ABSTRACT: We tested the hypothesis that the expansion of satellite cell

numbers, 24 h after maximal eccentric knee extensor exercise, is blunted in

older men. Muscle biopsies were obtained from the vastus lateralis of 10
young (23–35 years) and 9 older (60 –75 years) men. Satellite cells were
identified immunohistochemically using an antibody to neural cell adhesion
molecule. After 92 maximal eccentric contractions, the mean number of
satellite cells per muscle fiber increased to a greater extent among the
young men (141%; P ⬍ 0.001) than older men (51%; P ⫽ 0.002) from
preexercise levels. Similar results were obtained when satellite cells were
expressed as a proportion of all sublaminar nuclei. We conclude that a single
bout of maximal eccentric exercise increases satellite cell numbers in both
age groups, with a significantly greater response among the young men.
These data suggest that age-related changes in satellite cell recruitment
may contribute to muscle regeneration deficits among the elderly.
Muscle Nerve 33: 242–253, 2006

SATELLITE CELL NUMBERS IN YOUNG

AND OLDER MEN 24 HOURS
AFTER ECCENTRIC EXERCISE
HANS C. DREYER, PhD,1 CESAR E. BLANCO, PhD,2 FRED R. SATTLER, MD,1,3
E. TODD SCHROEDER, PhD,1,3 and ROBERT A. WISWELL, PhD1

1
Department of Biokinesiology and Physical Therapy, University of Southern California,
Los Angeles, California, USA
2
Alfred E. Mann Institute for Biomedical Engineering, University of Southern California,
Los Angeles, California, USA
3
Department of Medicine, Keck School of Medicine, University of Southern California,
Los Angeles, California, USA

Accepted 13 September 2005

Satellite cells provide the precursor cells necessary
for repair and hypertrophy of skeletal muscle. 11,23,40
In response to anabolic stimuli or muscle damage,
these cells divide and produce daughter cells, ulti-
mately providing new myonuclei.9,23,38,57 The addi-
tion of myonuclei to the existing multinucleated
muscle increases the capacity for protein synthesis
and allows for and maintains increases in cross-sec-
tional area (CSA).20 Indeed, if satellite cell prolifer-
ation is prevented, muscle growth and recovery of
lost muscle mass is inhibited.45 A diminished prolif-
eration of satellite cells (i.e., decreased rates of com-
mitment to myogenic differentiation) has been pro-
posed as a potential mechanism for the age-related
blunting of the regenerative process11 and for a re-
Abbreviations: CSA, cross-sectional area; NDS, normal donkey serum;
PCNA, proliferating cell nuclear antigen; TBS, Tris-buffered saline
Key words: aging; muscle regeneration; myogenic response; sarcopenia;
satellite cells
Correspondence to: H. C. Dreyer; e-mail: hcdreyer@utmb.edu
© 2005 Wiley Periodicals, Inc.
Published online 28 November 2005 in Wiley InterScience (www.interscience.
wiley.com). DOI 10.1002/mus.20461
duced response to anabolic stimuli18,33 in skeletal
muscle that may ultimately contribute to the devel-
opment of sarcopenia.
Progressive resistance training increases strength
and muscle mass in older adults, including nonage-
narians.16 However, the magnitude of the increases
in skeletal muscle mass observed in older adults may
be significantly less than that observed in younger
individuals.19,55 Animal studies have demonstrated
age-related attenuation in muscle mass accretion in
response to anabolic stimuli.6,8,32 The mechanisms
responsible for this apparent age-related attenuation
remains to be determined but could involve a re-
duced myogenic response during satellite cell prolif-
eration and phenotype commitment as demon-
strated in older rats.53
Few studies in humans have been conducted on
satellite cell proliferation in response to anabolic
stimuli.12,24,29,36,47,52 Of those studies, only two mea-
sured the potential age-related effects of resistance
exercise on satellite cell expansion.24,47 Their results
were based on muscle biopsies taken months apart
and did not include the immediate and early myo-

242 Acute Satellite Cell Response to Exercise MUSCLE & NERVE February 2006
genic response, which may occur within hours to
several days following the initial anabolic stimu-
lus.1,7,12,21,34,36,41 Indeed, in rodents a ⬃250% in-
crease in labeled satellite cells has been reported
24 h after running on a 18% decline treadmill13 and
level treadmill running resulted in a doubling of
satellite cells, also at the 24-h time point.26 In mice,
satellite cells become activated during the acute
phases of injury (⬍6 h) and increase in numbers
extensively within 48 h.17 In addition, Schultz et al.49
have shown that satellite cells are capable of migrat-
ing from distal regions of muscle cells to proximal
segments subjected to injury and that this response
was rapid, resulting in a 380% increase in satellite
cells within 20 h of injury. Thus, a potential contri-
bution of age-related differences in satellite cell re-
sponse may require sampling of the muscle tissue
shortly after the initial stimulus. Therefore, this
study was designed to determine whether satellite
cell numbers would increase 24 h after a single bout
of maximal eccentric exercise in young and older
men. We tested the hypothesis that older muscle
would respond to anabolic stimuli with an attenu-
ated increase in the number of satellite cells com-
pared to young muscle.
MATERIALS AND METHODS
Selection of Study Subjects. Potential subjects were
required to provide informed written consent that
was approved by our institutional review board. Sub-
jects had to be male, either 21–35 years or at least 60
years of age, and not currently participating in a
resistance training program. Each subject under-
went a complete history and physical examination,
and had screening blood tests performed to deter-
mine eligibility and to ensure the absence of under-
lying medical conditions or muscle inflammation
that could have confounded the results. Blood spec-
imens were processed and analyzed by our local
laboratory. Subjects with cardiac abnormalities
(heart failure, recent myocardial infarction, or an-
gina), resting blood pressure exceeding 180/94
mmHg, active inflammatory conditions, neuropathy,
untreated endocrine abnormalities (e.g., diabetes,
hypothyroidism), receiving anticoagulation, or in-
ability to perform lower-extremity exercise were ex-
cluded from participation. Men in the older group
were evaluated with a 12-lead EKG and blood pres-
sure–monitored stress test to achieve at least 85% of
their predicted maximal heart rate in order to ex-
clude subjects at risk for cardiac ischemia during
eccentric exercise. Shortly after the study was initi-
Acute Satellite Cell Response to Exercise
ated, the protocol was amended to test for serum
testosterone levels.
All procedures involving subjects were conducted
at our institutional General Clinical Research Center
(GCRC) except for the maximal eccentric loading
exercises, which were performed in our clinical ex-
ercise research center.
Muscle Biopsies. Two biopsies of the vastus lateralis
were obtained from the dominant leg. The first bi-
opsy (preexercise biopsy) served to generate base-
line measures for variables of interest including mus-
cle fiber type, fiber CSA determination, sublaminar
and centrally located myonuclei, and satellite cell
numbers. The preexercise biopsy was conducted
11–15 days prior to the second biopsy (postexercise
biopsy) in order to minimize the effects of the first
biopsy procedure on satellite cell counts performed
on the second sample. The postexercise biopsy was
preceded in time (24 h) by a single bout of unilateral
maximal eccentric exercise of the dominant quadri-
ceps muscle group. All subjects were instructed to
refrain from exercise for 24 h before and 72 h after
each biopsy.
Biopsies were obtained from the mid-portion of
the vastus lateralis muscle approximately 18 cm
proximal to the patella, approximating the midline
of the quadriceps muscle group. The postexercise
biopsy was performed at a distance of 2– 4 cm prox-
imal or distal (randomly assigned) to the preexercise
biopsy site. Biopsies were performed using a steril-
ized 5-mm Stille muscle biopsy needle (Micrins Sur-
gical, Lake Forest, Illinois) using applied suction.4
Approximately 100 –175 mg of muscle tissue was ob-
tained from each biopsy.
The presence of centrally located myonuclei is
indicative of recent skeletal muscle regeneration.9
From the preexercise biopsy tissue, we quantified the
number of centrally located nuclei to determine
whether baseline turnover was different between
groups. Central nuclei quantification was also per-
formed on the postexercise biopsy tissue to gauge
the effects of the previous biopsy procedure (harvest-
ing) on regeneration at that time point.
Maximal Eccentric Exercise. Single-leg maximal ec-
centric isokinetic loading was performed on the
dominant leg using a KinCom 500 dynamometer
(Kin-Com, Chattanooga, Tennessee) at 60°/s. Each
subject was familiarized with the equipment imme-
diately prior to the exercise. During the maximal
eccentric loading, a shoulder harness, hip restraint,
and thigh strap (exercised leg) were utilized to se-
cure the subject to the device. A strap was also se-
MUSCLE & NERVE February 2006 243
cured distally on the dominant leg at the level of the
load cell. The load cell was positioned 3 cm proximal
to the talocrural joint. The exercise intervention
consisted of an initial set of 12 maximal repetitions
with the subject performing the eccentric compo-
nent while the investigator performed the concen-
tric portion. This was followed by five additional sets
of 16 repetitions, for a total of six sets. Each eccentric
repetition was separated by the time it took one of
the investigators to manually bring the lever arm
back to 0° of knee flexion (i.e., starting position).
Each of the six sets was separated in time by 120 –180
s. Ample verbal encouragement was provided by the
investigator during each maximal eccentric contrac-
tion.
Muscle Biopsy Specimen Processing. Muscle tissue
samples were immediately rinsed with prechilled
normal saline and dissected free from connective
and adipose tissue. Muscle sections were mounted
(unfixed) on blocks of cork (1.0 ⫻ 1.0 ⫻ 0.5 cm;
length by width by height) with optimum cutting
temperature compound prechilled by ice water.
Once appropriate orientation was achieved, the tis-
sue block was quickly frozen in isopentane cooled to
freezing by liquid nitrogen. Cryoprotected tissue
samples were stored at ⫺80°C until processed. Each
biopsy sample was assigned a subject identifier num-
ber to minimize bias.
Cryostat Sectioning. Tissue sections were cut at
10-␮m thickness in a cryostat maintained at ⫺25°C,
and mounted on Fisherbrand Superfrost/Plus mi-
croscope slides (Fisher Scientific, Pittsburgh, Penn-
sylvania). Each cut of tissue that was included on the
same slide was obtained a minimum distance of 30
␮m apart. This was accomplished by placing the first
cut of tissue on slide A and the second on a separate
slide (slide B). After placing the third cut of tissue on
slide C, a fourth cut was discarded before placing the
fifth adjacent to the first cut on slide A. For satellite
cell analysis, each slide contained tissue sections
(2–10 ␮m thick) from the preexercise biopsy and the
postexercise biopsy for each subject. We included
the preexercise and postexercise biopsy from the
same individual on each slide to ensure that each
condition was processed identically. Additionally, we
included tissue from both a young and older subject
on each slide for identical processing. The inclusion
of preexercise and postexercise tissue from both a
young and older subject was known to the investiga-
tor performing the analysis, which was thus con-
ducted under partially blinded conditions.
244 Acute Satellite Cell Response to Exercise
Immunohistochemistry. Muscle tissues were washed
in 50 mM Tris-buffered saline (TBS; pH 7.40), fol-
lowed by incubation in TBS containing 0.3% Triton
X-100 with 4% normal donkey serum (NDS) for 1 h
in a humidified chamber at room temperature to
block nonspecific binding sites. For satellite cell im-
munohistochemistry, sections were subsequently
placed in a primary antibody medium containing
monoclonal mouse anti-CD56 antibody directed
against the neural cell adhesion molecule (NCAM)
located on satellite cells10,12,30,35,36,44 (identical to the
Leu 19 antigen25,31) (Clone MOC-1, DakoCytoma-
tion, Carpinteria, California; final dilution: 1:100
ng/ml), and polyclonal rabbit anti-laminin antibody
directed against laminin (extracellular matrix pro-
tein) (DakoCytomation; final dilution: 1:2,000 ng/
ml), in TBS containing 0.3% Triton X-100 with 2%
NDS overnight at room temperature. In addition to
the NCAM antibody, we unsuccessfully attempted to
double-stain satellite cell with two additional mark-
ers (CD34 and M-cadherin; Santa Cruz Biotechnol-
ogy, Santa Cruz, California). Similarly, we were un-
able to successfully utilize anti-MyoD1 (M3512;
DakoCytomation) as a potential marker of satellite
cell activation. For fiber type immunohistochemistry,
tissue cross-sections were processed as above, except
that a monoclonal mouse anti–myosin heavy chain-
slow (MyHCslow; final dilution: 1:2000 ng/ml) anti-
body replaced the NCAM antibody. The MyHCslow
antibody was purchased from Sigma (St. Louis, Mis-
souri; Clone NOQ7.5.4D). Lastly, one slide from
each experiment was incubated in a primary anti-
body medium lacking the anti-CD56, anti-MyHCslow
and anti-laminin antibodies to determine the degree
of nonspecific binding of the secondary antibodies.
Muscle tissue sections were subsequently washed
in TBS, followed by a 2-h incubation in 0.03% Triton
X-100 and 2% NDS containing Cy3-conjugated
donkey anti-mouse IgG (to visualize NCAM or
MyHCslow) and Cy2-conjugated donkey anti-rabbit
IgG (to visualize laminin; both at a final dilution of
1:330; Jackson Immuno Research, West Grove, Penn-
sylvania), at room temperature. The tissues were
then washed and incubated in a solution containing
the nuclear marker 4⬘, 6-diamidino-2-phenylindole,
dihydrochloride (DAPI; Molecular Probes, Eugene,
Oregon) in TBS for 5 min at room temperature,
followed by a final wash in distilled deionized water
and cover slipped while wet with Gel/Mount mount-
ing medium (Biomeda Corp., Foster City, Califor-
nia).
Image Capture. Muscle tissue sections were visualized
using a Nikon Eclipse E800 microscope equipped for
MUSCLE & NERVE February 2006

FIGURE 1. Digital microphotographs of young (left column) and
older muscle (right column). Top row, NCAM stain; middle row,
DAPI stain; bottom row, laminin stain with NCAM inset from top
row. Arrows indicate location of satellite cells in each image.
Image obtained at 40⫻ magnification. Calibration bar, 25 ␮m.
epifluorescence microscopy (Nikon, Tokyo, Japan)
connected to a Spot-2 digital camera (Diagnostics In-
strument, Sterling Heights, Michigan). Beginning at
the top left corner (as observed visually through the
eyepiece) of the tissue section, a set of three images of
the same visual field was digitized to visualize (Fig. 1)
NCAM or MyHCslow (first image; Cy3-fluorescence),
DAPI (second image; UV excitation), and laminin
(third image; Cy2-fluorescence). Next, the microscope
stage was repositioned horizontally (along the x-axis),
the distance of one image field, and a new set of three
(Cy3, UV, then Cy2) images were captured. This was
repeated until the end of the tissue cross-section was
reached horizontally. At that point, the microscope
stage was repositioned vertically to capture muscle cells
that were one image field below the previous set of
image fields. This process was repeated for each con-
dition. All image capture utilized the Spot-2 digital
camera and software interface in a way that allowed the
investigator to adjust the fine focus and microscope
stage (horizontally or vertically) while viewing the mus-
cle tissue images (real time) on a computer screen.
When it appeared to the investigator that a satellite cell
Acute Satellite Cell Response to Exercise
was present, verification by visual inspection through
the microscope eyepiece with the Cy3 filter in place
and fine focusing was performed in addition to alter-
nating between the Cy2 (laminin border) and UV (for
nuclei) filters (Fig. 1). To be counted as a satellite cell,
the following qualifications were required: (1) the cell
had to stain positively for NCAM; (2) each NCAM-
positive cell had to be encapsulated by the basal lam-
ina, demonstrated by the laminin antibody; and (3)
each NCAM-positive cell had to have a nucleus associ-
ated with it as demonstrated by DAPI. Each cell that
met these specifications was counted as a satellite cell.
Notes were compiled for each tissue section indicating
the presence or absence of satellite cells and were used
later during the analysis to confirm initial observations.
Primary satellite cell determination was made by visual
inspection of the tissue section with the microscope.
Histological Analysis. Images were analyzed using a
computer-assisted image analysis system (Meta-
Morph Imaging Software, Universal Imaging, Down-
ingtown, Pennsylvania). Muscle fiber boundaries
were traced by the operator using the laminin im-
munofluorescence as a template. Nuclei lying within
the laminin boundary (i.e., sublaminar nuclei and
centrally located nuclei) were determined to be myo-
nuclei. Sublaminar nuclei positive for NCAM immu-
nofluorescence were counted as satellite cells as in
published reports.10,12,25,27–29,35,48 There were no in-
cidences where a NCAM positive cell was located
outside the basal lamina border. The NCAM anti-
body stains both activated and quiescent satellite
cells,25,48,51 and has been used previously in human
tissue.10,12,30,35,36,44,51 For fiber typing, muscle cells
staining positive for the MyHCslow antibody were
counted as type I fibers and those muscle cells that
did not stain positive (e.g., MyHCslow-negative) were
counted as type II fibers. For fiber CSA determina-
tion, a calibration slide was imaged at the corre-
sponding magnification (40⫻ or 60⫻) to determine
the correction factor necessary to calculate fiber CSA
based on the number of pixels within the laminin
boundary for each muscle fiber.
Statistical Analysis. Data were analyzed using the Sta-
tistical Package for Social Sciences (SPSS) program,
version 11.0 for Windows (SPSS, Chicago, Illinois).
Repeated-measures analysis of variance (ANOVA) was
used to identify between- and within-group differences
and to determine whether an age by time interaction
effect occurred. When significance was obtained, inde-
pendent or paired t-tests (two-tailed) were used for
between- and within-group differences, respectively.
Baseline characteristics (blood samples and tissue val-
MUSCLE & NERVE February 2006 245
ues) were analyzed by independent t-test. Data are
reported as mean ⫾ SD. Significance was predeter-
mined at the P ⱕ 0.05 level.
RESULTS
Characteristics of the Study Groups. Twenty-one eli-
gible subjects were enrolled in the study. Adequate
muscle tissue was not available from either the pre-
exercise biopsy or postexercise biopsy in a single
subject from each group. Thus, a total of 19 subjects
were included for the analysis. Subjects in both
groups were generally comparable for physical char-
acteristics, blood counts, and chemistries, except
that the older group had significantly lower hemo-
globin (P ⬍ 0.036) and serum albumin (P ⬍ 0.01).
There was a trend for lower testosterone levels (P ⫽
0.12) in the older group, but only six values for the
young group were obtained and compared to all
nine older subjects. The serum creatine kinase, lac-
tate dehydrogenase, aspartate aminotransferase, and
alanine aminotransferase levels, each a marker of
inflammation, was normal and not different between
age groups. In addition, four subjects from the
young group and five from the older group regularly
exercised (more than twice weekly). Two of the
young subjects were runners, and two were cyclists.
In the older group, all four exercisers were runners.
However, none of the subjects performed resistance
exercise prior to or during the course of the study.
Design Characteristics. The number of days be-
tween the preexercise and postexercise biopsy was
not significantly different between groups (12 ⫾ 3
days and 12 ⫾ 1 days for young and older men,
respectively). Additionally, there was no significant
difference between groups for the time between the
maximal eccentric exercise and the postexercise bi-
opsy (24 ⫾ 0.5 h and 24 ⫾ 0.4 h) for young and older
men, respectively. Collectively, these data indicate
that each group was treated similarly.
Maximal Eccentric Resistance Exercise. We aver-
aged the maximal eccentric peak torques for sets 1
and 2, 3 and 4, and 5 and 6 for each subject to
determine whether differences between groups ex-
isted. As expected, differences occurred between
groups for the average of sets 1 and 2 (269 ⫾ 56 Nm
and 208 ⫾ 56 Nm; P ⫽ 0.028) and for the average of
sets 3 and 4 (268 ⫾ 50 Nm and 209 ⫾ 51 Nm), for
young and older men, respectively (P ⫽ 0.02). How-
ever, the average of sets 5 and 6 were not different
between young and older men (250 ⫾ 53 Nm and
206 ⫾ 48 Nm), respectively (Fig. 2). It should be
246 Acute Satellite Cell Response to Exercise
FIGURE 2. Averaged peak torque (Nm) values for sets 1 and 2,
3 and 4, and 5 and 6 by group. Filled columns represent young
subjects (n ⫽ 10) and clear columns, older subjects (n ⫽ 9).
*Significantly greater than older men.
noted that as a result of this protocol, one subject
from the young group experienced exertional rhab-
domyolysis that resolved without incident.
Muscle Fiber Type Distribution. Relative muscle fiber
type percentages are presented in Table 1. Indepen-
dent t-test revealed older subjects to have a greater
percentage of type I muscle fibers than the young
(Table 2). Additionally, older men had a lower per-
centage of type II muscle fibers than young subjects
(Table 2), similar to published reports.10,21
Muscle Fiber Cross-Sectional Area. Muscle fiber
type–specific CSA are presented in Table 2. There
was no significant difference between type I and II
CSA in the young subjects. Older subjects, however,
demonstrated fiber type–specific atrophy of the type
II fibers (Table 2). The type II CSA for older subjects
was also reduced relative to the young type II fibers,
whereas the type I CSA for young and older subjects
were not significantly different (Table 2).
Myonuclei/Muscle Fiber. Data for myonuclei are
presented in Table 1. The number of myonuclei/
muscle fiber cross-section was not different between
groups (Table 1) and did not change between pre-
exercise and postexercise biopsies.
Central Myonuclei/Muscle Fiber. There was no dif-
ference in the number of centrally located myonu-
clei per muscle fiber between groups (Table 1).
Additionally, there was no difference at preexercise
biopsy and no increase in the number of centrally
located myonuclei/muscle fiber at the second bi-
opsy. These data suggest that the preexercise biopsy
did not result in significant muscle regeneration
when utilizing “central nuclei” as a qualitative
marker.
MUSCLE & NERVE February 2006
 Table 1. Muscle tissue data by group and time.a
Central Mean change for
Time point (muscle Myonuclei/muscle myonuclei/muscle Satellite cell/muscle Satellite cell satellite cell
Group fibers analyzed) fiber fiber fiber cross-section proportionsf proportionsg
Young (n ⫽ 10) Preexercise biopsy 2.56 ⫾ 0.6 0.03 ⫾ 0.03 0.07 ⫾ 0.02 2.86 ⫾ 0.6 ⫹3.2 ⫾ 1.3d
(101 ⫾ 23)h
Postexercise biopsy 2.72 ⫾ 0.5 0.06 ⫾ 0.10 0.18 ⫾ 0.05b,c 6.05 ⫾ 1.4b,c
(108 ⫾ 33)h
Older (n ⫽ 9) Preexercise biopsy 2.34 ⫾ 0.4 0.03 ⫾ 0.02 0.07 ⫾ 0.02 2.81 ⫾ 0.7 ⫹1.3 ⫾ 0.6
(134 ⫾ 37)h
Postexercise biopsy 2.31 ⫾ 0.4 0.04 ⫾ 0.03 0.10 ⫾ 0.02b 4.12 ⫾ 1.0b
(132 ⫾ 17)e,h
a
Data are presented as mean ⫾ SD.
b
Significantly greater than preexercise biopsy (P ⬍ 0.001 and P ⫽ 0.002 for young and older men, respectively).
c
Significantly greater than older group at postexercise biopsy (P ⫽ 0.001).
d
Significantly greater than older group mean change for satellite cell proportions (P ⬍ 0.001).
e
Significantly greater number of muscle fibers analyzed than young at postexercise biopsy (P ⫽ 0.018).
f
Proportion of sublaminar nuclei that are satellite cells; calculation ⫽ [satellite cell/(satellite cell ⫹ myonuclei)] ⫻ 100.
g
Mean change ⫽ mean (postexercise biopsy ⫺ preexercise biopsy) for satellite cell proportions by age group.
h
Average number of muscle fibers analyzed per subject.

Satellite Cells.Comparative images for young and
older muscle are depicted in Figure 1. Addition-
ally, we observed several instances from postexer-
cise tissue samples where the NCAM-positive stain-
ing included two nuclei in apposition to each
other (i.e., doublets) within the same muscle cell
(Fig. 3A–D). In addition, we encountered single
muscle cells with two satellite cells located some
distance apart (Fig. 3E and F). Figure 4 shows
images of tissue sections demonstrating the robust
increase in the number of satellite cells that we
observed. Lastly, Figure 5 shows images of satellite
cells and NCAM-positive muscle fibers indica-
tive of recent muscle degeneration or regenera-
tion.25,48
Satellite Cells/Muscle Fiber Cross-Section. Satellite
cells/muscle fiber cross-section is presented in Ta-
ble 1. There was no group difference at the pre-
exercise biopsy, but a significant difference be-
tween groups existed at the postexercise biopsy
(P ⫽ 0.001). A significant increase in satellite
cells/muscle fiber cross-section at the postexercise
biopsy relative to the preexercise biopsy was ob-
served in both young (P ⬍ 0.001) and older (P ⫽
0.002) groups. The mean change in satellite cells/
muscle fiber was greater in magnitude in the
young (0.10 ⫾ 0.04) than older men (0.03 ⫾ 0.02;
P ⬍ 0.001).
Satellite Cell Proportions. Individual values for satel-
lite cell proportions are reported in Figure 6 and
group means are presented in Table 1. Satellite cell
proportions were calculated based on the formula
[satellite cells/(satellite cells ⫹ myonuclei)] ⫻ 100.
Satellite cell proportions were similar between
groups at the preexercise biopsy. An increase in
satellite cell proportions at the postexercise biopsy
relative to the preexercise biopsy was observed in
both the young (P ⬍ 0.001) and older (P ⬍ 0.001)
men, indicating that both groups responded posi-
tively to the exercise stimulus. The mean change in
satellite cell proportions demonstrated that the mag-
nitude of change was significantly greater in the
young relative to older men (P ⫽ 0.002; Table 1).

Table 2. Fiber type distribution (%) and fiber cross-sectional area of the vastus lateralis muscle.
Type I Type I CSA Type II Type II CSA
(%) (␮m2) (%) (␮m2)
Young (n ⫽ 10) 52 ⫾ 9 6,431 ⫾ 1,638 48 ⫾ 9 6,257 ⫾ 1,569
Older (n ⫽ 9) 66 ⫾ 16* 5,804 ⫾ 1,357 34 ⫾ 16† 4,714 ⫾ 1,195‡

Values are mean ⫾ SD. Data for percent fiber type and fiber type specific cross-sectional area generated from preexercise biopsy samples.
*Significantly greater than young group type I (P ⫽ 0.025) and older group type II.
†
Significantly less than young group type I (P ⫽ 0.033).
‡
Significantly less than young type II (P ⫽ 0.029) and older type I (P ⫽ 0.013).

Acute Satellite Cell Response to Exercise MUSCLE & NERVE February 2006 247
FIGURE 3. Digital microphotographs of postexercise muscle tissue showing potential “doublets” and two satellite cells within a single
muscle cell. Images (A–C) are from three older men (65, 75, and 65 years, respectively). Images (D–F) are from a 27-year-old man.
Arrows denote location of satellite cells. Images B, D, and E have one additional satellite cell in addition to the two within the inset. Color
images are magnified and include NCAM, laminin, and DAPI merge (A–C) and NCAM and laminin merge (D–F). Images A, C, D, and
F were obtained at 60⫻, while B and E were obtained at 40⫻ magnification. Calibration bar, 25 ␮m.

DISCUSSION was significantly greater in the young than older

Our results demonstrate that satellite cell numbers
are significantly increased within 24 h after a single
bout of 92 maximal eccentric contractions in both
young and older men. The magnitude of the change
in satellite cells/muscle fiber cross-section, however,
men (141% vs. 51%, respectively). Additionally,
when satellite cells were expressed relative to the
number of myonuclei (i.e., satellite cell propor-
tions), the magnitude of change was also greater for
young than older men (119% vs. 50%, respectively).

248 Acute Satellite Cell Response to Exercise MUSCLE & NERVE February 2006

FIGURE 4. Digital microphotographs of postexercise muscle tis-
sue showing examples of satellite cells. (A) is from a 65-year-old
man, and (B) and (C) are from two 27-year-old men. Arrows
denote location of satellite cells. Asterisk in C (bottom) demon-
strates variability in intensity of staining or exposure time. Notice
the variation in the size of the satellite cells (A vs. B; C, two
satellite cells at top left compared with the other satellite cells
from that image), which may be due to activation or sectioning
through varying regions of the satellite cell. The folded muscle
cells in the middle of C occurs during the transfer of tissue from
the microtome blade to the slide. All images at 40⫻ magnifica-
tion. Calibration bar, 25 ␮m.
These data indicate that, by 24 h after maximal
eccentric exercise, the vastus lateralis muscle from
both age groups demonstrated a significant increase
Acute Satellite Cell Response to Exercise
in satellite cells and that the increase was greater for
the young subjects.
The expansion in satellite cell numbers that we
observed was surprising considering satellite cell cy-
cle time has been reported to be approximately 32 h
in rats, albeit in the quiescent (unperturbed) state.50
However, a ⬃12 h satellite cell doubling time was
reported by Bischoff5 when cultured muscle fibers
were exposed to muscle extract and a similar (12.6
h) division time has been reported by Qu-Petersen et
al.42 when myogenic early preplate cells were grown
in culture containing proliferation medium. That
study42 also reported an average division time of
15.1 h for muscle-derived stem cells grown in culture
containing proliferation medium. In adult male
mice, proliferating cell nuclear antigen (PCNA) ex-
pression was significantly elevated 12 h after cardio-
toxin injection and remained elevated for 2 days and
was consistent with satellite cell activation (⬍6 h)
and robust proliferation within 48 h.17 Moreover, a
⬃250% increase in labeled satellite cells has been
observed within 24 h after rats ran down a 18%
incline.13 Also, a similar increase in satellite cells
from rats was observed 24 h after level treadmill
running (increasing from 0.06 to 0.12 satellite cells/
muscle fiber).26 These studies suggest that exercise
or possibly factors released during exercise (i.e., he-
patocyte growth factor released from the extracellu-
lar matrix54) induces a rapid response from the sat-
ellite cells and that this occurs within hours of the
perturbation.
A recent study by Crameri et al.12 showed that
satellite cells from the vastus lateralis muscle of eight
young (25 ⫾ 3 years) men increased in number 4
and 8 days after eccentric exercise. Although not
significant, satellite cell proportions at 2 days postex-
ercise (6.38 ⫾ 1.23), which are similar to our values
for young subjects, demonstrated a positive increase
from baseline.12 Their satellite cell numbers at day 2
may not have reached significance due to the smaller
sample size (n ⫽ 8) or the comparison of those
numbers to the control leg at day 2, which demon-
strated a nonsignificant increase, possibly a result of
the biopsy procedure performed on the control leg
at day 0.12 The intervention for that study required
subjects to perform three maximal-eccentric exer-
cises each separated in time by 5 min.12 The first
exercise consisted of 50 single-leg drop-down jumps
from a height of 45 cm, followed by 8 sets of 10
maximal eccentric knee extensions at 30°/s using a
KinCom isokinetic dynamometer, which was fol-
lowed by 8 more sets of 10 maximal-eccentric knee
extensions, this time at 180°/s.12 Despite the differ-
ence in number and speed of eccentric contractions
MUSCLE & NERVE February 2006 249
FIGURE 5. Digital microphotographs of postexercise muscle tissue showing NCAM-positive muscle fibers, central nuclei, and satellite cells. Top
row, 27-year-old man. Bottom row, 64-year-old man. Left image, black and white of Cy3 filter (NCAM-positive). Right image, merge image of
Cy3 (NCAM; red), Cy2 (laminin; green), and UV (nuclei; blue) filters. Asterisk, NCAM-positive muscle cells; circles surround central nuclei; white
arrows point to satellite cells. All images at 40⫻ magnification and color enhanced for clarity. Calibration bar, 25 ␮m.

performed in that study and ours (a total of 92), the
increase in satellite cell proportions between studies
was similar, suggesting that in young men, an in-
crease in satellite cell numbers may be achieved with
92 total maximal-eccentric contractions performed
at 60°/s and may not require or be augmented by
performing the additional 118 repetitions.
Our findings suggest that factors upstream to
satellite cell proliferation, such as satellite cell acti-
vation or precursor satellite cell involvement, or
both, may contribute to the age-related differential
response observed. Indeed, Barani et al.3 reported
an age-associated reduction in c-met receptor and
proliferating cell nuclear antigen expression in sat-
ellite cells stimulated in culture. A recent report by
Conboy et al.11 demonstrated diminished Delta ex-
pression, a ligand for the Notch-1 receptor impor-
tant for satellite cell activation, from satellite cells of
older relative to young mice after muscle injury.
That study clearly demonstrated reduced (by 75%)
satellite cell activation in older animals11 and pro-
vides insight as to a potential mechanism of the
age-specific diminished regenerative response. Alter-
natively, it is possible that older satellite cells are less
capable of rapidly proliferating,43 which may explain
differences observed in our study between young
and older men at 24 h. Additionally, the robust
increase in satellite cell numbers that we observed
may have been due, in part, to cell migration, which
has been shown49 to result in a 380% increase in
satellite cell numbers in the region of muscle ap-
proximating tissue damage at 20 h. It is also possible
that the activation of potential satellite cell progen-
itor cells2,14,15,56 is responsible, in part, for the robust
increase in NCAM-positive cells and moreover, the
potential contribution of these muscle-resident stem
cells may be altered or delayed with age. Regardless
of the origin of the additional NCAM-positive satel-
lite cells,51 our results demonstrate that older muscle
is capable of positively responding to maximal ec-

250 Acute Satellite Cell Response to Exercise MUSCLE & NERVE February 2006

FIGURE 6. Individual data points for satellite cells proportions for
preexercise biopsy and postexercise biopsy. Dashed lines with
filled squares represent young subjects (n ⫽ 10), and solid lines
with clear circles, older subjects (n ⫽ 9). Mean values for pos-
texercise biopsy are significantly greater than preexercise biopsy
for both young (P ⬍ 0.001) and older (P ⬍ 0.001) men. Mean
increase for young is significantly greater than mean increase for
older men (P ⫽ 0.002).
centric contractions but that that response is dimin-
ished when compared to young muscle at 24 h.
Baseline measures for satellite cell proportions
were not different between age groups, suggesting
that the number of satellite cells do not decline with
age. Similar findings have been reported by oth-
ers10,12,24,46 although two other studies suggest that a
significant decline in satellite cell numbers does oc-
cur with age.30,43
There were several potential limitations of this
study. We did not incorporate a prebiopsy and post-
biopsy control group. Thus, the potential effects of
the inflammatory/regenerative response from the
preexercise biopsy on satellite cell activation and
proliferation affecting counts performed on tissue
obtained at the postexercise biopsy may have influ-
enced our results. Indeed, a significant increase in
NCAM-positive cells has been reported in control
(after three biopsies without exercise) subjects
within 24 h.36 Moreover, the cell division times of
12–15 h mentioned above were calculated from in
Acute Satellite Cell Response to Exercise
vitro work which is not physiologic.5,17,42 Thus, we
cannot exclude the possibility that a portion of the
increase in satellite cell numbers is caused by preex-
ercise tissue harvesting or that the disruption caused
by the initial biopsy procedure potentiates or primes
the satellite cells within the vastus lateralis and that
initial stimulus was augmented by the 92 maximal
eccentric contractions performed later.
Another limitation was our utilization of a single
antibody to detect satellite cells (NCAM), which may
not discriminate between satellite cells originating
from extra-muscular sources (i.e., multipotential
stem cells) and merging with the muscle cell to
participate in regeneration of damaged tissue.51 Al-
though the contribution of nonmuscle stem cells to
normal skeletal muscle repair and regeneration is
reportedly minimal,9,11,39 future studies should elu-
cidate the potential contribution of muscle resident
stem cells (e.g., by utilizing antibodies directed
against the molecular markers CD45 and/or Pax7)
to myogenic precursor cells.2 The inclusion of pre-
exercise and postexercise biopsy tissue from the
same subjects on the same slide, as well as having
included both young and older tissue samples to
ensure identical processing, may have introduced
bias. As each subject was assigned a number, and
placement on the slide (first or second) was random,
we feel that bias was minimized, but postexercise
tissue was easy to identify due to the incidence of
“doublets” (Fig. 3A–D), the occurrence of two satel-
lite cells within a single muscle fiber (Fig. 3E and F),
and increased numbers of satellite cells per tissue
section (Fig. 4B and C). Additionally, although not
quantified, we observed an increased incidence of
NCAM-positive muscle cells in the postexercise tissue
samples (Fig. 5) relative to the preexercise sample,
indicating recent muscle degeneration or regenera-
tion25,48 and thus indicated that the tissue section
was postexercise. Testosterone levels were similar in
the two age groups, possibly due to small number in
the young group as testosterone levels in older sub-
jects are generally lower than in younger sub-
jects.22,37 Whether differences in endogenous testos-
terone levels affect satellite cell activation during a
bout of maximal eccentric exercise is unknown but
evidence indicates that exogenous testosterone acti-
vates satellite cells.52 Lastly, future studies should
incorporate multiple postexercise biopsies spread
out over several days which would allow for satellite
cell number determination at later time points to see
whether older subjects make up the difference in
satellite cell numbers that we observed at 24 h.
MUSCLE & NERVE February 2006 251
This work was supported in part by a grant from the Foundation
for Physical Therapy Promotion of Doctoral Studies II (PODS II),
the National Institutes of Health MOI RR00043 and AG18169,
and by a General Clinical Research Center (GCRC) grant, 5 M01
RR-00043, from the National Center for Research Resources,
NIH.
REFERENCES
1. Adams GR, Haddad F, Baldwin KM. Time course of changes
in markers of myogenesis in overloaded rat skeletal muscles.
J Appl Physiol 1999;87:1705–1712.
2. Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA. Myo-
genic specification of side population cells in skeletal muscle.
J Cell Biol 2002;159:123–134.
3. Barani AE, Durieux AC, Sabido O, Freyssenet D. Age-related
changes in the mitotic and metabolic characteristics of mus-
cle-derived cells. J Appl Physiol 2003;95:2089 –2098.
4. Bergstrom J. Percutaneous needle biopsy of skeletal muscle in
physiological and clinical research. Scand J Clin Lab Invest
1975;35:609 – 616.
5. Bischoff R. Proliferation of muscle satellite cells on intact
myofibers in culture. Dev Biol 1986;115:129 –139.
6. Blough ER, Linderman JK. Lack of skeletal muscle hypertro-
phy in very aged male Fischer 344 x Brown Norway rats. J Appl
Physiol 2000;88:1265–1270.
7. Cameron-Smith D. Exercise and skeletal muscle gene expres-
sion. Clin Exp Pharmacol Physiol 2002;29:209 –213.
8. Carson JA, Alway SE. Stretch overload-induced satellite cell
activation in slow tonic muscle from adult and aged Japanese
quail. Am J Physiol 1996;270:C578 –C584.
9. Charge SB, Rudnicki MA. Cellular and molecular regulation
of muscle regeneration. Physiol Rev 2004;84:209 –238.
10. Charifi N, Kadi F, Feasson L, Denis C. Effects of endurance
training on satellite cell frequency in skeletal muscle of old
men. Muscle Nerve 2003;28:87–92.
11. Conboy IM, Conboy MJ, Smythe GM, Rando TA. Notch-
mediated restoration of regenerative potential to aged mus-
cle. Science 2003;302:1575–1577.
12. Crameri RM, Langberg H, Magnusson P, Jensen CH, Schro-
der HD, Olesen JL, et al. Changes in satellite cells in human
skeletal muscle after a single bout of high intensity exercise.
J Physiol (Lond) 2004;558:333–340.
13. Darr KC, Schultz E. Exercise-induced satellite cell activation
in growing and mature skeletal muscle. J Appl Physiol 1987;
63:1816 –1821.
14. De Angelis L, Berghella L, Coletta M, Lattanzi L, Zanchi M,
Cusella-De Angelis MG, et al. Skeletal myogenic progenitors
originating from embryonic dorsal aorta coexpress endothe-
lial and myogenic markers and contribute to postnatal muscle
growth and regeneration. J Cell Biol 1999;147:869 – 878.
15. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stor-
naiuolo A, Cossu G, et al. Muscle regeneration by bone mar-
row-derived myogenic progenitors. Science 1998;279:1528 –
1530.
16. Fiatarone MA, Marks EC, Ryan ND, Meredith CN, Lipsitz LA,
Evans WJ. High-intensity strength training in nonagenarians.
Effects on skeletal muscle. JAMA 1990;263:3029 –3034.
17. Goetsch SC, Hawke TJ, Gallardo TD, Richardson JA, Garry DJ.
Transcriptional profiling and regulation of the extracellular
matrix during muscle regeneration. Physiol Genomics 2003;
14:261–271.
18. Grounds MD. Age-associated changes in the response of skel-
etal muscle cells to exercise and regeneration. Ann NY Acad
Sci 1998;854:78 –91.
19. Hakkinen K, Kallinen M, Izquierdo M, Jokelainen K, Lassila
H, Malkia E, et al. Changes in agonist-antagonist EMG, mus-
cle CSA, and force during strength training in middle-aged
and older people. J Appl Physiol 1998;84:1341–1349.
252 Acute Satellite Cell Response to Exercise
20. Hall ZW, Ralston E. Nuclear domains in muscle cells. Cell
1989;59:771–772.
21. Hameed M, Orrell RW, Cobbold M, Goldspink G, Harridge
SD. Expression of IGF-I splice variants in young and old
human skeletal muscle after high resistance exercise. J Physiol
(Lond) 2003;547:247–254.
22. Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR.
Longitudinal effects of aging on serum total and free testos-
terone levels in healthy men. Baltimore Longitudinal Study of
Aging. J Clin Endocrinol Metab 2001;86:724 –731.
23. Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to
molecular biology. J Appl Physiol 2001;91:534 –551.
24. Hikida RS, Eriksson A, Holmner S, Thornell LE. Is hypertro-
phy limited in elderly muscle fibers? A comparison of elderly
and young strength-trained men. Basic Appl Myol 1998;8:
419 – 427.
25. Illa I, Leon-Monzon M, Dalakas MC. Regenerating and de-
nervated human muscle fibers and satellite cells express neu-
ral cell adhesion molecule recognized by monoclonal anti-
bodies to natural killer cells. Ann Neurol 1992;31:46 –52.
26. Jacobs SC, Wokke JH, Bar PR, Bootsma AL. Satellite cell
activation after muscle damage in young and adult rats. Anat
Rec 1995;242:329 –336.
27. Kadi F, Eriksson A, Holmner S, Thornell LE. Effects of ana-
bolic steroids on the muscle cells of strength-trained athletes.
Med Sci Sports Exerc 1999;31:1528 –1534.
28. Kadi F. Adaptation of human skeletal muscle to training and
anabolic steroids. Acta Physiol Scand Suppl 2000;646:1–52.
29. Kadi F, Thornell LE. Concomitant increases in myonuclear
and satellite cell content in female trapezius muscle following
strength training. Histochem Cell Biol 2000;113:99 –103.
30. Kadi F, Charifi N, Denis C, Lexell J. Satellite cells and myo-
nuclei in young and elderly women and men. Muscle Nerve
2004;29:120 –127.
31. Lanier LL, Testi R, Bindl J, Phillips JH. Identity of Leu-19
(CD56) leukocyte differentiation antigen and neural cell ad-
hesion molecule. J Exp Med 1989;169:2233–2238.
32. Lowe DA, Lund T, Alway SE. Hypertrophy-stimulated myo-
genic regulatory factor mRNA increases are attenuated in fast
muscle of aged quails. Am J Physiol 1998;275:C155–C162.
33. Lowe DA, Alway SE. Stretch-induced myogenin, MyoD, and
MRF4 expression and acute hypertrophy in quail slow-tonic
muscle are not dependent upon satellite cell proliferation.
Cell Tissue Res 1999;296:531–539.
34. MacDougall JD, Gibala MJ, Tarnopolsky MA, MacDonald JR,
Interisano SA, Yarasheski KE. The time course for elevated
muscle protein synthesis following heavy resistance exercise.
Can J Appl Physiol 1995;20:480 – 486.
35. Maier F, Bornemann A. Comparison of the muscle fiber
diameter and satellite cell frequency in human muscle biop-
sies. Muscle Nerve 1999;22:578 –583.
36. Malm C, Nyberg P, Engstrom M, Sjodin B, Lenkei R, Ekblom
B, et al. Immunological changes in human skeletal muscle
and blood after eccentric exercise and multiple biopsies.
J Physiol (Lond) 2000;529:243–262.
37. Morley JE, Kaiser FE, Perry HM, Patrick P, Morley PM,
Stauber PM, et al. Longitudinal changes in testosterone, lu-
teinizing hormone, and follicle-stimulating hormone in
healthy older men. Metabolism 1997;46:410 – 413.
38. Moss FP, Leblond CP. Satellite cells as the source of nuclei in
muscles of growing rats. Anat Rec 1971;170:421– 435.
39. Partridge TA. Cells that participate in regeneration of skeletal
muscle. Gene Ther 2002;9:752–753.
40. Phelan JN, Gonyea WJ. Effect of radiation on satellite cell
activity and protein expression in overloaded mammalian
skeletal muscle. Anat Rec 1997;247:179 –188.
41. Phillips SM, Tipton KD, Aarsland A, Wolf SE, Wolfe RR.
Mixed muscle protein synthesis and breakdown after resis-
tance exercise in humans. Am J Physiol 1997;273:E99 –E107.
42. Qu-Petersen Z, Deasy B, Jankowski R, Ikezawa M, Cummins J,
Pruchnic R, et al. Identification of a novel population of
MUSCLE & NERVE February 2006
 muscle stem cells in mice: potential for muscle regeneration.
J Cell Biol 2002;157:851– 864.
43. Renault V, Piron-Hamelin G, Forestier C, DiDonna S, Decary
S, Hentati F, et al. Skeletal muscle regeneration and the
mitotic clock. Exp Gerontol 2000;35:711–719.
44. Renault V, Rolland E, Thornell LE, Mouly V, Butler-Browne
G. Distribution of satellite cells in the human vastus lateralis
muscle during aging. Exp Gerontol 2002;37:1513–1514.
45. Rosenblatt JD, Yong D, Parry DJ. Satellite cell activity is re-
quired for hypertrophy of overloaded adult rat muscle. Mus-
cle Nerve 1994;17:608 – 613.
46. Roth SM, Martel GF, Ivey FM, Lemmer JT, Metter EJ, Hurley
BF, et al. Skeletal muscle satellite cell populations in healthy
young and older men and women. Anat Rec 2000;260:351–
358.
47. Roth SM, Martel GF, Ivey FM, Lemmer JT, Tracy BL, Metter
EJ, et al. Skeletal muscle satellite cell characteristics in young
and older men and women after heavy resistance strength
training. J Gerontol A Biol Sci Med Sci 2001;56:B240 –B247.
48. Schubert W, Zimmermann K, Cramer M, Starzinski-Powitz A.
Lymphocyte antigen Leu-19 as a molecular marker of regen-
eration in human skeletal muscle. Proc Natl Acad Sci USA
1989;86:307–311.
49. Schultz E, Jaryszak DL, Valliere CR. Response of satellite cells
to focal skeletal muscle injury. Muscle Nerve 1985;8:217–222.
50. Schultz E. Satellite cell proliferative compartments in growing
skeletal muscles. Dev Biol 1996;175:84 –94.
Acute Satellite Cell Response to Exercise
51. Sinanan AC, Hunt NP, Lewis MP. Human adult craniofacial
muscle-derived cells: neural-cell adhesion-molecule (NCAM;
CD56)-expressing cells appear to contain multipotential stem
cells. Biotechnol Appl Biochem 2004;40:25–34.
52. Sinha-Hikim I, Roth SM, Lee MI, Bhasin S. Testosterone-
induced muscle hypertrophy is associated with an increase in
satellite cell number in healthy, young men. Am J Physiol
Endocrinol Metab 2003;285:E197–E205.
53. Tamaki T, Uchiyama S, Uchiyama Y, Akatsuka A, Yoshimura
S, Roy RR, et al. Limited myogenic response to a single bout
of weight-lifting exercise in old rats. Am J Physiol Cell Physiol
2000;278:C1143–C1152.
54. Tatsumi R, Hattori A, Ikeuchi Y, Anderson JE, Allen RE.
Release of hepatocyte growth factor from mechanically
stretched skeletal muscle satellite cells and role of pH and
nitric oxide. Mol Biol Cell 2002;13:2909 –2918.
55. Welle S, Totterman S, Thornton C. Effect of age on muscle
hypertrophy induced by resistance training. J Gerontol A Biol
Sci Med Sci 1996;51:M270 –M275.
56. Young HE, Steele TA, Bray RA, Hudson J, Floyd JA, Hawkins
K, et al. Human reserve pluripotent mesenchymal stem cells
are present in the connective tissues of skeletal muscle and
dermis derived from fetal, adult, and geriatric donors. Anat
Rec 2001;264:51– 62.
57. Zammit P, Beauchamp J. The skeletal muscle satellite cell:
stem cell or son of stem cell? Differentiation 2001;68:193–204.
MUSCLE & NERVE February 2006 253