sports Med 21:
13–16, 2000.
8. Darr KC and Schultz E. Exercise-induced satellite cell activa-
tion in growing and mature skeletal muscle. J Appl Physiol 63:
1816–1821, 1987.
9. DeFazio A, Leary JA, Hedley DW, and Tattersall MH.
Immunohistochemical detection of proliferating cells in vivo.
J Histochem Cytochem 35: 571–577, 1987.
10. Esser KA and White TP. Mechanical load affects growth and
maturation of skeletal muscle grafts. J Appl Physiol 78: 30–37,
1995.
11. Ferry A, Amiridis I, and Rieu M. Glycogen depletion and
resynthesis in the rat after downhill running. Eur J Appl Physiol
64: 32–35, 1992.
12. Gibson MC and Schultz E. Age-related differences in absolute
numbers of skeletal muscle satellite cells. Muscle Nerve 6: 574–
580, 1983.
13. Grounds MD. Towards understanding skeletal muscle regen-
eration. Pathol Res Pract 187: 1–22, 1991.
14. Hennig R and Lomo T. Firing patterns of motor units in
normal rats. Nature 314: 164–166, 1985.
15. Jacobs SCJM, Wokke JHJ, Bar PR, and Bootsma AL.
Satellite cell activation after muscle damage in young and adult
rats. Anat Rec 242: 329–336, 1995.
16. Kannus P, Jozsa L, Jarvinen TLN, Kvist M, Vieno T, Jarvi-
nen TAH, Natri A, and Jarvinen M. Free mobilization and
low- to high-intensity exercise in immobilization-induced muscle
atrophy. J Appl Physiol 84: 1418–1424, 1998.
17. Kelly AM. Satellite cells and myofiber growth in the rat soleus
and extensor digitorum longus muscles. Dev Biol 65: 1–10, 1978.
18. Komulainen J, Kytola J, and Vihko V. Running-induced
muscle injury and myocellular enzyme release in rats. J Appl
Physiol 77: 2299–2304, 1994.
19. Kopriwa BM and Moss FP. A radioautographic technique for
whole mounts of muscle fibers. J Histochem Cytochem 19: 51–55,
1971.
20. McCormick KM and Schultz E. Role of satellite cells in
altering myosin expression during avian skeletal muscle hyper-
trophy. Dev Dyn 199: 52–63, 1994.
21. McCormick KM and Thomas DP. Exercise-induced satellite
cell activation in senescent soleus muscle. J Appl Physiol 72:
888–893, 1992.
22. Moss FP and Leblond CP. Satellite cells as the source of nuclei
in the muscles of growing rats. Anat Rec 170: 421–436, 1971.
23. Mozdziak PE, Pulvermacher PM, and Schultz E. Unloading
of juvenile muscle results in a reduced muscle size 9 wk after
reloading. J Appl Physiol 88: 158–164, 2000.
24. Mozdziak PE, Truong Q, Macius A, and Schultz E. Hind-
limb suspension reduces muscle regeneration. Eur J Appl
Physiol 78: 136–140, 1998.
25. Newham DJ, Jones DA, and Clarkson PM. Repeated high-
force eccentric exercise: effects on muscle pain and damage.
J Appl Physiol 63: 1381–1386, 1987.
26. Roberts P and McGeachie JK. The effects of pre- and post-
transplantation exercise on satellite cell activation and the re-
generation of skeletal muscle transplants: a morphometric and
autoradiographic study in mice. J Anat 180: 67–74, 1992.
27. Rosenblatt JD, Yong D, and Parry DJ. Satellite cell activity
is required for hypertrophy of overloaded adult rat muscle.
Muscle Nerve 17: 608–613, 1994.
28. Schultz E. Quantification of satellite cells in growing muscle
using electron microscopy and fiber whole mounts. In: Muscle
Regeneration, edited by Mauro A. New York: Raven, 1979, p.
131–135.
29. Schultz E. Satellite cell proliferative compartments in growing
skeletal muscles. Dev Biol 175: 84–94, 1996.
30. Schultz E, Darr KC, and Macius A. Acute effects of hindlimb
unweighting on satellite cells of growing skeletal muscle. J Appl
Physiol 76: 266–270, 1994.
1414 EXERCISE-ENHANCED ACCRETION OF NEW MYONUCLEI

31. Schultz E, Jaryszak DL, and Valliere CR. Response of satellite
cells to focal skeletal muscle injury. Muscle Nerve 8: 217–222, 1985.
32. Schultz E and McCormick KM. Skeletal muscle satellite cells.
Rev Physiol Biochem Pharmacol 123: 213–257, 1994.
33. Smith HK, Plyley MJ, Rodgers CD, and McKee NH. Skeletal
muscle damage in the rat hindlimb following single or repeated
daily bouts of downhill exercise. Int J Sports Med 18: 94–100, 1997.
34. Smith HK, Plyley MJ, Rodgers CD, and McKee NH. Expres-
sion of developmental myosin and morphological characteristics
in adult rat skeletal muscle following exercise-induced injury.
Eur J Appl Physiol 80: 84–91, 1999.
35. Snow MH. Satellite cell response in rat soleus muscle undergo-
ing hypertrophy due to surgical ablation of synergists. Anat Rec
227: 437–446, 1990.
36. Tapscott SJ, Lassar AB, Davis RL, and Weintraub H.
5-Bromo-2⬘-deoxyuridine blocks myogenesis by extinguishing ex-
pression of MyoD1. Science 245: 532–536, 1989.
37. Umnova MM and Seene TP. The effect of increased functional
load on the activation of satellite cells in the skeletal muscle of
adult rats. Int J Sports Med 12: 501–504, 1991.
38. Wanek LJ and Snow MH. Activity-induced fiber regeneration
in rat soleus muscle. Anat Rec 258: 176–185, 2000.

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (115.186.033.248) on May 17, 2018.

Copyright © 2001 American Physiological Society. All rights reserved.