Limited myogenic response to a single bout of weight-lifting
exercise in old rats
Tetsuro
Tamaki1,2,
Shuichi
Uchiyama3,
Yoshiyasu
Uchiyama4,
Akira
Akatsuka5,
Shinichi
Yoshimura6,
Roland R.
Roy7, and
V. Reggie
Edgerton2,7
5 Laboratory for Structure and Function Research,
Division of Human Structure and Function, Departments of
1 Physiology,
4 Orthopedics, and
6 Molecular Life Science, Tokai University
School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193;
3 Tokai University School of Physical Education,
Hiratsuka, Kanagawa 259-12, Japan; and
7 Brain Research Institute and
2 Department of Physiological Science, University
of California, Los Angeles, California 90095
 |
ABSTRACT |
The purpose of the present study was to compare the
myogenic response of hindlimb muscles in young (14-20 wk of age)
and old (>120 wk of age) rats with a single exhaustive bout of heavy
resistance weight lifting. [3H]thymidine and
[14C]leucine labeling were monitored for up to
2 wk after the exercise bout to estimate serial changes in mitotic
activity and the level of amino acid uptake and myosin synthesis.
Histological, histochemical, and immunohistochemical
[anti-5-bromo-2'-deoxyuridine and myogenic determination
genes (MyoD)] analyses of whole muscles and analysis of
muscle-specific gene expression (MyoD) using Western blotting and
RT-PCR were performed. Old rats showed significant muscle atrophy and a
lower exercise capacity than young rats. Exercise-induced muscle
damage, as assessed in histological sections, and increases in serum
creatine kinase activity were evident in both young and old exercised
groups. Mitotic activity was increased in young, but not old, rats 2 days after exercise. There was a biphasic increase in
[14C]leucine uptake during the 14 days
postexercise (peaks at 1-4 and 10 days) in young rats: only the
first peak was observed in old rats. There was a lower uptake of
[14C]leucine in the myosin fraction and an
impaired expression of MyoD at the protein (immunohistochemistry and
Western blotting) and mRNA (RT-PCR) levels in old rats throughout the
postexercise period. These results demonstrate a reduced reparative
capability of muscle in response to a single bout of exercise in old
compared with young rats.
satellite cell; mitotic activity; amino acid uptake; gene
expression; myogenic determination gene; rat skeletal muscle
 |
INTRODUCTION |
AGING IS COMMONLY ASSOCIATED with a decrement in motor
function due, in part, to a loss in muscular strength and fatigue
resistance. In turn, these decrements are reflected in detrimental
adaptations in some of the mechanical, biochemical, and morphological
properties of the skeletal musculature (9, 12, 13, 20). Some of these
adaptations appear to be related to a reduction in use, i.e., decreased
levels of neuromuscular activity (26), as is typical of muscles in the
lower extremities of old people and animals (9). These adaptations,
however, also have been associated with intrinsic age-related changes
in the muscles themselves (12).
Skeletal muscles in old individuals are responsive to increased levels
of neuromuscular activity. For example, progressive resistance training
results in strength gains due to either muscle hypertrophy (5, 25)
and/or neural factors (24) in older people. The capacity for muscle
hypertrophy is attenuated in older subjects (33). The reasons for the
attenuated cellular and biochemical responses of atrophied muscles of
older individuals to resistance exercise remain unclear. Heavy
resistance exercise can induce inflammation and/or damage in the
muscle, and the associated degeneration-regeneration responses can be
beneficial for a subsequent increase in muscle mass (19). In addition,
there appears to be an age-related impairment in myogenic potential as
indicated by a deficiency in the capacity for skeletal muscle
reinnervation in old rats (8, 23).
Heavy resistance exercise, such as weight lifting, is easy to apply in
humans but is very difficult to apply in laboratory animals. Human
studies on the cellular responses to resistance training are limited
due to the invasive nature of muscle biopsies and to the risk of using
old people as subjects, as well as the more limited control of
lifestyles. To circumvent these problems, we have been using a
weight-lifting protocol designed for rats (30). Recently, we have
reported morphological and biochemical evidence of muscle fiber
hyperplasia associated with muscle damage and regeneration in the
plantaris (Plt) muscle after a single exhaustive session of weight
lifting in previously nontrained young rats (28).
The purpose of the present study was to compare the myogenic response
of hindlimb muscles in young (14-20 wk of age) and old (>120 wk
of age) rats with a single intense session of weight-lifting exercise.
We hypothesized that the myogenic potential of old rats after a single
bout of exercise is less than that in young rats. Serum creatine kinase
(CK) activity was used as a measure of muscle damage. Changes in the
incorporation of [3H]thymidine and
[14C]leucine in selected hindlimb extensor and
flexor muscles after the weight-lifting session were followed for 2 wk
to determine the level of mitotic activity and amino acid uptake,
respectively. The expression of myogenic determination genes (MyoD;
protein and mRNA levels) and 5-bromo-2'-deoxyuridine (BrDU)
labeling also were used as indicators of cell proliferation and
cellular mitotic activity.
 |
MATERIALS AND METHODS |
Animals.
The following two groups of Wistar male rats (specific pathologen free)
were studied: 1) young (14-20 wk old; 380-520 g body mass; total n = 46) rats and 2) old (>120 wk old;
375-540 g body mass; total n = 37) rats. Exercised (see
below) and nonexercised subgroups were studied in both age groups. In
addition, 3-wk-old rats (85-95 g body mass; total n = 12)
were used as positive controls for the analysis of muscle-specific gene
expression (see below). The animals were housed in standard cages and
were provided food and water ad libitum. The room temperature
was kept at 23 ± 1°C, and a 12:12-h light-dark cycle was
maintained throughout the experiment. All experimental procedures were
conducted in accordance with the Japanese Physiological Society Guide
for the Care and Use of Laboratory Animals as approved by the Tokai
University School of Medicine Committee on Animal Care and Use and
followed the American Physiological Society Animal Care Guidelines.
Exercise protocol.
The hindlimbs of the rats in the exercise groups were trained for one
intense exercise session using a weight-lifting technique described in
detail elsewhere (28, 30). The exercise was performed preferentially by
the plantarflexors, with a minimal usage of the dorsiflexors. The
session involved multiple sets of 10 repetitions (lifts) per set with
~1 min rest between each set. The first set of lifts was with a 500-g
load. In the subsequent sets, an additional 500-g load was added until
the rat could not complete 10 repetitions. The load then was adjusted
in 100-g increments and/or decrements until the maximum load at which
10 repetitions could be completed, i.e., the 10 repetition maximum (10 RM), was determined. The 10 RM was repeated until the rat could not
complete the set, and then the load was decreased by 500 g. This
procedure was followed until the rat failed to complete three
consecutive sets, even when the load was being reduced. The total time
of the exercise bout was ~30-40 min for the young and
~20-30 min for the old rats. The 10 RM (g), number of sets, and
total amount of load lifted (number of lifts × load
lifted by different groups in absolute values expressed as kg) were recorded.
Measurement of serum CK activity.
Blood samples (0.2 ml) were obtained from the caudal vein before and 30 and 60 min after the exercise session. Serum CK activity was measured
using a standard kit (Monotest CK-NAC; Boehringer, Mannheim, Germany)
and was used to estimate exercise-induced muscle damage. The activities
were expressed as international units per milliliter.
Muscle fiber number.
To determine whether a decrease in fiber number occurred during aging,
the number of total and branched fibers of the Plt muscles in both
groups were counted using a nitric acid digestion method (29, 30).
Briefly, the frozen muscle was thawed in distilled water for a few
minutes and then immersed in 15% nitric acid (Wako Pure Chemical,
Osaka, Japan) for 2-3 h at room temperature. The muscle was washed
in cooled distilled water for 60 min at 4°C. The solution was
changed from distilled water to 0.01 M PBS (pH = 7.4) and was stored at
4°C. Subsequently, a few muscle fiber bundles were transferred to a
silicone-coated petri dish containing 0.01 M PBS (pH = 7.4) at room
temperature. All fibers were teased free, and the total number of
straight (nonbranched) and branched fibers was carefully and precisely
counted under a dissection microscope (×10-15). These
procedures were repeated until all fibers, nonbranched and branched, in
each muscle were counted.
Histochemical and immunohistochemical analyses.
Histochemical and immunohistochemical analyses were performed 48 h
after the exercise session in exercised (n = 4) and
nonexercised (n = 2) rats in both groups. BrDU (Takeda
Chemical, Osaka, Japan), a nonradioactive marker for DNA synthesis, was
injected (100 mg/kg ip) 1 h before sampling. This procedure is useful
for labeling proliferating cells in muscle (28, 29). The rats were
killed with an overdose of pentobarbital sodium (60 mg/kg ip), and the soleus (Sol) and Plt muscles of both hindlimbs were excised within 10 min. The Sol and Plt muscles then were wet weighed after trimming excess connective tissue, were immediately frozen in isopentane precooled by liquid nitrogen, and were stored at
80°C. After equilibration at
20°C, each muscle was divided
longitudinally into three (Sol) or six (Plt) portions to allow full
characterization of the entire muscle (29). Serial 10-µm-thick
transverse sections (10-20 sections) were cut from each muscle
portion. Staining of hematoxylin-eosin and actomyosin ATPase (mATPase)
after acid (pH 4.3) preincubation was performed on four to eight
sections to examine the general morphology and the fiber type
characteristics of the muscles.
For the remaining 6-12 sections, immunohistochemical staining was
performed using BrDU (monoclonal anti-BrDU from mouse;
Becton-Dickinson, San Jose, CA) and MyoD (monoclonal anti-MyoD1 from
mouse, 5.8A; Dako, Carpinteria, CA) antibodies to identify and localize
proliferating cells (BrDU) and myoblasts (MyoD) in the muscles.
For the BrDU staining, the sections were fixed in 70% ethanol for 15 min at 4°C and were treated with 100% methanol containing 0.3%
H2O2 to inhibit endogenous peroxidase for 30 min at room temperature and then were treated with 2 N HCl for 60 min
at room temperature for DNA denaturation followed by washing in 0.01 M PBS (pH = 7.4). The sections were treated with 10% normal sheep serum
(NSS) in PBS for 30 min and were incubated for 60 min with the primary
monoclonal antibody diluted 1:50 in PBS (pH = 7.4) containing 1% BSA
(Seikagaku Kogyo, Tokyo, Japan). The sections were washed in PBS,
treated with 10% NSS for 20 min, and incubated with anti-mouse IgG
F(ab')2 combined with sheep horseradish peroxidase (Amersham International, Buckinghamshire, UK) diluted 1:75 in PBS
containing 1% BSA for 60 min at room temperature. Finally, the
sections were washed in PBS, visualized with 0.02%
3,3-diaminobenzidine (Wako Pure Chemical)/0.05 M
Tris · HCl buffer, pH = 7.4, containing 0.005%
H2O2 for 5-10 min, and counterstained with eosin.
For the immunohistochemical staining of MyoD, the sections were fixed
in 4% paraformaldehyde/0.05 M phosphate buffer (pH 7.4) for 15 min at
room temperature. Anti-MyoD was used as the primary antibody diluted
1:50 in PBS containing 1% BSA. The same procedures described for the
BrDU staining were used, except for the treatment of 2 N HCl.
Western blotting procedures.
To determine the level of MyoD expression, the Sol and Plt muscles from
two nonexercised rats from the young and old groups were prepared for
Western blotting. In addition, muscles from two 3-wk-old rats were
analyzed as a positive control. Each muscle was minced using scissors
at 4°C on an ice-cooled glass plate. The samples were immersed in a
homogenizing buffer containing 1% Nonidet P-40, 0.5% sodium
deoxycholate, and 0.1% SDS in PBS, and 0.1 mM phenylmethylsulfonyl
fluoride was added at the time of use at 4°C. Muscle tissues were
homogenized (×10) with a glass homogenizer and were incubated for
30 min at 4°C. Each homogenate was transferred to microfuge tubes
and centrifuged at 15,000 g for 20 min at 4°C, and the
supernatant was obtained. Cell lysate with an equal volume of
electrophoresis sample buffer (0.15 M Tris · HCl, 4%
SDS, 10 mM EDTA, 40% glycerol, and 10% 2-mercaptoethanol, pH 6.7) was
boiled for 5 min, separated by 10% SDS-PAGE, and blotted for 1.5 h
with a constant current of 150 mA on a polyvinylidene difluoride
membrane (ATTO, Tokyo, Japan). In this step, the total protein
concentration of each sample was adjusted preliminarily. The membrane
was blocked with a blocking buffer (5% nonfat milk powder in 0.01 M
PBS, pH 7.4, containing 0.1% Tween 20) overnight at 4°C. Primary
antibody [polyclonal anti-MyoD (C-20) from rabbit; Santa Cruz
Biotechnology, Santa Cruz, CA] was applied in a dilution of 1:100
with blocking buffer for 1.5 h at 37°C. The blot was washed for 1.5 h with several changes of PBS containing 0.1% Tween 20 (PBS-Tween) at
room temperature and then was incubated with a second antibody
[anti-rabbit IgG F(ab')2 combined with sheep horseradish peroxidase; Amersham International] diluted 1:5,000 with blocking buffer for 1.5 h at 37°C. The blot was washed with PBS-Tween for 2 h. Antibody binding was visualized by the enhanced chemiluminescence (ECL) technique (ECL+plus System; Amersham
International) and X-ray film (BioMax MS-1; Kodak, Tokyo, Japan). The
immunoblots were photographed and scanned with a scanning densitometer
(Ultrascan laser; Pharmacia LKB, Uppsala, Sweden) for quantification.
RT-PCR analyses.
Total RNA was extracted from nonexercised young (n = 2), old
(n = 2), and 3-wk-old (n = 10) rats using the guanidine
isothiocyanate technique (10). The Sol and Plt muscles were dissected
from each rat, the tendons were removed, and the muscles were minced using scissors at 4°C on an ice-cooled glass plate under the
RNase-free condition. Each muscle was lysed in guanidine isothiocyanate
buffer and homogenized using a polytron (Kinematica Littau/Luzen,
Switzerland). The lysate was layered on a CsCl gradient buffer (5.7 M
CsCl/3 M sodium acetate, pH 6, sterile), spun in an ultracentrifuge at 179,000 g (SW55; Beckman, Fullerton, CA) for 21 h at 20°C,
and then prepared for extraction of intact total RNA. RNA quantity was
determined by an optical density measurement at 260 nm. Samples were
stored at
80°C. Primer pairs and the nucleotide position used for PCR amplification of MyoD mRNA were as follows: MyoD-forward, 5'-ACCATGGAGCTACTATCGCCGCCA-3', nucleotide 2590-2611
(32); MyoD-reverse, 5'-TCTCGAGCACCTGGTAAATCGGATT-3',
complementary to nucleotide 4287-4307 (32). RT-PCR was performed
using an RNA-PCR kit (Perkin-Elmer, Cetus, CT). To enable a comparison
between muscles from young, old, and 3-wk-old rats, 25, 30, and 35 PCR
cycles were chosen.
Analyses of mitotic activity and amino acid uptake.
Our previous data indicate that in vivo
[3H]thymidine and
[14C]leucine labeling is a useful method to
detect the mitotic activity and amino acid uptake in the muscles after
the exercise (28). Analyses of mitotic activity and muscle amino acid
uptake were performed at rest and 1-4, 7, 10, and 14 days after
exercise in young (n = 31, 3-6/time point) and old
(n = 24, 3/time point) rats.
[3H]thymidine (methyl-3H;
15.5 MBq/kg ip; specific activity 247.9 GBq/mmol; NEN Life Science
Products, Boston, MA) and [14C]leucine
(U-14C; 1.15 MBq/kg ip; specific activity 13.6 GBq/mmol;
NEN Life Science Products) were injected 1 and 3 h before sampling to
label proliferating cells and proteins that use leucine during protein
synthesis, respectively. The rats were overdosed with pentobarbital
sodium (60 mg/kg ip), and the following muscles were removed
bilaterally: primary extensors [Sol, Plt, and gastrocnemius
(Gas)] and primary flexors [tibialis anterior (TA) and
extensor digitorum longus (EDL)]. After excess connective tissue
and fat were removed, each muscle was wet weighed and homogenized in
0.02 M phosphate buffer (pH = 7.4) at a 1:20 dilution at 4°C. In
the next step, 1 ml of the homogenate from each muscle sample was added
to 5 ml of 10% TCA and mixed well. This mixture was centrifuged (2,050 g for 10 min), and the upper solution (TCA) was removed. This
procedure was repeated five times, and the remaining TCA-insoluble
material was collected and dried with 70% ethanol. The dried material
was treated overnight with 1 ml of dissolving solution (Solvable; Packard, Meriden, CT) at 45°C, and then a 10-ml liquid
scintillation cocktail (Atomlight; Packard) was added to count
radioactivity (Beckman LS4800). The total protein concentration in each
homogenate was measured, and the radioactivity of each sample was
expressed as disintegrations per minute per milligram protein.
We have previously reported that the uptake of thymidine and leucine
into individual muscles within a rat and for an individual muscle
across rats varies widely (28), most likely reflecting varying levels
of recruitment of each muscle during the weight-lifting task. In all
cases, the pooled values for the extensors (Sol, Plt, and Gas) had a
higher amino acid uptake than the pooled values for the flexors (TA and
EDL). Thus, to minimize the effects of intra- and intermuscle
variability on the effects of exercise on thymidine and leucine uptake,
the difference in the uptake between the extensor and flexor muscles in
both legs of each rat is reported.
Analysis of myosin synthesis.
The determination of myosin synthesis was performed at the same time
points and for the same groups as for the analysis of mitotic activity
and amino acid uptake. Myosin was extracted with 0.6 M KCl solution (50 ml) from a 1-ml homogenate for 15 min at 4°C and was filtered with
three sheets of gauze. The myosin-extracted KCl solution was diluted
with cool distilled water (1:20), which resulted in the reappearance of
myosin deposits. The diluted solution was passed through an omnipore
nondissolving membrane filter (10 µm aperture and 47 mm diameter;
Nihon Millipore, Yonezawa, Japan). The membrane containing the deposits
was dried, cut into several pieces, and soaked in a dissolving solution
overnight at 45°C. The radioactivity was counted using the same
procedures employed for the mitotic activity and protein synthesis
analyses. Values were expressed as disintegrations per minute per
milligram protein.
Statistical analyses.
All data are expressed as means ± SE. Differences in body mass,
muscle mass, fiber numbers, and [3H]thymidine
and [14C]leucine uptake levels between young
and old rats were determined using Student's t-tests. ANOVA
was used to determine overall differences, and Duncan's post hoc
analyses were used for individual group differences for the preexercise
and serial postexercise data for [3H]thymidine,
[14C]leucine, and myosin fraction uptake.
Standard regression analysis and Pearson's product correlation
procedures were used to determine the relationship between
[3H]thymidine uptake and CK activity.
Differences were considered statistically significant at either the
0.05 or the 0.01 levels.
 |
RESULTS |
Exercise capacity.
The total load lifted during the single exhaustive exercise bout was
lower in old than young rats. The total number of sets, the 10 RM, and
the total amount of load lifted were 24 ± 3 and 13 ± 5 sets, 2,664 ± 38 and 2,083 ± 42 g, and 417 ± 86 and 209 ± 26 kg
for the young and old rats, respectively. All values were significantly
higher in the young than the old rats (P < 0.01).
Body mass, muscle mass, and total muscle fiber number.
The mean final body mass was not significantly different between the
two groups (Table 1). However, it should be
noted that the body masses of the old rats had increased to
650-750 g and then gradually decreased with advancing age. The
mean absolute masses of the TA, Plt, and Gas and relative masses of TA,
Sol, Plt, and Gas were significantly smaller in old than young rats. The mean total fiber number in the Plt muscle was significantly lower
(17%) in old than young rats (Table 2).
Although the absolute number of branched fibers was 24% (P > 0.05) higher in the old than the young rats, the incidence of branched
fibers relative to nonbranched fibers was low in both young (0.26%)
and old (0.38%) rats.
Serum CK activity.
The mean preexercise serum CK levels were similar in young (n = 25) and old (n = 21) rats (Fig. 1).
These levels were significantly increased in both groups 30 or 60 min
after the exercise session. The postexercise values were significantly
lower in the old than the young rats at both time points.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
Mean serum creatine kinase (CK) activities at rest and 30 and 60 min
after a single exhaustive resistive bout of exercise. Bars are SE. IU,
international units. Rest, preexercise. * P < 0.05 (young vs. old
rats); P < 0.05 and  P < 0.01 (rest vs.
postexercise).
|
|
Muscle morphology and immunostaining.
In contrast to the typical mosaic pattern of fiber types (slow fibers
dark, based on mATPase staining at a pH 4.3 preincubation) in the Plt
muscle of young rats (Fig. 2A),
"fiber type grouping," i.e., an abnormal number of fibers of the
same ATPase type that are adjacent, was observed in the Plt of old rats
(Fig. 2B). In addition, fibers having unusual cross-sectional
shapes, e.g., small angulated fibers (Fig. 2C),
were evident in all muscles of old, but not young, rats.

View larger version (99K):
[in this window]
[in a new window]
|
Fig. 2.
Representative cross-sectional profiles of the plantaris muscles of a
young (A) and an old (B and C) rat. A
and B: actomyosin ATPase at a preincubation of pH 4.3. C: hematoxylin and eosin (HE) staining. Fiber type grouping
(B) and small angulated fibers (arrowheads in C) were
evident in muscles of old, but not young (A), rats. The
appearance of small angulated fibers is consistent with degenerating
fibers that are not reinnervated (3, 21, 22). Fiber type grouping of
slow fibers in older animals (14) has been interpreted as evidence for
the reinnervation of some of the fast fibers by motoneurons innervating
slow muscle fibers (12, 16). Calibration bar is equal to 100 µm.
Magnifications: A and B, ×24; C,
×120.
|
|
Necrotic fibers were scattered throughout the Plt muscle cross section
2 days after exercise in both young (Fig.
3A) and old (Fig. 3B) rats.
The overall mean number of these necrotic fibers for both exercised
groups was 29 ± 10 per cross section, i.e., occupying
~0.5-0.6% of the cross section. Note that there were almost no
necrotic fibers in young nonexercised rats and only a few in old
nonexercised rats.

View larger version (124K):
[in this window]
[in a new window]
|
Fig. 3.
Representative cross-sectional profiles of the plantaris (A-F)
and soleus (G and H) muscles of a young (A,
C, E, and G) and old (B, D,
F, and H) rat. A and B: HE. C
and D: anti-5-bromo-2'-deoxyuridine (BrDU). E-H:
anti-myogenic determination gene (MyoD) staining. Arrowheads in
A and B indicate necrotic (damaged) fibers, arrowheads
in D indicate cells showing positive staining for BrDU (cell
proliferation), and arrowheads in F and H indicate
positive staining for MyoD (myoblast). Calibration bars are equal to
100 µm. Magnifications: A and B, ×100;
C, D, G, and H, ×120; E
and F, ×110.
|
|
Immunostaining patterns for anti-BrDU (Fig. 3, C and D)
and anti-MyoD 2 days after the exercise session are shown for the Plt
(Fig. 3, E and F) and Sol (Fig. 3, G and
H) muscle of a representative young (Fig. 3, C,
E, and G) and old (Fig. 3, D, F, and
H) rat. An increase in proliferating cells occurred in and/or
near necrotic fibers and around some normal-appearing fibers in the
muscle of young rats (Fig. 3C). Only one or two anti-BrDU
positive cells were observed near the necrotic fibers in the muscles of
old rats (Fig. 3D). Similar differences between the two age
groups were observed for anti-MyoD staining. Generally, in the young
rats, the localization and number of anti-MyoD positive cells were
similar to that of the anti-BrDU positive cells. A large number of MyoD positive cells was observed in the Plt of young rats (Fig. 3E), but only a few were seen in old (Fig. 3F) rats. This tendency was similar in the Sol muscles of young (Fig. 3G) and old (Fig. 3H) rats. Positive reactions were rarely observed in
nonexercised rats of either age group. Edema (increased extracellular
space) after weight-lifting exercise was evident histologically in the muscles of both young and old rats.
Expression of MyoD protein and mRNA.
An immunoreactive band of 35 kDa corresponded to the predicted
molecular mass of the rat MyoD protein (Fig.
4). The lowest levels of MyoD protein were
observed in both the Sol and Plt of old rats. To examine the age
dependence of the MyoD expression, we compared the nonexercised young
and old rats with 3-wk-old rats. MyoD protein tended to be higher in
the 3-wk-old than in the young rats for the Plt but not the Sol
muscles. The expression of MyoD mRNA levels in both the Sol and Plt was
highest in the 3-wk-old rats and lowest in the old rats (see 25 and 30 cycles; Fig. 5). The expression levels were
lowest in the Sol of old rats (Fig. 4). It also appeared that the
expression of MyoD mRNA levels were higher in the Plt than the Sol
muscle in all groups. We confirmed that the nucleotide sequence of the
RT-PCR product was completely matched to the MyoD mRNA (data not
shown).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 4.
Resting levels of MyoD protein contained in the soleus (Sol) and
plantaris (Plt) muscles of a representative old, young (Y), and
3-wk-old (3w) rat analyzed by Western immunoblot. An immunoreactive
band of ~35 kDa (K) corresponded to the predicted molecular mass of
the rat MyoD protein. The density of each band was expressed relative
to the background density (considered to be 1.0). These values were
2.97, 9.67, and 8.41 for the Sol and 1.30, 3.43, and 14.18 for the Plt
in old, Y, and 3w rats, respectively.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
MyoD mRNA in the Sol and Plt muscles of old, young (Y), and 3-wk-old
(3w) rats measured by RT-PCR. Kb, kilobase.
|
|
Thymidine and amino acid uptake rates.
The flexor muscles (TA and EDL) of old rats had a significantly lower
uptake of [3H]thymidine compared with young
rats in the resting state (Table 3). The extensor muscles (Sol,
Plt, and Gas) in young and old rats were similar in thymidine uptake.
There was no significant difference between young and old rats in
[14C]leucine uptake in either the extensor or
flexor muscles. Significant increases in the uptakes of
[3H]thymidine and
[14C]leucine in the extensors of young rats
were observed at 2 and 10 days after exercise, respectively, whereas
there was no significant change in the extensors of the old rats
(compare Figs. 6 and
7). In addition, no significant changes in
the uptake of either [3H]thymidine or
[14C]leucine were observed in the flexor
muscles of young or old rats after the single bout of exercise (Figs. 6
and 7). Because there was no exercise effect on the overall mean
uptakes for the flexor muscles for either group (Figs. 6 and 7), we
then normalized the exercise effects of the extensors to the flexor
muscles and compared the response of young and old rats (Fig.
8). This procedure minimized the
intra-animal variability. The uptake of
[3H]thymidine was elevated significantly at 2 days after exercise in young rats (Fig. 8A). In contrast, there
was a small insignificant peak at 1 day after exercise in old rats
(Fig. 8A). [14C]leucine uptake in the
young rats was elevated up to 10 days after exercise (significant
differences at 3 and 10 days) and then returned to rest levels at 14 days. In the old rats, the uptake pattern was similar to that of the
young rats for up to 7 days but returned to resting levels 10 days
after exercise and remained at that level up to 14 days (Fig.
8B).
View this table:
[in this window]
[in a new window]
|
Table 3.
[3H]thymidine and [14C]leucine
uptake in the flexor and extensor muscles of young and old rats
at resting state
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Absolute change in [3H]thymidine (A)
and [14C]leucine (B) uptake in
extensors and flexors during the 2 wk after a single exhaustive bout of
weight lifting in young rats. Values are expressed as means ± SE.
Extensors, Sol, Plt, and gastrocnemius; flexors, tibialis anterior and
extensor digitorum longus. Rest, values from nonexercised control rats.
** Significantly different from rest at P < 0.01.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Absolute change in [3H]thymidine (A)
and [14C]leucine (B) uptake in
extensors and flexors during the 2 wk after a single exhaustive bout of
weight lifting in old rats. Values are expressed as means ± SE.
Extensors, Sol, Plt, and gastrocnemius; flexors, tibialis anterior and
extensor digitorum longus. Rest, values from nonexercised control rats.
Note that there are no significant changes during the 2 wk after the
exercise bout.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
Changes in [3H]thymidine (A) and
[14C]leucine (B) uptake during the 2 wk
after a single exhaustive bout of weight lifting. Uptake values were
calculated as (mean value of extensors) (mean value of flexors)
in both legs and are expressed as means ± SE. Extensors, Sol, Plt,
and gastrocnemius; flexors, tibialis anterior and extensor digitorum
longus. Rest, values from nonexercised control rats. * and
** Significantly different from rest at P < 0.05 and P < 0.01, respectively.
|
|
Fraction of amino acid uptake for myosin.
The uptake of [14C]leucine for the myosin
fraction for the extensor muscles was elevated significantly at 10 days
after exercise for the young rats, whereas there were no changes at any
other postexercise time point for the young or old rats (Fig.
9A). When the uptake of
[14C]leucine for the myosin fraction on the
extensor muscles was expressed relative (%) to total uptake, there was
a progressive decrease in old rats that reached significance at 7, 10, and 14 days postexercise (Fig. 9B). The resting levels for the
relative uptake of [14C]leucine for the myosin
fraction were similar in the young and old rats, i.e., 29 ± 3 and 26 ± 4%, respectively.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 9.
Changes in the absolute [14C]leucine uptake for
the contractile component (myosin) of the extensor muscles (A)
and its fraction (%) relative to total uptake for the muscle
(B) during the 2 wk after a single exhaustive bout of weight
lifting. Rest, values from nonexercised control rats. Values are
expressed as means ± SE. * and
** Significantly different from rest at P < 0.05 and P < 0.01, respectively.
|
|
 |
DISCUSSION |
Muscle damage and appearance of proliferating cells and myoblasts.
Although the serum CK activities 30-60 min after the exercise bout
were increased significantly in both the young and old rats, the
increase was two- to threefold higher in the young rats (Fig. 1). With
the use of the same weight-lifting model, the degree of CK leakage
clearly reflected the severity of damage in exercised muscles of young
rats (28). In the present study, in the young rats, these changes were
closely associated with the incidence of degenerated fibers in the
histological sections of the Plt and Sol muscles (Fig. 3, A and
G) and by the increased uptake of
[3H]thymidine and
[14C]leucine in the extensor, but not the
flexor, muscles postexercise. These changes were not present or were of
a lesser magnitude in the old rats. The similarities in the number and
location of anti-BrDU and MyoD positive cells in the Plt and Sol
muscles 2 days after exercise in young rats suggest that some of the
cells that were proliferating were myogenic cells. The number of these
cells at the same time point was lower in old than young rats (Fig. 3, C-H), indicating a reduced proliferating capacity of myogenic cells in muscles of old rats. The lower levels of MyoD protein (Western
blots) and mRNA (RT-PCR) in the Sol and Plt of old compared with young
and 3-wk-old rats are consistent with a decreased reparative capacity
in old rat muscles (Figs. 4 and 5).
Thymidine uptake and activity of myogenic cells.
There was a significant peak in [3H]thymidine
uptake 2 days after the exercise bout in young, but not old, rats (Fig.
8A). Recently, we demonstrated that this time point also shows
peak proliferation of satellite cells for the regeneration associated with damaged muscle fibers, to include fiber hyperplasia, after a
weight-lifting bout in young rats (28). Furthermore, there was a
significant correlation between the serum CK activity 30-60 min
after the exercise bout and the uptake of
[3H]thymidine 2 days after the exercise bout in
young, but not old, rats (Fig. 10). These
results also indicate that the ability of the muscle to initiate
reparative processes is reduced in old compared with young rats. An
impaired capacity for muscle regeneration in old animals also has been
reported after bupivacaine injection (8), contraction-induced injury
(12), ischemic necrosis (31), eccentric contraction-induced injury (4),
and transplantation of muscle grafts (7).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 10.
Relationship between serum CK activity and
[3H]thymidine uptake in the extensor muscles in
young and old rats after a single exhaustive bout of weight lifting.
Abscissa, CK leakage 30-60 min after exercise; ordinate,
[3H]thymidine uptake 2 days after exercise. The
regression line and correlation for the young rats are shown, P < 0.01. There was no relationship between these two variables in old
rats.
|
|
The more limited myogenic potential of muscles in old rats may reflect
a more limited plasticity of the nervous system. Carlson and Faulkner
(6, 8) suggested that a reduced capacity for reinnervation in muscles
of old rats may be a contributing factor to the decreased muscle
regenerative capacity. Innervation is an essential factor for the
differentiation from a myotube to a myofiber during muscle regeneration
(1). The results of the present study clearly demonstrate that the
impaired regenerative capacity of muscles in the old rats was
associated with a reduction in, or the lack of, proliferating
capability of the satellite and/or stem cells.
In contrast to the in vivo studies noted above, some tissue culture
studies suggest that satellite cells from muscles of old rats maintain
the capacity to replicate (27, 34) and that the rate of proliferation
is not decreased with age (11, 17). These cells in culture appear to
have only an increased "lag phase" before the onset of
proliferation (11, 17, 27). These differences in results obtained from
in vivo and in vitro studies emphasize the importance of taking into
account the experimental paradigms when comparing the responses of
cellular and system studies.
Amino acid uptake and ability for muscle hypertrophy.
Young rats showed a biphasic response in
[14C]leucine uptake, i.e., peaks at 1-4
and at 10 days after the exercise bout (Fig. 8B). The first
peak most likely reflects increased protein synthesis (hypertrophy) in
the primary tissues (contractile proteins, connective tissue,
revascularization, activation of satellite cells, etc.), whereas the
second peak may reflect the synthesis of the contractile components of
regenerated and/or de novo muscle fibers (hyperplasia; see Ref. 28).
Previous studies have demonstrated that muscle protein synthesis
accelerates after the formation of myotubes to allow for accumulation
of the contractile components within the cytoplasm and differentiation
of the myofibers (15). The old rats showed a similar peak in
[14C]leucine uptake at 1 wk but no peak at 2 wk
after the exercise bout (Fig. 8B). Furthermore, the
postexercise data for [14C]leucine uptake for
myosin (Fig. 9A) in the extensor muscles and for the fraction
of myosin uptake relative (%) to the total uptake (Fig. 9B)
suggest that the increase in amino acid uptake during the first week in
old rats was used primarily for the synthesis of noncontractile protein
such as connective tissue. An age-related decrease of myosin heavy
chain synthesis rate has been reported by Balagopal et al. (2),
suggesting a decreased capacity for hypertrophy in muscles of old rats.
Combined with the small increase in
[3H]thymidine uptake in old rats, these results
support the view that there is a lower muscle capacity for hypertrophy,
including hyperplasia, in old than in young rats.
Perspective
There was a lower reparative capacity of skeletal muscles in older than
in younger rats that was related to a reduction in myogenic cell
(satellite cell) activity in vivo. Most of the myogenic cells in the
muscles of old rats did not enter the cell cycle, based on the low
number of BrDU and MyoD positive cells after the weight-lifting
exercise bout. This loss of myogenic cell activation and proliferation
potential may be one of the critical factors in the reduction of the
function of skeletal muscle in the later stages of life. Although the
reduced effects of the exercise bout in old rats could have been due to
a lower level of overload in old compared with young rats, this is
unlikely for two reasons. First, markers for "muscle damage" were
significantly elevated in old rats after the exercise bout. Second, the
same criteria for exhaustive exercise was used for both young and old
rats. Thus it appears that not only does the muscle in the aged rat have a reduced performance potential but its ability to respond to
functional perturbations and maintain a homeostatic state is markedly reduced.
 |
ACKNOWLEDGEMENTS |
We thank K. Nakane, Facilities for Radioisotope Research, Tokai
University School of Medicine, for assistance with animal care and use.
We also thank M. Tokunaga, Laboratory for Structure and Function
Research, Tokai University School of Medicine, and Jung Kim, Department
of Physiological Science, University of California Los Angeles, for
photographic technical assistance.
 |
FOOTNOTES |
This work was supported in part by Meiji Life Foundation of Health and
Welfare and The Nakatomi Foundation.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. Tamaki, Dept.
of Physiology, Division of Human Structure and Function, Tokai Univ.
School of Medicine, Bohseidai, Isehara, Kanagawa 259-11 Japan
(E-mail: tamaki{at}is.icc.u-tokai.ac.jp).
Received 18 June 1999; accepted in final form 21 December 1999.
 |
REFERENCES |
1.
Albrook, D.
Skeletal muscle regeneration.
Muscle Nerve
4:
234-245,
1981[ISI][Medline].
2.
Balagopal, P,
Rooyackers OE,
Adey DB,
Ades PA,
and
Nair KS.
Effects of aging on in vivo synthesis of skeletal muscle myosin heavy-chain and sarcoplasmic protein in humans.
Am J Physiol Endocrinol Metab
273:
E790-E800,
1997[Abstract/Free Full Text].
3.
Banker, BQ,
and
Engel AG.
Basic reactions of muscle.
In: Myology, edited by Engel A G.,
and Franzini-Armstrong C.. New York: McGraw-Hill, 1986, p. 832-888.
4.
Brooks, SV,
and
Faulkner JA.
Contraction induced injury: recovery of skeletal muscles in young and old mice.
Am J Physiol Cell Physiol
258:
C436-C442,
1990[Abstract/Free Full Text].
5.
Brown, AB,
McCartney N,
and
Sale DG.
Positive adaptations to weight-lifting training in the elderly.
J Appl Physiol
69:
1725-1733,
1990[Abstract/Free Full Text].
6.
Carlson, BM,
and
Faulkner JA.
Reinnervation of long-term denervated rat muscle freely grafted into an innervated limb.
Exp Neurol
102:
50-56,
1988[ISI][Medline].
7.
Carlson, BM,
and
Faulkner JA.
Muscle transplantation between young and old rats: age of host determines recovery.
Am J Physiol Cell Physiol
256:
C1262-C1266,
1989.
8.
Carlson, BM,
and
Faulkner JA.
The regeneration of noninnervated muscle grafts and marcaine-treated muscles in young and old rats.
J Gerontol. Biol Sci
51A:
B43-B49,
1996[Abstract].
9.
Carmeli, E,
and
Reznick AZ.
The physiology and biochemistry of skeletal muscle atrophy as a function of age.
Proc Soc Exp Biol Med
206:
103-113,
1994[Abstract].
10.
Davis, LG,
Dibner MD,
and
Battey JF.
Guanidine isothiocyanate preparation of total RNA.
In: Basic Methods in Molecular Biology, edited by Davis L G.,
Dibner M. D.,
and Battey J. F.. New York: Elsevier, 1986, p. 130-135.
11.
Dodson, MV,
and
Allen RE.
Interaction of multiplication stimulating activity/rat insulin-like growth factor II with skeletal muscle satellite cells during aging.
Mech Ageing Dev
39:
121-128,
1987[ISI][Medline].
12.
Faulkner, JA,
Brooks SV,
and
Zerba E.
Muscle atrophy and weakness with aging: Contraction-induced injury as an underlying mechanism.
J Gerontol Series A
50A:
124-129,
1995[ISI].
13.
Grimby, G.
Muscle performance and structure in the elderly as studied cross-sectionally and longitudinally.
J Gerontol Series A
50A:
17-22,
1995[ISI].
14.
Grimby, G,
and
Saltin B.
The aging muscle.
Clin Physiol
3:
209-218,
1993.
15.
Grounds, MD.
Towards understanding skeletal muscle regeneration.
Pathol Res Pract
187:
1-22,
1991[ISI][Medline].
16.
Gutmann, E,
and
Hanzlikova V.
Motor unit in old age.
Nature
209:
921-922,
1966[ISI][Medline].
17.
Johnson, SE,
and
Allen RE.
Proliferating cell nuclear antigen (PCNA) is expressed in activated rat skeletal muscle satellite cells.
J Cell Physiol
154:
39-43,
1993[ISI][Medline].
18.
Larsson, L,
and
Ansved T.
Effects of aging on the motor unit.
Prog Neurobiol
45:
397-458,
1995[ISI][Medline].
19.
Leadbetter, WB.
An introduction to sports induced soft-tissue inflammation.
In: Sports-Induced Inflammation, edited by Leadbetter W B.,
Buchwalter J. A.,
and Gordon S. L.. Bethesda, MD: Am. Acad. Orthop. Surg., 1989, p. 3-23.
20.
Lexell, J.
Human aging: Muscle mass, and fiber type composition.
J Gerontol Series A
50A:
11-16,
1995[ISI].
21.
Lexell, J,
Taylor CC,
and
Sjostom M.
What is the cause of the aging atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-year-old men.
J Neurol Sci
84:
275-294,
1988[ISI][Medline].
22.
Lin, M,
and
Nonaka I.
Facioscapulohumeral muscular dystrophy: muscle fiber type analysis with particular reference to small angular fibers.
Brain Dev
13:
331-338,
1991[ISI][Medline].
23.
Marsh, DR,
Criswell DS,
Hamilton MT,
and
Booth FW.
Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats.
Am J Physiol Regulatory Integrative Comp Physiol
273:
R353-R358,
1997[Abstract/Free Full Text].
24.
Moritani, T,
and
deVries HA.
Potential for gross muscle hypertrophy in older men.
J Gerontol A Biol Sci Med Sci
35:
672-682,
1980.
25.
Pyka, G,
Lindenberger S,
Charette S,
and
Marcos R.
Muscle strength and fiber adaptations to a year-long resistance training program in elderly men and women.
J Gerontol Med Sci
149:
M22-M27,
1994.
26.
Roy, RR,
Baldwin KM,
and
Edgerton VR.
The plasticity of skeletal muscle, effects of neuromuscular activity.
In: Exercise and Sports Sciences Reviews, edited by Holloszy J. Baltimore: Williams and Wilkins, 1991, p. 269-312.
27.
Schultz, E,
and
Lipton BH.
Skeletal muscle satellite cells: changes in proliferation potential as a function of age.
Mech Ageing Dev
20:
377-383,
1982[ISI][Medline].
28.
Tamaki, T,
Akatsuka A,
Tokunaga M,
Ishige K,
Uchiyama S,
and
Shiraishi T.
Morphological and biochemical evidence of muscle hyperplasia following weight-lifting exercise in rats.
Am J Physiol Cell Physiol
273:
C246-C256,
1997[Abstract/Free Full Text].
29.
Tamaki, T,
Akatsuka A,
Tokunaga M,
Uchiyama S,
and
Shiraishi T.
Characteristics of compensatory hypertrophied muscle in the rat. I. Electron microscopic and immunohistochemical studies.
Anat Rec
246:
325-334,
1996[ISI][Medline].
30.
Tamaki, T,
Uchiyama S,
and
Nakano S.
A weight-lifting exercise model for inducing hypertrophy in the hindlimb muscles of rats.
Med Sci Sports Exerc
24:
881-886,
1992[ISI][Medline].
31.
Ullman, M,
Ullman A,
Sommerland H,
Skottner A,
and
Oldfors A.
Effects of growth hormone on muscle regeneration and IGF-I concentration in old rats.
Acta Physiol Scand
140:
521-525,
1990[ISI][Medline].
32.
Vaidya, TB,
Rhodes SJ,
Moore JL,
Sherman DA,
Konieczny SF,
and
Taparowsky EJ.
Isolation and structural analysis of the rat MyoD gene.
Gene
116:
223-230,
1992[ISI][Medline].
33.
Welle, S,
Totterman S,
and
Thornton C.
Effect of age on muscle hypertrophy induced by resistance training.
J Gerontol.
51A:
M270-M275,
1996[ISI].
34.
Wright, WE.
Myoblast senescence in muscular dystrophy.
Exp Cell Res
157:
343-354,
1985[ISI][Medline].
Am J Physiol Cell Physiol 278(6):C1143-C1152
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society