J Orthop Sci (2003) 8:213–217
Expression of heat shock protein 72 in rat quadriceps muscles
following anterior cruciate ligament resection
Ryoko Okuyama1,2, Masanori Honda2, Hiroto Fujiya3, Katsumasa Goto2, Takao Sugiura4,
and Tatsuo Akema2
1
Department of Orthopaedic Surgery, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki 216-8511, Japan
2
Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Japan
3
Laboratory of Sports Medicine, St. Marianna University School of Medicine, Kawasaki, Japan
4
Faculty of Education, University of Yamaguchi, Yamaguchi, Japan
Abstract This study investigated the expression of heat shock
protein 72 (HSP 72) in quadriceps femoris muscle following
anterior cruciate ligament (ACL) resection and running
training in rats. A group of 15 Wistar strain male rats (10
weeks old) were divided into three groups: sham-operated (S),
ACL resection (A), and running training following ACL
resection (R). In the A and R groups, the ACL of the right
knee was resected using microsurgical techniques. Rats in the
R group were subjected to running training for 9 weeks on
a motor-driven treadmill for rodents 1 week after ACL
resection. At 10 weeks after ACL resection, glycerinated
single fibers were prepared from vastus lateralis (VL), vastus
medialis (VM), rectus femoris (RF), and semimembranosus
(SM) muscles. To determine the levels of HSP72 (HSP-
inducible) in each muscle, we used the Western blotting
technique. HSP72 expression in VL and VM increased
following ACL resection (P ⬍ 0.05) but not in RF or SM.
Exercise training caused an elevation in HSP72 expression in
VL and VM. There were no significant changes in HSP72
expression in RF or SM following exercise training. These
results suggest that some stress would be generated by ACL
resection in VM and VL and might cause atrophy in VM and
VL.
Key words Anterior cruciate ligament · Quadriceps femoris
muscle · Heat shock protein
Introduction
Rupture of the anterior cruciate ligament (ACL) is one
of the more common sports injuries of the knee.1,5
Patients with a complete tear of the ACL are expected
to experience some secondary dysfunction of the knee
joint as a result.6,26,27 Meniscal injury, instability of the
knee, and arthrosis have all been associated with ACL
injury.7 Atrophy and weakness of the quadriceps
Offpring requests to: R. Okuyama
Received: July 29, 2002 / Accepted: October 18, 2002
femoris muscle is a frequent clinical finding in patients
with long-standing ligamentous instability of the knee,
but it is unclear about the details of the weakness.5,6,18
Previous studies had demonstrated that ACL rupture
caused diminution of the size, weight, and enzyme
activities related to energy metabolism of thigh
muscles.5,28 However, the stress on the quadriceps
femoris muscle is not elucidated.
Stress induces the expression of stress proteins, such
as heat shock proteins (HSPs). This response, observed
in most cells, is called the heat shock response. HSPs
bind to denatured proteins and are involved in the
assembly of protein complexes,20 so they are also known
as “molecular chaperones.”7 HSPs are classified into
families according to their molecular weight.15 The most
abundant and widely studied family is the 70-kDa
family, which contains the constitutive HSP73 and
inducible HSP72 forms. HSP72 expression is induced by
increased temperature,19 ischemia,17 protein degrada-
tion,3 hypoxia,8 and acidosis.29 These factors are charac-
teristic of contracting mammalian skeletal muscle cells.
Therefore, HSP72 would be up-regulated in the skeletal
muscle of rodents in response to exercise.24,25 These
observations suggest that HSP72 expression may have
protective responses against cellular stress, such as
super oxide, ischemia, and metabolites (lactate, H⫹, Pi)
during contraction of skeletal muscle fibers, but the
precise mechanism of exercise-induced HSP72 expres-
sion is not known. It has been well documented that
quadriceps muscles undergo some stress after ACL
resection, but it is not clear whether the ACL resection
induced stress in quadriceps muscle.
In the present study, we investigated the expression
of HSP72 to evaluate the level of stress in the
quadriceps muscle following ACL resection. Exercise
training was used for rehabilitation following ACL
injuries. The effects of exercise training on HSP72
expression in quadriceps femoris muscle following ACL
resection were also evaluated.
214
Materials and methods
A group of 15 Wistar strain male rats (10 weeks old)
were used. We placed five rats in each of three groups
— sham-operated (S), ACL resection (A), running
training following ACL resection (R) — so the average
weights for the three groups were approximately the
same. In the A and R groups, the ACLs of the right
knees of rats were resected using microsurgical
technique under intraperitoneal anesthesia with sodium
pentobarbital (2.5 mg/100 g body weight). After ACL
resection, the anterior instability of the knee joint was
checked. The physical condition of the rats was
carefully observed for 1 week after the operation. The
five rats in the R group were subjected to running
training once a day 5 days/week for 9 weeks on a motor-
driven treadmill for rodents. The running speed and
times were progressively increased until 4 weeks after
training started. After 4 weeks, both the running speed
and times were maintained constant at 30 m/min and
90 min, respectively, for 4 weeks (gradient 0°C). Water
and food were supplied to all groups ad libitum.
Animals were housed in a temperature-controlled
animal facility (temperature 22.5° ⫾ 0.5°C; humidity
55% ⫾ 5%).
Electrophoresis
After 4 weeks of training the vastus lateralis (VL),
vastus medialis (VM), rectus femoris (RF), and
semimembranosus (SM) muscles were rapidly removed
from the experimental hindlimb. Glycerinated single
fibers were prepared from each muscle.10 Muscle
bundles of each muscle were stored at ⫺20°C in 50%
(v/v) glycerol solution for up to 2 months prior to use.
On the day of the experiment, single muscle fibers were
carefully dissected from each bundle. Each single
muscle fiber was then solubilized in 0.03 ml of a sodium
dodecyl sulfate (SDS)-dithiothreitol (DTT) buffer
consisted of 5 mM ethylenediaminetetraacetic acid
(EDTA), 10% SDS, 50 mM DTT, and 0.1 M Tris-HCl
(pH 8.8) and boiled for 3 min. The solublized buffer was
mixed with 0.01 ml of 50% glycerol solution (50%
glycerol, 0.1% bromophenol blue) at a 1 : 1 ratio and
used for SDS-polyacrylamide gel electrophoresis (SDS-
PAGE). The electrophoresis was carried out on a
12.5% polyacrylamide [biascrylamide/acrylamide, 1 : 20
(w/w)] slab gel (60 ⫻ 85 ⫻ 1 mm) containing 0.5% SDS
at a constant current of 20 mA for 90 min according to
the method of Laemmli.12
Western blotting
We investigated the expression of HSP72 (HSP70
inducible) in each muscle following ACL resection with
R. Okuyama et al.: HSP72 induced by ACL resection
or without running training. Following SDS-PAGE,
proteins were transferred to polyvinylidene difluoride
(PVDF) membranes (pore size 0.2 µm) (Bio-Rad,
Hercules, CA, USA) using a Bio-Rad mini trans-blot
cell at a constant voltage of 100 V for 60 min at 4°C.
After transferring, the membranes were blocked for 1 h
using a blocking buffer [5% skim milk in Tris-buffered
saline (TTBS), pH 7.5]. Then the membranes were
incubated for 1 h with a polyclonal antibody for HSP72
(SPA-812; StressGen, Victoria, BC, Canada) and then
reacted with a secondary antibody (goat anti-mouse
immunogloblin G conjugated to alkaline phosphatase)
(BioRad) for 2 h. The membranes were subsequently
reacted with bromochloroindolyl phosphate-nitro blue
tetrazolium substrate. The bands from immunoblots
were quantified using computerized densitometry (NIH
Image). Standard curves were constructed during
preliminary experiments to ensure linearity.
Statistical analysis
All values are expressed as means ⫾ SE. The values
were analyzed using two-factor functional ANOVA
followed by Scheffé’s post hoc test.
Results
Body weight
The body weights of the groups were 471.8 ⫾ 35.9 g for
S group, 479.0 ⫾ 46.2 g for A group, and 417.6 ⫾ 13.7 g
for R group. There was no significant difference in the
body weights of the S and A groups. The body weight
for the R group was significantly lower than those for
the S and A groups (P ⬍ 0.05).
HSP72 expression
The levels of HSP72 expression in VM, VL, RF, and SM
in the sham-operated (S) group are shown in Figs. 1–4.
There were no significant differences in the levels of
HSP72 expression among the four muscles.
Expression of HSP72 in VM is shown in Fig. 1. HSP72
expression in the ACL resection (A) group was signi-
ficantly higher (91%) than in the sham-operated (S)
group (P ⬍ 0.05). HSP72 expression in VM also was
significantly higher in the R group (164%) than in the S
group (P ⬍ 0.05). HSP72 expression in VM was highest
in the R group, lowest in the S group, and intermediate
in the A group. HSP72 expression in VM was signifi-
cantly higher in the R group (38%) than in the A group
(P ⬍ 0.05).
Figure 2 shows the changes in HSP72 expression in
VL. In VL, the HSP72 expression was significantly
R. Okuyama et al.: HSP72 induced by ACL resection
Fig. 1. Representative Western blotting to determine heat
shock protein 72 (HSP72) levels in vastus medialis muscle
(VM). Sham-operated (S), anterior cruciate ligament (ACL)
resection (A), or running training following ACL resection
(R) groups. HSP72 expressions in the A and R groups were
significantly higher than those in the S group (P ⬍ 0.05).
HSP72 expression was significantly higher in the R group than
in the A group (P ⬍ 0.05). Values are means ⫾ SE.
*#Significantly different (P ⬍ 0.05) from *S or #A
Fig. 2. Representative Western blotting to determine HSP72
levels in vastus lateralis muscle (VL). HSP72 expression was
significantly higher in the A and R groups than in the S group
(P ⬍ 0.05). HSP72 expression was significantly higher in the R
group than in the A group (P ⬍ 0.05). Values are means ⫾ SE.
*#Significantly different (P ⬍ 0.05) from *S or #A
215
Fig. 3. Representative Western blotting to determine HSP72
levels in rectus femoris muscle (RF). There were no significant
differences in HSP expression among the three groups (P ⬍
0.05). Values are means ⫾ SE
higher in the ACL resection (A) group (39%) than in
the sham-operated (S) group (P ⬍ 0.05). HSP72
expression in VL was also significantly higher in the R
group (108%) than in the S group (P ⬍ 0.05). HSP72
expression in VL was highest in the R group, lowest in
the S group, and intermediate in the A group. HSP72
expression in VL (50%) was significantly higher in the
R group (50%) than in the A group (P ⬍ 0.05).
Figures 3 and 4 show HSP72 expression in RF and
SM, respectively. HSP72 expression in RF was 66% and
68% higher, respectively, after ACL resection (A)
and exercise training following ACL resection (R) than
in the sham-operated (S) group (Fig. 3). There were no
significant differences in HSP72 expression among the
three groups (P ⬎ 0.05). Neither ACL resection nor
exercise training following ACL resection had any
effect on HSP72 expression in SM (Fig. 4).
Discussion
We investigated the effects of ACL resection (with and
without exercise training) on HSP72 expression. To our
knowledge, this is the first report on the changes in
HSP72 expression in the quadriceps femoris muscle
following ACL resection and exercise training. ACL
resection caused an increase in HSP72 expression in
VM and VL but not in RF or SM. HSP expression
increased after exercise training following ACL resec-
tion in VM and VL but not in RF or SM.
216
Fig. 4. Representative Western blotting to determine HSP72
levels in semimenbranosus muscle (SM). There were no
significantly differences in HSP expression among the three
groups. P ⬍ 0.05. Values are means ⫾ SE
It has been reported that ACL resection induces
atrophy in thigh muscles.1,2,4,5,16,27,28 Clinically, thigh
muscle atrophy occurs frequently after ACL injuries,7,28
but the precise mechanism of the ACL resection-
induced muscular atrophy is unknown. It has been
thought that some muscular properties in atrophied
quadriceps femoris muscle following ACL resection
were similar to those in atrophied muscles due to disuse.
On the other hand, it has been reported that HSP72
expression decreases in skeletal muscle during disuse
atrophy.21,22,24
In the present study, HSP72 expression increased in
VL and VM but not in RF or SM after ACL resection.
VM and VL insert into the patella, the former on the
medial side and the latter on the lateral side. In rats, the
extent of the adhesion areas around VM and VL is
wider than that of humans. Therefore, after ACL
resection the effects of knee instability on VM and VL
may be more influential than that on RF and SM.
It has been reported that HSP72 expression is fiber
type-specific. There are no differences in the fiber-type
composition among these four muscles in rats.
Quadriceps muscles in rats consist of more than 95% of
fast-twitch fibers.5 These results suggested that some
stress might be generated in VM and VL following ACL
resection, though the kind of stress was still unclear.
It has been reported that ACL resection caused
shortening of the quadriceps femoris muscle. It is
thought that ACL resection-induced muscular atrophy
R. Okuyama et al.: HSP72 induced by ACL resection
might be caused by shortening of the quadriceps
femoris muscle. However, there have been no reports
showing that HSP72 expression is induced by the
muscular length shortening. On the other hand,
mechanical stretch could cause an increase in HSP72
expression. It is unclear that mechanical stretch is
generated in VM and VL.
In the present study, exercise training following ACL
resection caused an increase in HSP72 expression in
VM and VL but not in RF and SM. The responses of
HSP72 expression in RF and SM to ACL resection were
different from those in VM and VL. These observations
suggest that the activation patterns in RF and SM were
different from those in VL and VM. An increase in
slow-twitch fibers was observed after ACL resection in
VM and VL but not in RF or SM.5 HSP72 expression
was higher in slow-twitch fibers than in fast-twitch
fibers. Therefore, increased HSP72 expression in VM
and VL might depend on transformation of the fiber
types induced by ACL resection. It is still unclear,
however, what stimuli caused the fiber-type composi-
tion to change following ACL resection.
The existence of mechanoreceptors in the ACL has
been suggested, but the function of a mechanoreceptor
has not been clarified. A mechanoreceptor works as a
sensor for mechanical stress on the ACL to maintain the
stability of a knee joint.2,11,18,23 After ACL resection,
afferent signals from mechanoreceptors would be
absent. The absence of afferent signals from mecha-
noreceptors in the ACL would cause instability of the
knee joint. However, the interrelation of mechanore-
ceptors and HSP72 expression is unclear. Another
approach is needed to elucidate this matter.
The responses of HSP72 expression to disuse atrophy
are not the same as those in quadriceps femoris muscle
after ACL resection. Therefore, atrophy in quadriceps
femoris muscle might not be derived from the decre-
ment in its activity after ACL resection. Such stress
might cause atrophy of the quadriceps femoris muscle.
It has been reported that HSP72 expression increases
after a single bout of exercise and exercise training.
Hyperthermia is one of the factors that induce the stress
response. Because muscular temperature rises during
exercise, HSP72 expression might be induced by the
heat shock response. Other reports showed that
exercise-induced HSP72 is independent of the elevated
body temperature.25 Metabolic products also induce
HSP72, which may be responsible for HSP72 expression
in exercised muscle. A recent study suggested that
depletion of adenosine triphosphate (ATP) during
metabolic stress was one of the main signals for HSP70
induction.9,25 Unfortunately, the precise mechanisms for
exercise-induced HSP72 expression is unknown. It is
suggested that the HSP72 response to training seems to
be dependent on exercise intensity.13,14
R. Okuyama et al.: HSP72 induced by ACL resection
The elevated HSP72 expression in VM and VL
induced by exercise training after ACL resection was
significantly higher than that induced by ACL resection
alone (Figs. 1, 2). These results suggest that the stress
caused by ACL resection might be different from that
induced by exercise training. We did not investigate the
effect of exercise training on HSP72 expression in
quadriceps femoris muscle in the sham-operated knees
in the present study. The difference in HSP72
expression between the A and R groups might depend
only on the exercise stimulus.
Conclusions
Resection of the ACL caused increased HSP72
expression in VL and VM but not in RF and SM.
Running exercise following ACL resection also induced
an elevation in HSP72 expression in VL and VM. There
were no significant changes in HSP72 expression in RF
or SM following exercise training. Some stress would be
derived from the ACL resection itself and would cause
muscular atrophy in VL and VM. The kind of stress
caused by ACL resection is unclear. Expression of
stress proteins such as HSP72 may be a useful tool for
exploring the effects of ACL resection on the function
of the quadriceps femoris muscle.
Acknowledgments. We especially thank Professor H. Aoki of
the Department of Orthopaedic Surgery at St. Marianna
University School of Medicine for his encouragement and
many valuable comments. We also thank Vice President T.
Yoshioka of Aomori University of Health and Welfare for his
valuable criticism and advice. This study was supported
in part by a Grant-in-Aid for Encouragement of Young
Scientists (B) from The Ministry of Education, Culture,
Sports, Science and Technology (MEXT) to K.G.
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