Full text of DOI:10.1093/gerona/56.12.B510

Journal of Gerontology: BIOLOGICAL SCIENCES
001, Vol. 56A, No. 12, B510–B519
Copyright 2001 by The Gerontological Society of America
2
Fiber-Type Transitions and Satellite Cell Activation in
Low-Frequency-Stimulated Muscles of Young and
Aging Rats
1
2
2
1
ˇ
2
Charles T. Putman, Karim R. Sultan, Thomas Wassmer, Jeremy A. Bamford, Dejan Skorjanc,
and Dirk Pette2
1Skeletal Muscle Research Group, Faculty of Physical Education, University of Alberta, Edmonton, Canada.
2Department of Biology, University of Konstanz, Germany.
We examined satellite cell content and the activity of satellite cell progeny in tibialis anterior
muscles of young (15 weeks) and aging (101 weeks) Brown Norway (BN) rats, after they were
exposed for 50 days to a standardized and highly reproducible regime of chronic low-frequency
electrical stimulation. Chronic low-frequency electrical stimulation was successful in inducing
fast-to-slow fiber-type transformation, characterized by a 2.3-fold increase in the proportion of
IIA fibers and fourfold and sevenfold decreases in the proportion of IID/X and IIB fibers in both
young and aging BN rats. These changes were accompanied by a twofold increase in the satellite
cell content in both the young and aging groups; satellite cell content reached a level that was
significantly higher in the young group (p ꢀ .04). The total muscle precursor cell content (i.e.,
satellite cells plus progeny), however, did not differ between groups, because there was a greater
number of satellite cell progeny passing through the proliferative and differentiative compart-
ments of the aging group. The resulting 1.5-fold increase in myonuclear content was similar in
the young and aging groups. We conclude that satellite cells and satellite cell progeny of aging
BN rats possess an unaltered capacity to contribute to the adaptive response.
GING is accompanied by a loss of functional ability
and a restricted adaptive potential. At the level of skel-
etal muscle fibers, the major changes associated with aging
include a decline in mass and strength caused by fiber atro-
phy, fiber loss, and a shift toward slower phenotypes (for re-
views, see 1–3). Furthermore, aging muscle has been shown
to display lower adaptive (4) and regenerative capacities
fibers, CLFS obviously enhanced fiber regeneration in ag-
ing muscle, thus decreasing the fraction of COX-deficient
fibers.
A
The formation of new fibers in aging muscle obviously
disagrees with findings on a reduced regenerative capacity
and decrease in satellite cell content and proliferative poten-
tial in old muscle. Our observations, however, are in line
with the notion that neuromuscular activity has an impact
on satellite cell content. We, therefore, undertook the
present study to compare the effects of enhanced neuromus-
cular activity on satellite cell content and proliferative activ-
ity of a fast-twitch muscle in young and aging rats. Because
it is impossible to create conditions under which young and
aging animals perform voluntary exercise at high and iden-
tical intensities, CLFS was chosen as a standardized and re-
producible regimen of increased neuromuscular activity.
CLFS activates all motor units of the stimulated muscle,
even those normally not recruited by exercise training. It
imposes higher levels of activity over time than any exer-
cise regimen and, therefore, challenges the adaptive poten-
tial of the target muscle to its limits. These attributes make
CLFS an ideal experimental model for studying effects of
enhanced contractile activity on various structural, func-
tional, metabolic, and molecular properties.
(
5), caused by decreases in satellite cell content (6) and pro-
liferation potential (7).
Another age-related change, especially seen in muscles
abundant in mitochondria, is the appearance of muscle fi-
bers with point and/or deletion mutations of mitochon-
drial DNA (for reviews, see 8 and 9). At the protein level,
these changes may result in defects of mitochondrially en-
coded proteins such as cytochrome c-oxidase (COX), a mul-
timeric enzyme with three mitochondrially encoded sub-
units. Thus, COX-deficient fibers are typically found in
aging muscle (10,11). In a previous study, we investigated
effects of enhanced contractile activity by chronic low-fre-
quency electrical stimulation (CLFS) on the incidence of
COX-deficient fibers in muscles of young and aging rats
(
12). In stimulated young muscle, we observed an increase
in COX-deficient fibers concomitant with the increase in
mitochondrial content. In aging muscle with a similar in-
crease in mitochondrial content, CLFS, however, led to a
decrease in the number of COX-deficient fibers. As indi-
cated by the appearance of some very small COX-positive
The same experimental animals and experimental condi-
tions were applied as in our preceding studies (12,13):
Briefly, the left tibialis anterior (TA) muscles of 15-week-
B510

SATELLITE CELLS IN ELECTROSTIMULATED AGING MUSCLE
B511
old and 101-week-old male rats of the genetically pure-bred
Brown Norway (BN) strain were exposed to CLFS at 10 Hz,
isoforms were used: MHCI [NOQ7.5.4D (25)]; MHCIIa
[SC-71 (26)], and all fast MHC isoforms with the ex-
ception of MHCIIx/d [BF-35 (26)]. Embryonic MHC
(MHCemb) was detected on frozen sections by using anti-
MHCemb clone 47A (27). Mouse monoclonal anti-Ki-67
(clone MIB-5; 18,19) was obtained from Dianova (Ham-
burg, Germany). Anti-dystrophin monoclonal antibody
(clone DYS2, Dy8/6C5) directed against the carboxyl ter-
minus (28) was obtained from Novocastra Laboratories
(Newcastle, UK). Monoclonal anti-desmin (clone DE-B-5)
and monoclonal anti-vimentin (clone V9) were obtained
from Boehringer Mannheim (Mannheim, Germany) and
Sigma (Diesenhofen, Germany), respectively. Goat poly-
clonal anti-M-cadherin (N-19) and rabbit polyclonal anti-
myogenin (M-225) antibodies were obtained from Santa
Cruz Biochemicals (Santa Cruz, CA). Rabbit polyclonal
anti-laminin (IgG) was obtained from ICN Biochemicals
(Costa Mesa, CA).
1
0 hours daily, for 50 days. The contralateral, unstimulated
muscles served as an intra-animal control. The 15-week-old
rats were considered to be young adults, whereas the 101-
week-old animals were considered to be aging. This assign-
ment was based on the average life span (ꢀ120 weeks),
which represents the 50% survival rate and has traditionally
been regarded as the point at which BN rats are considered
old or senescent (14).
Changes in myosin heavy chain (MHC)-based fiber-type
distribution and fiber size were assessed immunohistochem-
ically on serial cross sections of the stimulated and con-
tralateral unstimulated muscles. Quiescent satellite cells
were identified immunohistochemically (15,16) by using an
antibody specific for M-cadherin, a calcium-dependent
transmembrane glycoprotein, which anchors satellite cells
to the sarcolemma (17). As a way to identify proliferating
satellite cell progeny, sections were stained for Ki-67, a nu-
clear nonhistone protein that is expressed in all phases of
the cell cycle, except the quiescent Go phase (18,19). Satel-
lite cell progeny, newly fused to adjacent fibers, were iden-
tified by their intense immunohistochemical reaction for the
muscle-specific transcription factor myogenin, which is
strongly upregulated during terminal differentiation (20–
Biotinylated horse-anti-mouse-IgG (rat-absorbed, affin-
ity-purified), biotinylated goat-anti-rabbit-IgG, and bio-
tinylated horse-anti-goat-IgG were obtained from Vector
Laboratories, Inc. (Burlingame, CA). Nonspecific control
mouse-IgG, goat-IgG, and rabbit-IgG antibodies were ob-
tained from Santa Cruz Biochemicals.
2
2). Intrafiber myonuclei were distinguished from interfiber
nuclei by staining for hematoxylin and laminin, whereas
changes in capillary density were assessed by laminin stain-
ing only (23).
Immunohistochemistry for Myosin, Ki-67, Dystrophin,
Vimentin, and Desmin
Frozen, 12-ꢃm-thick sections of TA muscles were air
dried, washed once in phosphate-buffered saline (PBS) with
0.1% Tween-20 (PBS-Tween) and twice in PBS, and then
incubated for 30 minutes in 3% H O in methanol. Sections
METHODS
2
2
Animals and Low-Frequency Stimulation
were subsequently washed and incubated at room tempera-
ture for 1 hour in a blocking solution (BS-1: 1% bovine se-
rum albumin, 10% horse serum, and 0.1% PBS-Tween, pH
7.4). Excess BS-1 was removed and sections were incu-
bated (15 minutes) in Avidin-D blocking solution (Vector
Laboratories), rinsed with PBS, incubated (15 minutes) with
Biotin blocking solution (Vector Laboratories), and washed
with PBS. Primary anti-mouse-IgG monoclonal antibodies
were diluted in BS-1 as follows: NOQ7.5.4D, culture super-
natant 1:400; SC-71, 3.8 ꢃg/ml; BF-35, 1.7 ꢃg/ml; DYS2,
culture supernatant 1:8; V9, 4 ꢃg/ml; DE-B-5, 6 ꢃg/ml.
Culture supernatants containing anti-MHCemb (clone 47A)
and anti-Ki-67 (clone MIB-5) were applied neat. All pri-
mary antibodies were applied for 1 hour at room tempera-
ture, except anti-Ki-67, which was applied overnight at 4ꢁC.
Control sections were completed in parallel in which (a) the
primary antibody was substituted with nonspecific control
mouse IgG, and (b) the primary antibody was omitted. Sec-
tions were washed as before and reacted for 30 minutes with
biotinylated horse-anti-mouse-IgG. Sections were then
washed, incubated with Biotin-Avidin-horse-radish peroxi-
dase (HRP) complex for 60 minutes, washed again, and re-
acted for 5 minutes with a substrate solution containing
Five young adult and five aging male BN rats (TNO Pre-
vention and Health Center for Ageing Research, Leiden,
The Netherlands) were utilized in this study. Young and ag-
ing rats were 15 and 101 weeks of age at the beginning and
2
2 and 108 weeks of age at the end of the stimulation pe-
riod, respectively. All experiments were approved by the lo-
cal government (Regierungspräsidium Freiburg, Germany)
and rats were treated in accordance with established princi-
ples of care and use. Rats were housed in the Animal Re-
search Center of the University of Konstanz in a thermally
controlled room maintained at 22ꢁC with 12-hour dark cy-
cles alternated with 12-hour light cycles. All rats received
food (Altromin 1314, Altromin GmbH, Lage, Germany)
and water ad libitum. Electrodes were implanted laterally to
the peroneal nerve of the left hind limb (24). After 1 week
of recovery, CLFS was initiated (continuously for 10 hours
daily at 10 Hz; impulse width of 0.3 milliseconds) and pro-
ceeded for 50 consecutive days. Following completion of
the stimulation period, rats were euthanized and the TA
muscles of the stimulated (left) and contralateral control
(
(
right) legs were excised, weighed, and frozen in melting
ꢂ159ꢁC) isopentane. Muscles were stored in liquid nitro-
gen until they were analyzed.
DAB, H O , and NiCl in 50 mM Tris-HCl, pH 7.5 (Vector
2 2 2
Laboratories). Reactions were stopped by washing with dis-
tilled water. After dehydration in ethanol, sections were
cleared and mounted with Entellan (Merck, Darmstadt, Ger-
many).
Antibodies
To examine changes in fiber-type distribution, the fol-
lowing monoclonal antibodies directed against adult MHC

B512
PUTMAN ET AL.
Immunohistochemistry for Myogenin, M-cadherin,
and Laminin
Sections that were 12 ꢃm thick were air dried and then
fixed for 10 minutes in cold acetone (ꢂ20ꢁC); excess ace-
tone was allowed to evaporate. Sections were then washed
once in PBS-Tween and twice in PBS, incubated for 30
minutes in 3% H O in methanol, washed again, and incu-
2 2
bated for 1 hour in a blocking solution. BS-1 was used for
M-cadherin staining, whereas goat serum was substituted
for horse serum in a second blocking solution (BS-2) and
used for myogenin and laminin staining. Endogenous Avi-
din and Biotin binding were blocked as previously de-
Figure 1. Representative photographs of myosin heavy chain (MHC) immunostains of control (A, C, E, G) and 50-day stimulated (B, D, F,
H) tibialis anterior muscles of an aging rat. A, B: immunostains for MHCI (clone NOQ7.5.4D); C, D: immunostains for MHCIIa (clone SC-71);
E, F: immunostains for all MHCs except MHCIId/x (clone BF-35); G, H: IgG control. Bar is 150 ꢃm.

SATELLITE CELLS IN ELECTROSTIMULATED AGING MUSCLE
B513
scribed. Primary polyclonal antibodies were diluted as fol-
lows: anti-myogenin to 0.3 ꢃg/ml in BS-2; anti-M-cadherin
to 0.7 ꢃg/ml in BS-1; anti-laminin 1:300 in BS-2. Excess
blocking solution was removed, and primary antibodies
were overlaid on sections and incubated overnight at 4ꢁC.
Sections were then washed once in PBS-Tween, washed
twice in PBS, and reacted for 30 minutes with either bio-
tinylated goat-anti-rabbit-IgG (for anti-myogenin M-225
and anti-laminin) or horse-anti-goat-IgG (for anti-M-cad-
herin N19). After washing, sections were incubated for 60
minutes with Biotin-Avidin-HRP, washed, and reacted for 6
minutes with the substrate solution (previously described).
Immunolocalization of laminin was visualized with Vector
Fast Red (Vector Laboratories). After the reaction was
stopped with distilled water, those sections stained for lami-
nin were counterstained with Mayer’s Hemalum (Merck)
and used to evaluate the total number of muscle nuclei and
capillary density. All sections were subsequently dehy-
drated, cleared, and mounted in Entellan.
ences between the young and aging groups were assessed
by an independent samples Student’s t test, whereas differ-
ences between control and stimulated conditions were as-
sessed by a paired dependent samples Student’s t test. Be-
cause changes in fiber-type distribution and relative satellite
cell content were predicted a priori, these data were evalu-
ated with one-tailed tests. Differences between individual
fiber cross-sectional areas were detected by a two-way anal-
ysis of variance, with repeated measures within animals
(i.e., stimulated vs control). When a significant F ratio was
found, differences were located by using the Newman-
Keuls post hoc analysis. Differences were considered sig-
nificant at p ꢀ .05.
RESULTS
Fiber-Type Transitions
Fiber-type transitions were assessed immunohistochemi-
cally on serial sections (Figure 1), in both the deep and su-
perficial regions of each TA muscle. Because no differences
were detected between the two experimental groups in ei-
Immunohistochemical Analyses
Serial sections, stained for the various MHC isoforms,
were examined by using a computer program (28) to deter-
mine the relative proportion of muscle fiber types. A similar
number of fibers were examined from two distinct areas
(
superficial and deep) for each of the stimulated (total fi-
bers: 431 ꢄ 28 fibers/muscle) and control legs (total fibers:
80 ꢄ 25 fibers/muscle). A total of 8094 fibers were exam-
3
ined for these analyses: aging control, 1838 fibers; aging
stimulated, 2151; young control, 1943; young stimulated,
2
162. A minimum of 25 and a maximum number of 1680 fi-
bers were examined to assess differences in fiber cross-sec-
tional area, according to fiber type and experimental condi-
tion. Because the number of fibers staining positive for
MHCemb was small by comparison, they were not included
in these analyses but were evaluated separately over the en-
tire cross-sectional area of each muscle.
Serial sections stained for vimentin, desmin, and dystro-
phin were examined to determine the number of damaged
fibers after 50 days of CLFS (28,29). The criteria for identi-
fying a damaged fiber were as follows: vimentin positive,
dystrophin negative, and/or an altered pattern of desmin
staining (i.e., absence of staining or foci of positivity); the
entire cross section of each muscle was examined. Subse-
quently, the total number of fibers expressing MHCemb was
determined for each muscle. Each of these analyses encom-
2
passed an area in excess of 25 mm for each of the 20 mus-
cles studied. The mean cross-sectional areas examined for
M-cadherin (i.e., quiescent satellite cell), total nuclei (i.e.,
Mayer’s Hemalum and laminin), Ki-67 (i.e., proliferating
satellite cell progeny), and myogenin (i.e., terminally differ-
entiating satellite cell progeny) stains were 25.5 ꢄ 2.5,
2
5
.3 ꢄ 0.4, 19.5 ꢄ 2.2, and 16.3 ꢄ 2.3 mm , respectively.
Capillaries were identified according to their pattern of
laminin staining (23), and the number per unit area was
evaluated over the entire cross section of each muscle.
Figure 2. Percentage of distribution of immunohistochemically de-
fined pure and hybrid fiber types in control and stimulated tibialis an-
terior muscles of young and aging rats after 50 days of chronic low-
frequency stimulation. a ꢅ different from contralateral control;
MHC ꢅ myosin heavy chain.
Statistical Analyses
Data are presented as mean ꢄ standard error of the mean
and analyzed as follows. Unless otherwise stated, differ-

B514
PUTMAN ET AL.
cance was, however, only observed in the young group (p ꢀ
05), in which the total proportion of MHCI-expressing fi-
.
bers increased from 3.5% to 10.9% (Figure 2).
The mean cross-sectional area of fibers from contralateral
2
control muscles of the young group was 2034 ꢄ 92 ꢃm ,
and it was slightly larger (p ꢀ .05) in the aging rats at
2
2
203 ꢄ 576 ꢃm . Further examination of the fiber-type-
specific cross-sectional areas revealed that the difference
was primarily due to significantly larger IIA fibers in the
aging group before (young vs aging: 1563 ꢄ 354 vs 1654 ꢄ
2
4
1
06 ꢃm ; p ꢀ .01) and after CLFS (young vs aging: 1155 ꢄ
2
44 vs 1450 ꢄ 320 ꢃm ; p ꢀ .0001). CLFS had similar ef-
fects on both groups; that is, it reduced (df ꢅ 4822; F ꢅ
50; p ꢀ .00001) the mean cross-sectional areas of the stim-
1
2
ulated muscles to 1626 ꢄ 382 ꢃm in the young group and
to 1689 ꢄ 411 ꢃm in the aging group. Stimulated fibers re-
2
mained larger in the muscles of aging rats compared with
those in the young stimulated group (p ꢀ .02).
Figure 3. Example of a regenerating fiber that is stained for vimen-
Structural Morphology, Capillaries
tin (A), dystrophin (B), desmin (C), and IgG control (D). Note the
presence of vimentin in A, the absence of dystrophin (B), and a small
foci of desmin positivity (C) within the fiber marked with an arrow.
Bar is 50 ꢃm.
Examination of serial sections for damaged fibers, identi-
fied as vimentin positive, dystrophin negative, and/or an al-
tered pattern of desmin staining (Figure 3), revealed no sig-
nificant differences between young and aging muscles. In
the 20 muscles examined, only 25 fibers were detected that
met the described criteria. What is more, only 3 were ob-
served in all of the control muscles, whereas the remaining
22 were equally distributed between the stimulated muscles
of the young and aging groups.
Examination of the incidence of small myotubes or very
small fibers expressing MHCemb revealed 10- and 50-fold
elevated (p ꢀ .05) values in the control and stimulated TA
muscles of the aging rats, respectively (Figure 4). Despite
the much greater incidence in aging muscles, the absolute
numbers of myotubes or small regenerating fibers amounted
ther of the stimulated or control conditions, the resulting fi-
ber-type distributions of the two regions were averaged.
Fifty days of CLFS induced similar fast-to-slow fiber-type
transitions in young and aging rats, namely approximately
twofold increases in the proportion of IIA fibers and ap-
proximately fourfold and sevenfold decreases in the propor-
tions of IID(X) and IIB fibers, respectively (Figure 2). The
proportion of fibers expressing MHCI (i.e., type I ꢆ type
I/IIA) increased by 1.6- and 3.1-fold as a result of CLFS, in
the aging and young groups, respectively. Statistical signifi-
Figure 5. Representative photographs of immunostains of M-cad-
herin (A), Ki-67 (B), laminin and hematoxylin (C), and myogenin
(D). These stains were used to identify A, satellite cells (arrow); B,
proliferating satellite cell progeny (arrow); C, intrafiber muscle nuclei
(arrow) and capillaries (asterisk); and D, satellite cell progeny com-
mitted to terminal differentiation (arrow). Bar is 25 ꢃm.
Figure 4. Number of fibers per unit area staining positive for em-
bryonic myosin heavy chain (MHC) in control and stimulated tibialis
anterior muscles of young and aging rats after 50 days of chronic low-
frequency stimulation. a ꢅ different from contralateral control; b ꢅ
aging different from young.

SATELLITE CELLS IN ELECTROSTIMULATED AGING MUSCLE
B515
Figure 6. Number of satellite cells per unit area in control and
stimulated tibialis anterior muscles of young and aging rats after 50
days of chronic low-frequency stimulation. a ꢅ different from con-
tralateral control; b ꢅ aging different from young.
Figure 8. Number of proliferating satellite cell progeny (Ki-67 pos-
itive) per unit area in control and stimulated tibialis anterior muscles
of young and aging rats after 50 days of chronic low-frequency stimu-
lation. a ꢅ different from contralateral control; b ꢅ aging different
from young.
to only 10 and 50 over the entire section, or less than 0.3%
and 2% of the muscle fibers present, respectively.
than twofold increases in the satellite cell contents in both
groups (Figure 6), reaching a 30% higher level in the young
muscles than in the aging muscles (p ꢀ .04). The absolute
increases in satellite cells per unit area for stimulated young
CLFS induced similar 1.8-fold increases in the number of
capillaries per unit area in the young and aging groups (Fig-
ure 5). In the young group the capillary density increased
2
2
2
from 340 ꢄ 5/mm before to 614 ꢄ 26/mm after 50 days of
CLFS (p ꢀ .05). Similarly, in the aging group the number
of capillaries per square millimeter increased from 382 ꢄ
and aging muscles were 13 ꢄ 1.6/mm and 10.0 ꢄ 0.7/
2
mm , respectively. However, when the total number of mus-
cle precursor cells (mpc) was calculated by addition of sat-
ellite cells, proliferating satellite cell progeny, and termi-
nally differentiating satellite cell progeny (Figure 7), there
was no apparent difference between the young and aging
groups.
Fifty days of CLFS resulted in elevated numbers of pro-
liferating satellite cell progeny (Ki-67 positive) and termi-
nally differentiating satellite cell progeny (myogenin posi-
tive), both reaching higher values in the aging than in the
young muscles. The number of proliferating satellite cell
progeny (Figure 8) was 1.8- and 1.6-fold elevated in the
young and aging rats, respectively. Similarly, the number of
terminally differentiating satellite cell progeny (Figure 9)
increased by twofold and threefold in the muscles of aging
and young rats, respectively.
3
0 to 677 ꢄ 62 after CLFS (p ꢀ .05).
Myonuclear Content and Satellite Cell Activity
The number of satellite cells per unit area, as determined
by immunohistochemical patterns of M-cadherin staining
(
Figure 5), was ꢀ20% greater in control muscles of young
rats than in those of aging rats, but it did not reach statistical
significance (p ꢅ .32). Fifty days of CLFS induced more
The total number of intrafiber nuclei observed per unit
area was similar in the young and aging control muscles
(
5
Figure 10) and was elevated 1.5-fold in both groups after
0 days of CLFS. However, the proportion of satellite cells
relative to the total number of intrafiber myonuclei was
greater (p ꢀ .05) in young stimulated muscles (4.5 ꢄ 0.3%)
compared with the same condition in the aging group (3.4 ꢄ
0
.3%). In both groups, the stimulated muscles contained a
higher percentage of satellite cells than their respective con-
tralateral controls (p ꢀ .05), namely 3.1 ꢄ 0.3% in the
young and 2.5 ꢄ 0.3% in the aging muscles. In contrast, the
proportion of total mpc, relative to the total myonuclear
content, was similar between young and aging muscles be-
fore and after CLFS (aging, stimulated vs control: 4.9 ꢄ 0.6
vs 3.7 ꢄ 0.6%; young, stimulated vs control: 5.3 ꢄ 0.2 vs
3.7 ꢄ 0.2%).
Figure 7. Number of total muscle precursor cells per unit area in
control and stimulated tibialis anterior muscles of young and aging
rats after 50 days of chronic low-frequency stimulation. a ꢅ different
from contralateral control.

B516
PUTMAN ET AL.
as well as the potential of aging satellite cells to proliferate
and terminally differentiate, in vivo, in response to 50 days
of CLFS. This model appeared to be appropriate for two
reasons: First, CLFS is known to induce increases in satel-
lite cell activity and content that are temporally and spa-
tially correlated with a large fast-to-slow fiber-type trans-
formation (21,22,28). Second, it has been demonstrated
that satellite cells isolated from aging rat skeletal muscle
possess significantly lower proliferation potential in cul-
ture (7).
Age- and CLFS-Related Changes in Fiber Types
With the use of sensitive and highly specific immunohis-
tochemical methods, age-related fast-to-slow phenotypic
changes were not detectable in either the deep or superficial
regions of control TA muscles of 108-week-old BN rats
(
Figures 1 and 2). Our results are in agreement with previous
Figure 9. Number of differentiating satellite cell progeny (myoge-
studies that used classical histochemical myosin adenosine
triphosphate methods to examine age-related phenotypic
changes in control muscles of young and old rats (30–33).
Fifty days of CLFS induced fast-to-slow fiber-type tran-
sitions in young and aging TA muscles that were quantita-
tively and qualitatively similar. We interpret this as evi-
dence that aging BN rats possess an adaptive range that is
comparable with that of their young counterparts. Both
groups demonstrated 2.3-fold increases in the proportion of
pure IIA fibers that were in excess of 80% of all fibers ex-
amined. Increases in the less fast IIA fibers were primarily
offset by reductions in the proportions of the faster pure
IID/X and IIB fibers. Differences in the findings of this
study and our previous one (13) relate to different analyti-
cal methods used and muscles studied. In our previous
study (13), quantitative electrophoretic analysis revealed
an increased proportion of MHCIId/x and decreased
MHCIIb in the control extensor digitorum longus (EDL)
muscle of the aging group. This differed from the present
study, in which we used a semiquantitative analytical ap-
proach and studied the TA muscle, which experiences a
lower postural load than the EDL and may therefore be less
susceptible to age-related changes in fiber-type profile and
MHC complement.
nin positive) per unit area in control and stimulated tibialis anterior
muscles of young and aging rats after 50 days of chronic low-fre-
quency stimulation. a ꢅ different from contralateral control.
DISCUSSION
The present study investigated the effects of 50 days of
CLFS on fiber-type transitions and accompanying changes
in satellite cell activity in young and aging TA muscles. We
investigated aging rather than senescent skeletal muscle be-
cause the latter is associated with an intrinsic state of irre-
versible deterioration (4). In contrast, skeletal muscles of
mammals that are late middle-aged to old begin to demon-
strate significant functional and structural decline, but they
may retain all or part of their adaptive potential (13). Thus
we were interested in determining if factors intrinsic to the
aging satellite cell population limited the adaptive potential
of skeletal muscle or if extrinsic factors associated with an
age-related decline in physical activity limited the potential
adaptive contributions of satellite cells and their progeny.
To that end, a standardized and reproducible model of
muscle training was used to examine satellite cell number
The CLFS-induced phenotypic changes were almost
identical in the young and aging groups, but they did differ
somewhat with respect to the proportion of fibers express-
ing MHCI, which was slightly greater in the young group.
Despite this small difference, these data clearly demonstrate
that CLFS was successful in inducing fiber-type transitions
in the fast-to-slow direction. This was especially evident in
the superficial region of stimulated TA muscles, where IIA
fibers were rare under normal control conditions but were in
abundance within the deep region of control and stimulated
rat TA muscles. Our inclusion of the superficial region in
the present analyses thus explains the larger relative CLFS-
induced increase in IIA fibers compared with our previous
paper (12), which focused entirely on the deep region of the
same TA muscles.
Figure 10. Number of intrafiber muscle nuclei per unit area in con-
trol and stimulated tibialis anterior muscles of young and aging rats
after 50 days of chronic low-frequency stimulation. a ꢅ different from
contralateral control.
Age- and CLFS-Related Increase in Small Myotubes
The 10-fold increase in the number of small MHCemb
-
positive myotubes observed in the aging versus young con-

SATELLITE CELLS IN ELECTROSTIMULATED AGING MUSCLE
B517
trol muscles (Figure 4) is consistent with the development
of an age-related increase in regenerating fibers. Interest-
ingly, CLFS induced a further fivefold increase in the ap-
pearance of small myotubes. The reason for the age- and
CLFS-related increases in regenerating myotubes is not
readily obvious, but it may relate to a mechanical vulnera-
bility of aging muscle (34–36). It is also probable that the
increases resulted from neurodegenerative changes (37),
leading to denervation and fiber atrophy. These events re-
sult in a rearrangement of the fast motor units, increasing
their innervation ratios and areas (37). To accommodate for
the loss of fibers, the surviving motor units tend to be re-
cruited more frequently, which may facilitate the transfor-
mation of the remaining fast fibers toward a slower, more
efficient phenotype and/or lead to fiber hypertrophy. The in-
crease in the cross-sectional area of the IIA fibers observed
in the present study may be explained by such events.
satellite cells remains unclear. The work of Rosenblatt and
colleagues (42,43) suggests that the recruitment of satellite
cells may not be necessary for a limited amount of transfor-
mation within the subpopulations of fast-twitch fibers. Fur-
ther examination of this point is, however, required because
there are fundamental limitations of the ablation experimen-
tal model used (44). Sterilization of the satellite cell pool
was not confirmed in those studies. Furthermore, it has been
demonstrated that the dose of ꢇ-irradiation used (i.e., 25
Gy) is sufficient to disrupt mitotic activity of the satellite
cell pool for only 6 days. The data of those studies were,
however, collected 14 days after ablation and irradiation, a
full 8 days after the satellite cell pool is known to begin re-
covery (44). Finally, the magnitude of the stimulus provided
by the ablation experimental model used is significantly
lower and qualitatively different than that provided by
CLFS. The best evidence of this lies in the degree of fast-to-
slow transformation, which is much more extensive after
CLFS.
Satellite Cells
In response to increased contractile activity (22) or
chronically increased load bearing (38), satellite cells be-
come activated and leave their quiescent Go state. A portion
of the new progeny proliferates, proceeds toward terminal
differentiation, and fuses to adult fibers or with one another
to form new fibers; the remaining progeny reenter the qui-
escent Go phase to replenish the satellite cell pool. Al-
though the exact nature of their long-term contribution(s) to
the adaptive response to CLFS remains uncertain, two dis-
tinct possibilities exist. First, during the fast-to-slow pheno-
typic transformation, slow satellite cell lineages are prefer-
entially recruited to transforming fast fibers, where they
override the native phenotype. Indeed, Bischoff (39) has re-
ported that exogenously added satellite cells imposed local
phenotypic changes on adult fibers in culture. A second and
more plausible explanation is that both slow and fast satel-
lite cell lineages are recruited and their phenotype is regu-
lated by local factors released from transforming fibers or
their associated nerves. Such plasticity of the satellite cell
population has been demonstrated in vitro (40), which sug-
gests that nuclear accreditation is the primary role of satel-
lite cells during phenotypic transformation. In this regard,
satellite cells may be considered as important accessories to
the transformation process (41).
We have observed that during 20 days of CLFS, trans-
formation in the fast-twitch IIB fiber population follows
the time-dependent increase in proliferation and differenti-
ation (21,22) of satellite cell progeny, leading to an in-
crease in myonuclear density (28), which approaches lev-
els typically found in slow-twitch fibers. Most notable
was that satellite cell progeny selectively fused to the fast-
est IIB fiber population in the early phase of adaptation
to CLFS (i.e., 5 days), followed by the appearance of
MHCemb and a slower MHC isoform (i.e., MHCIIa) in the
undamaged transforming IIB fiber population 15 days later
We suggest that the primary contribution of the satellite
cell population in rat skeletal muscle undergoing CLFS-
induced fast-to-slow fiber-type transformation is to provide
a necessary and sufficient myonuclear density to first allow
and later maintain the transformed state. In the present study
the myonuclear density increased by the same absolute (i.e.,
2
ꢀ200 new nuclei/mm ) and relative (i.e., ꢀ50%) amounts
in both groups, indicating that the satellite cell pools of
young and aging BN rats were equally capable of contribut-
ing to the myonuclear production during CLFS. Interest-
ingly, the extent of CLFS-induced fiber-type transformation
and the relative increase in myonuclear content reported
here after 50 days are similar to those observed in our previ-
ous experiments after 20 days of CLFS. These observations
are consistent with the notion that a threshold myonuclear
density is probably achieved within the first 20 days of
CLFS, allowing transformation to proceed. Further, the
higher myonuclear density may then be sufficient to main-
tain the newly transformed state.
From previous studies it is apparent that the satellite cell
population plays a significant and central role in mediating
the adaptive response and maintaining muscle-fiber integ-
rity during growth and adaptation (21,22,28,45,46). It is not
clear, however, how much of the developing motor frailty
associated with aging results from intrinsic changes to the
satellite cell population. Previous studies have reported that
the absolute and relative number of satellite cells and the
satellite cell proliferation potential decrease in rat fast-
twitch muscles with advancing age (6,7). It has also been
suggested that these changes may underlie much of the re-
duction in adaptive capacity of aging skeletal muscle. In the
present study, the selection of aging 25-month-old rats was
significant because this age is associated with a large reduc-
tion in proliferation potential in culture (7) and might be ex-
pected to underlie a reduced adaptive potential in vivo.
Clearly this did not occur in our study, as the capacities of
satellite cell progeny for self-renewal and terminal differen-
tiation were not compromised in our group of aging rats af-
ter 50 days of CLFS.
(
21,22). These findings point to the primary role of satel-
lite cells as providing a source of new myonuclei that is
probably both necessary and sufficient for the adaptive re-
sponse to CLFS.
Whether CLFS-induced fast-to-slow phenotypic transfor-
mation is impeded by the absence of a viable population of
In view of the fact that satellite cells and their progeny
may exist in three compartments, we used a panel of anti-

B518
PUTMAN ET AL.
bodies that recognize proteins uniquely expressed in satel-
lite cells and their progeny during the quiescent stage (15),
during proliferation (18,19), or during terminal differentia-
tion (20). CLFS resulted in similar increases in the total
number of muscle precursor cells present in the young and
Conclusions
We examined fiber-type transitions, changes in satellite
cell content, and the number of mpc in the proliferative and
differentiative compartments after 50 days of CLFS in the
fast-twitch TA muscles of young and aging BN rats. Al-
though the extent of the CLFS-induced fast-to-slow fiber type
transitions was similar in the young and aging groups, a
greater number of small myotubes expressing MHCemb was
present in the latter group, which is suggestive of regenera-
tion secondary to age-related changes in ꢈ-motor neurons.
Fifty days of CLFS induced a significantly greater increase in
the number of satellite cells in the young group. However, the
total increase in the number of muscle precursor cells present
(i.e., satellite cells plus their progeny) did not differ between
the young and aging groups, because a greater number of
progeny passed through the proliferative and differentiative
compartments of the aging group. Thus satellite cells of the
aging BN rat retain their capacities to produce progeny and
contribute to the adaptive response. Satellite cells of aging
skeletal muscles do, however, seem to pass through the pro-
liferative and differentiative compartments more frequently
because of new fiber formation. The formation of new myo-
tubes also appears to explain the CLFS-induced reduction in
the number of COX-deficient fibers by regenerated small
COX-positive type I fibers reported in our preceding study
(12). We conclude that satellite cells of aging BN rats are not
limited by intrinsic factors but are dependent on extrinsic fac-
tors that are determined by the quantity and quality of con-
tractile activity imposed on the muscle. Forced contractile ac-
tivity thus appears to have a positive effect on aging skeletal
muscle by reducing the number and proportion of fibers dis-
playing point mutations of mitochondrial DNA.
2
aging groups; they increased from ꢀ15/mm in the control
2
conditions to ꢀ30/mm after CLFS. Despite displaying sim-
ilar numbers of total mpc, the young and aging groups dif-
fered in the proportion of mpc that was actively cycling
through the proliferative and differentiative compartments.
Approximately 68% of the mpc population was quiescent in
the aging muscle, whereas the remaining 32% were evenly
distributed between the proliferative and differentiative
compartments. By comparison, the fraction of quiescent
mpc was higher in young muscles at 85%, whereas the re-
maining 15% were actively cycling and also evenly distrib-
uted between the proliferative and differentiative compart-
ments. Thus, although CLFS did increase the absolute
numbers of quiescent, proliferating, and differentiating mpc
in each group, it did not alter their proportions within the
three compartments. Whether the same can be said of earlier
time points can not be established from the present study.
From the results of our previous studies (21,22), however, it
is possible to speculate that the satellite cell progeny of the
young group may have entered the proliferative and differ-
entiative compartments much earlier in response to CLFS
(
e.g., 5–10 days), possibly as a result of differences in the
availability of biofactors regulating those events. This ex-
planation would also account for the lower proportion of
satellite cell progeny actively cycling in our young group
after 50 days of CLFS.
The results of the present study indicate that satellite cells
of aging BN rats are not limited by intrinsic factors in vivo.
It seems plausible, therefore, that differences in the avail-
ability of extrinsic factors between young and aging muscle
might underlie differences in satellite cell content in our
control muscles and others (4,6), as well as possible differ-
ences in proliferation potential during exercise training and
regeneration (5). Whether the potential difference in extrin-
sic factors arises secondary to an age-related decline in en-
docrine, paracrine or autocrine factors remains uncertain.
Carlson and Faulkner (47) have demonstrated that, during
muscle transplantation, the regenerative potential of skeletal
muscle graft is dependent on the age of the host and not the
age of the graft and its satellite cell population. Others have
noted that decreases in growth factors such as bFGF (48)
and IGF-I (49), which regulate mpc proliferation and differ-
entiation, parallel the decline in satellite cell content in old
skeletal muscle, whereas restoration of these factors (48,50)
in old rats to levels found in young controls can restore the
regenerative potential of aging skeletal muscle. Moreover,
our recent finding that CLFS enhances the expression of
FGF-1 and FGF-2, and their receptors in stimulated TA
muscles in vivo and in primary myotubes in vitro (46), is
most relevant. Taken together with the results of the present
study, this finding suggests that changes in the concentra-
tion or availability of extrinsic factors associated with
forced contractile activity (i.e., CLFS) may elicit similar re-
sponses in aging skeletal muscle satellite cells, if the stimu-
lus is of sufficient intensity and duration.
Acknowledgments
This study was supported by research grants from the Deutsche For-
schungsgemeinschaft Du 260/2-2 and Fonds der Chemischen Industrie (to
D. Pette), the Natural Sciences and Engineering Research Council of Can-
ada (to C.T. Putman), the Alberta Heritage Foundation for Medical Re-
search (to C.T. Putman), and the Universities of Konstanz and Alberta.
We thank Elmi Leisner for excellent technical assistance and Professors
Schiaffino (University of Padova, Italy) and Merrifield (University of
Western Ontario, Canada) for providing some of the MHC antibodies.
Address correspondence to Dr. Charles T. Putman, Assistant Professor,
AHFMR Medical Scholar, E-417 Van Vliet Centre, University of Alberta,
Edmonton, Alberta, Canada, T6G 2H9. E-mail: tputman@ualberta.ca
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Received February 5, 2001
Accepted July 17, 2001
Decision Editor: John A. Faulkner, PhD