Apoptosis of Skeletal Muscle on Steroid-Induced Myopathy in Rats

Abstract

Recently apoptotic cell death has been reported in differentiated skeletal muscle, where apoptosis was generally assumed not to occur. To investigate whether apoptosis may contribute to the steroid-induced myopathy, rats treated with triamcinolone acetonide (TA) for 9 days were sacrificed for detecting apoptosis by in situ end labeling (ISEL) and electron microscopy in the soleus muscles. Immunohistochemical stainings of Fas antigen and p53 protein were performed to examine whether apoptosis-related proteins were present in the myopathy. Muscle fiber necrosis and apoptotic myonuclei appeared in the soleus muscles following administration of TA, while control muscles showed no evidences for apoptosis. Fas antigen was not detected in control muscles, but expressed in the soleus muscles of steroid-induced myopathy. Some of the Fas antigen-expressing muscle fibers were positive for ISEL. p53 protein was not detected in any muscle fibers. These findings indicate that TA can induce apoptosis in differentiated skeletal muscles, and Fas antigen might be partly related to apoptotic muscle death in steroid-induced myopathy.

Full text

INTRODUCTION
Long-term administration of glucocorticoids in human
and animals has been reported to induce steriod myopathy
(1-3). Although any of the commonly available glucocor-
ticoid preparations can cause myopathy, the fluorinated
steroids, e.g., triamcinolone, betamethasone, and dexam-
ethasone, seem more likely to produce muscle weakness.
The typical findings of steroid myopathy are selective atro-
phy of type II muscle fibers and necrotic changes (4, 5). It
has been suggested that glucocorticoid-induced mitochon-
drial damage can lead to muscle fiber necrosis (4, 6-8).
It is widely accepted that there are two major distinct
modes of death in eukaryotic cells, i.e., necrosis and apopto-
sis (9-11). Apoptosis in response to a variety of injurious
stimuli has been demonstrated in several organs (12-16). In
the lymphoid tissue and brain, glucocorticoids are known
to easily induce apoptosis (17-19). Although differentiated
skeletal muscles usually undergo necrotic death in response
to injury, apoptotic cellular death occurs in dystrophin-defi-
cient muscle (20, 21) and differentiated skeletal muscle
after treatment with anabolic steroids (22).
Apoptosis is stimulated or inhibited by several mediator
proteins. Fas antigen, which mediates apoptosis of lym-
phoid cells (23, 24), has been identified in diseased muscles
(25-27). p53 protein was proven to stimulate apoptosis (28, 29).
This study investigated whether apoptosis might con-
tribute to the death of differentiated skeletal muscle after
administration of glucocorticoid. Two assays for the detec-
tion of apoptosis were employed, i.e., in situ end labeling
(ISEL) and electron microscopic examinations (30, 31).
Immunohistochemical stainings of Fas antigen and p53
protein were performed to examine the roles of these pro-
teins in apoptosis of skeletal muscle in steroid-induced
myopathy.
MATERIALS AND METHODS
Development of steroid-induced myopathy and tissue
sampling
Twenty female Sprague-Dawley rats weighing between
180-210 g used in this study were maintained under stan-
dard condition. Rats were divided into two groups. Each
group was given daily intraperitoneal injection of either
physiologic saline (n=5) or triamcinolone acetonide (TA) at
a dose of 5 mg/kg body weight for 9 days (n=15). Ten days
after the completion of the injections, the soleus muscles
from both legs were taken under ether anesthesia, and the
weight was measured. A 5-10 mm thick cross section was
taken from the midbelly of the right soleus, and mounted
on cork in a manner such that the muscle fibers were per-
pendicular to the cork surface. The sections were quick-
Myung Ju Lee, Ji Shin Lee*,
Min Cheol Lee
�
Department of Plastic and Reconstructive Surgery,
College of Medicine, Chosun University,
Department of Pathology, College of Medicine,
Seonam University*, and Department of Pathology,
Chonnam National University Medical School and
Research Institute of Medical Sciences
�
, Kwangju,
Korea
Received : 13 October 2000
Accepted : 23 April 2001
Address for correspondence
Min Cheol Lee, M.D.
Department of Pathology, Chonnam National
University Medical School, 5 Hak-dong, Dong-ku,
Kwangju 501-190, Korea
Tel : +82.62-220-4300, Fax : +82.62-227-3429
E-mail : mclee@chonnam.ac.kr
467
J Korean Med Sci 2001; 16: 467-74
ISSN 1011-8934 Copyright

The Korean Academy
of Medical Sciences
Apoptosis of Skeletal Muscle on Steroid-Induced Myopathy in Rats
Recently apoptotic cell death has been reported in differentiated skeletal mus-
cle, where apoptosis was generally assumed not to occur. To investigate
whether apoptosis may contribute to the steroid-induced myopathy, rats treated
with triamcinolone acetonide (TA) for 9 days were sacrificed for detecting apop-
tosis by in situ end labeling (ISEL) and electron microscopy in the soleus mus-
cles. Immunohistochemical stainings of Fas antigen and p53 protein were per-
formed to examine whether apoptosis-related proteins were present in the
myopathy. Muscle fiber necrosis and apoptotic myonuclei appeared in the
soleus muscles following administration of TA, while control muscles showed no
evidences for apoptosis. Fas antigen was not detected in control muscles, but
expressed in the soleus muscles of steroid-induced myopathy. Some of the Fas
antigen-expressing muscle fibers were positive for ISEL. p53 protein was not
detected in any muscle fibers. These findings indicate that TA can induce apop-
tosis in differentiated skeletal muscles, and Fas antigen might be partly related
to apoptotic muscle death in steroid-induced myopathy.
Key Words : Myopathy, Steroid-Induced; Apoptosis; Fas Antigens, CD95; Protein p53; Muscular Dis-
eases

468 M.J. Lee, J.S. Lee, M.C. Lee
frozen in isopentane cooled by liquid nitrogen. The tissues
were cut in 10 m thick serial sections in a cryostat main-
tained at -20

and mounted on probed microscopic slides
(FisherBiotech, Pittsburgh, PA, U.S.A.). The frozen sections
were used for histological studies, ISEL, and immunohisto-
chemical studies. Remainder of the soleus muscles were
used for electron microscopic examination.
In situ end labeling (ISEL)
Apoptotic cells were visualized using the ApopTag

Plus
In Situ Apoptosis Detection Kit (Oncor, Gaithersburg,
MD, U.S.A.). Briefly, frozen sections were fixed in 10%
neutral buffered formalin for 10 min and postfixed in
ethanol: acetic acid (2:1) for 5 min at -20

After washing
in phosphate-buffered saline (PBS; 50 mM sodium phos-
phate, pH 7.4, 200 mM NaCl), sections were digested with
proteinase K (20 g/mL in PBS; Sigma, St. Louis, MO,
U.S.A.) for 15 min at room temperature and washed with
distilled water. Slides were then put into 3% H2O2for 5
min to quench the endogenous peroxidase activity and
washed with PBS. After adding the equilibration buffer for
1 min, terminal deoxynucleotidyl transferase (TdT) enzyme
was pipetted onto the sections, which were then incubated
in a humidified chamber at 37

for 1 hr. The reaction was
stopped by putting sections in a stop/wash buffer. After
washing, anti-digoxigenin-peroxidase was added to the
slides. Slides were washed, stained with diaminobenzidine
substrate, and counterstained with hematoxylin. A speci-
men known to be positive for apoptotic cells was used as a
positive control for staining. Substitution of TdT with dis-
tilled water was used as a negative control. Positive muscle
fibers were counted in all sections.
Immunohistochemistry for Fas antigen and p53 pro-
tein
Frozen sections were fixed with the ethanol/acetic acid
fixative solution for 10 min. The slides were immunostained
with the avidin-biotin-peroxidase complex method (32).
The endogenous peroxidase activity was blocked by incu-
bating 1.5% H2O2in PBS. The slides were treated in the
10% normal in PBS for 30 min in order to block charged
sites on tissue surfaces and then incubated with anti-Fas
antigen (titer 1:50; clone UB-2; MBL, Watertown, MA,
U.S.A.) or anti-p53 protein (titer 1:500; clone Ab-7; Onco-
gene, Cambrige, MA, U.S.A.) overnight at 4

The strep-
tavidin-horseradish peroxidase (Research Genetics, Hunts-
ville, Alabama, U.S.A.) detection system was then applied.
After treatment with 1% avidin-biotinylated horseradish
peroxidase for 1 hr at room temperature, the tissue sections
were prepared for chromogen reaction with 3-amino-9-ethyl
carbazole (Biomeda, Foster, CA, U.S.A.). The sections were
counterstained with hematoxylin and mounted on Crys-
tal/Mount (Biomeda). Positive muscle fibers were counted
in all sections.
Electron microscopy
The soleus muscles used for electron microscopy were ini-
tially kept in a slightly stretched state, via pins passed
through their tendons into dental wax, while they were
fixed for 1 hr in 2.5% glutaraldehyde in 0.2 M sodium
cacodylate buffer, pH 7.2. Three pieces of muscle, measur-
ing 2×2×3 mm, were then excised and left for several
hours in fresh fixative. They were then postfixed in 1%
osmiun tetroxide for 1 hr, dehydrated in graded alcohols,
cleared in propylene oxide, and embedded in epoxy resin.
Sections were cut 80 nm in thickness by an ultramicro-
tome, collected on uncoated grids, stained with uranyl
acetate and lead citrate, and examined with a JEM 1200
EX II (JEOL).
RESULTS
Gross and histologic examinations of myopathy
Control rats gained weight during the experimental peri-
ods. In contrast, rats treated with TA showed loss of body
weight (average weight loss, 26%). The excised soleus mus-
cle in TA-treated rats weighed 11% less than in control
rats.
In light microscopic examinations, the muscle fibers of
control rats were arranged in bundles or fascicles, surround-
ed by perimysial connective tissue. There was little varia-
tion in muscle fiber size or shape. Cytoplasmic staining was
uniform, and small peripherally located nuclei were abun-
dant (Fig. 1A). Variation in fiber size and shape was seen in
the TA-treated soleus muscles. Atrophic fibers were noted
occasionally (Fig. 1B). Some fibers had pale-staining cyto-
plasm. Necrotic fibers invaded by numerous macrophages
were seen (Fig. 1C). A few small eosinophilic fibers with
increased nuclei, indicative of atrophic change, were obser-
ved (Fig. 1D).
In situ end labeling (ISEL) and electron microscopy
for apoptosis
No ISEL-positive signal was noted in the soleus muscles
of control group (Fig. 2A). The TA-treated soleus muscles
had 1 to 4 muscle fibers having apoptotic myonuclei (Fig.
2B). Myonuclei labeled with ISEL were found in muscle
fibers, which had pale or intact staining cytoplasms (Fig.
2C and D). Necrotic fibers were negative for ISEL (Fig. 2E
and F).
In electron microscopic examinations, the nuclei of con-
trol soleus muscles appeared just beneath the plasma mem-

Apoptosis in Steroid-Induced Myopathy 469
brane. They were elongated and ovoid in shape, with the
chromatin distributed towards the periphery (Fig. 3A).
In the TA-treated soleus muscles, a few apoptotic myonu-
clei characterized by irregularly condensed chromatin near
the myonuclear membrane with intact sarcoplasm were
noted (Fig. 3B). There were no discernible signs of degener-
ation or necrosis in fibers that contained apoptotic nuclei.
Some muscle fibers contained swollen sarcoplasmic reticu-
lum and mitochondria. Necrotic muscle fibers, with focal
loss of myofilaments and swollen nuclei containing dis-
persed chromatin, were also observed.
Immunohistochemical staining of Fas antigen and
p53 protein
Immunohistochemical staining of Fas antigen was not
detected in the normal soleus muscles (Fig. 4A). Fas antigen
was detected in the TA-treated soleus muscles and was
mainly localized in the cytoplasm with granular pattern
(Fig. 4B). Number of Fas antigen-positive muscle fibers
ranged from 1 to 8 per each muscle bundle. Some of the Fas
antigen-expressing muscle fibers were positive for ISEL
(Fig. 4C and D). Necrotic muscle fibers invaded by
CD
AB
Fig. 1. Steroid myopathy on the soleus muscle (H&E). There is little variation in size or shape of muscle fibers in control group (A, ×200).
Triamcinolone acetonide (TA)-treated soleus muscle reveals increased variation in size and shape and a few atrophic fibers (B, ×200),
necrotic muscle fiber invaded by macrophages (C, ×400), and atrophic fibers with increased nucleation (D, ×200).

470 M.J. Lee, J.S. Lee, M.C. Lee
macrophages demonstrated no expression of Fas antigen.
p53 protein was not detected in any muscle fibers of the
normal or TA-treated soleus muscles.
DISCUSSION
Several of synthetic glucocorticoids have been developed
and used in a wide range of medical and surgical fields.
Despite their therapeutic benefits, the long-term adminis-
tration of glucocorticoids in humans has often resulted in
progressive muscle weakness and steroid myopathy. Charac-
teristic biopsy findings from patients with steroid myopa-
thy are selective atrophy of type II muscle fibers and occa-
sional random fiber necrosis (1-3). As in human disease,
steroid myopathy has been induced in a variety of experi-
mental animals using a number of different steroids. The
fluorinated steroids, e.g., triamcinolone, betamethasone,
and dexamethasone, appear more likely to produce steroid
myopathy (4, 5). Necrotic lesions are easily induced in the
rat soleus muscle by steroid administration (4). In this
experiment, steroid-induced myopathies in rats were devel-
CD
AB
Fig. 2. In situ end labeling (ISEL) for apoptotic cells. None of myonuclei in control muscles show positive reaction (A, ×200). Positive
staining in myonuclei of TA-treated soleus muscle is seen (B, ×200). Muscle fiber having pale cytoplasm (C, ×400) is positive by ISEL
(D). (Fig. 2 continued next)

Apoptosis in Steroid-Induced Myopathy 471
oped by daily intraperitoneal injections of TA for 9 days. In
the steroid-induced myopathy, necrotic changes were found
in frozen sections prepared for histological study and in
electron microscopical examination. We thought that our
experimental protocol (i.e. species, drug, duration) might
be suitable for the study of this specific muscle lesion. Glu-
cocortoid-induced mitochondrial damage leading to muscle
fiber necrosis has been suggested in both ultrastructural and
biochemical studies (4, 6-8). The soleus muscle, which
showed necrotic change in this experiment, consists mainly
of type I fibers, being rich in mitochondrial oxidative
enzymes (33).
Apoptosis is a type of cell death that has been shown to
differ from necrosis by defined ultrastructural and biochem-
EF
Fig. 2. (Continued from the previous page) In situ end labeling (ISEL) for apoptotic cells. Necrotic muscle fiber invaded by macrophages
(E, H&E, ×400) is negative for ISEL (F).
AB
Fig. 3. Electron micrograph of a nucleus of the normal soleus muscle displays condensed chromatin along the nuclear membrane.
Other organelles in the sarcoplasm appear normal (A, ×4,000). Apoptotic change in the TA-treated soleus muscle shows irregularly
clumped chromatin of the nucleus without sarcoplasmic degeneration (B, ×10,000).

472 M.J. Lee, J.S. Lee, M.C. Lee
ical features (9-11). Through the implementation of ISEL
and electron microscopy, we examined the possibility that
apoptosis might contribute to the death of differentiated
skeletal muscles of steroid myopathy. The technique of ISEL
introduced by Gavrieli et al. (30) detects fragmented DNA
via TdT reactions. This technique not only labels apoptotic
cells already in progress but also potentially detects any
cells where DNA strand breaks occurred without morpho-
logical changes. In addition, this technique can be per-
formed on histologic sections so that both the quantifica-
tion of apoptosis and the location of apoptotic nuclei can be
determined. Morphological changes in cell structure repre-
senting apoptotic cell death can be identified by electron
microscopy (31).
Glucocorticoids are known to easily induce apoptosis in
some tissue. Apoptosis of thymocytes (17) and the intestinal
intraepithelial lymphocytes (18) has been experimentally
induced by treatment with glucocorticoids. Hassan et al.
have also demonstrated the exacerbation of apoptosis in the
dentate gyrus of aged rats induced by dexamethasone (19).
Differentiated skeletal muscle cells of rats underwent
apoptosis in response to glucocorticoid injury. Some myonu-
CD
AB
Fig. 4. Immunohistochemical staining of Fas antigen and ISEL. Fas antigen is not detected in control muscle (A, ×200), and is positive
in the cytoplasm of the TA-treated soleus muscle (B, ×400). Fas antigen-positive muscle fiber (C, ×400) observed in the TA-treated
soleus muscle is also positive by ISEL (D, ×400).

Apoptosis in Steroid-Induced Myopathy 473
clei were stained positively by ISEL. Peripheral condensation
of nuclear chromatin, which appears at early stages of apop-
tosis and is distinct from necrotic cells, was observed by elec-
tron microscopy. Contrary to a widespread belief that differ-
entiated skeletal muscle undergoes only necrotic death,
apoptotic cellular death has been demonstrated in dys-
trophin-deficient muscle (20, 21) and differentiated skeletal
muscle after treatment with anabolic steroids (22).
Apoptosis in any particular cell lineage is mediated by
exogenous influences, such as survival factors and the genes
of the susceptible cell. One signal which leads to cell death
by apoptosis is the Fas gene product (23, 24). The Fas
molecule, synonymously referred to as APO-1, contains a
cytoplasmic death domainshared with the type I tumor
necrosis factor receptor (34). To date, apoptosis induced by
Fas antigen has been extensivley investigated in the lym-
phoid systems (23, 24). Immunohistochemical studies
demonstrated that Fas antigen was expressed on muscle
fibers from patients with various muscle wasting diseases,
but not in normal muscle cells (26, 27). However, whether
the muscle fiber that expressed the Fas antigen is involved
in apoptotic processes has not been fully investigated. In
this study, Fas antigen was expressed only in the TA-treated
soleus muscles. Fas antigen was mainly detected in the
cytoplasm of muscle fibers. Some Fas antigen-positive mus-
cle fibers showed positive reaction for ISEL, indicating
apoptosis. These findings suggest that Fas antigen expres-
sion might be related to apoptotic muscle death in steroid
myopathy. The mechanism of the Fas antigen activation in
the apoptotic process in steroid myopathy needs to be fur-
ther investigated.
The role of tumor suppressor gene p53 in the process of
apoptosis induced by different stimuli has been studied.
Specifically, radiation-induced apoptosis has been demon-
strated to be strictly p53-dependent (29). However, glu-
cocorticoid-triggered apoptosis in immature thymocytes
was independent of p53 (35). We could not find the evi-
dences for involvement of p53 protein in the apoptotic pro-
cess in steroid myopathy.
In this study, the number of apoptotic myonuclei ranged
from 1 to 4. Considering the facts that apoptosis is a rapid
process and that ISEL method can only detect apoptotic
nuclei for approximately 1 to 3 hr following the onset of
apoptosis (30), the small number of apoptotic nuclei in this
study is not an indicator of insignificance of apoptosis in
steroid myopathy. The apoptotic cell death in steroid
myopathy is mediated by Fas antigen expression in this
study.
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