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B-crystallin distribution and its
shifts with T3 and PTU treatments in rat hindlimb
muscles
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-8902 Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Changes in 
B-crystallin content in
adult rat soleus and extensor digitorum longus (EDL) were examined
after 8 wk of 3,5,3'-triiodothyronine (T3) and
propylthiouracil (PTU) treatments. Cellular distributions of
B-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray
level of reactivity to specific anti-B-crystallin antibody. B-crystallin content in both soleus and EDL muscles was
significantly decreased after T3, and that in EDL was
significantly increased over twofold after PTU treatment. In both
control soleus and EDL muscles, the gray level of type I fibers was
higher than that of type II fibers. B-crystallin expression among
type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx 
IIa in soleus muscle.
The relation was basically unchanged in both muscles after
T3 treatment and was, in particular, well maintained in EDL
muscle. Under hypothyroidism conditions with PTU, the mean
B-crystallin levels of type IIa and IIx fibers were significantly
lower than levels under control conditions. Thus the
relation between fiber type and the expression manner of stress protein
B-crystallin is muscle specific and also is well regulated under
thyroid hormone, especially in fast EDL muscle.
stress protein; immunohistochemistry; thyroid hormone; propylthiouracil; 3,5,3'-triiodothyronine
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THERE ARE MANY TYPES of heat shock proteins (HSPs) that
are constitutively expressed in both prokaryotic and eukaryotic cells. HSPs play fundamental roles, such as transcriptional regulation, nascent protein folding, and protein transport to endoplasmic reticulum
and/or mitochondria (28). B-crystallin is one of the small HSPs
(sHSPs) and can function as a molecular chaperone (20). B-crystallin
not only is a major lens protein but also is expressed in other tissues
such as heart, skeletal muscle, and kidney (5, 7, 13, 15, 23, 25). In
skeletal muscle, some reports indicated that HSP70 (31) and
B-crystallin are constitutively expressed at levels higher in the
slow-twitch muscles than in the fast-twitch muscles (4, 25). This
higher expression of HSPs in the slow-twitch muscles might be related
to their higher metabolic and protein turnover rates compared with
fast-twitch muscles (34). Locke et al. (29) reported that
the shift in the type I fiber composition to type II composition in rat
hindlimb muscle after 3,5,3'-triiodothyronine (T3)
treatment or incapacitation of synergistic muscles was accompanied by
changes in HSP72 content, which suggests a significant relationship
between HSP72 content and the composition of the type I muscle fiber
myosin heavy chain (MHC). However, there is no study showing a
precise relationship between the fiber type and the
expression of stress proteins in the skeletal muscle.
In skeletal muscle, B-crystallin is expressed at a higher
level in slow-twitch soleus muscles and heart compared with
fast-twitch plantaris and extensor digitorum longus (EDL) muscles (4,
5, 22). We have found that B-crystallin specifically decreases in
atrophied soleus muscle, but the expression of B-crystallin can
be sustained if the muscle is passively stretched (3, 4). Recently, it
was reported that the expression of B-crystallin is related to
maintaining the stability of the cytoskeleton (2, 23, 33).
B-crystallin localizes at Z bands in skeletal muscle (5)
where many cytoskeleton-relating proteins assemble and mechanical
stress is intensively transferred. If we consider the function of
stress protein as a chaperone, possible fiber-type-dependent expression of B-crystallin might indicate differences in its structure and function related to cytoskeletons, depending on the differences in the actomyosin contracting system in the
skeletal muscle.
In this study, the relationship between B-crystallin expression and
the muscle fiber type was precisely investigated by means of an
immunostaining technique using anti-myosin monoclonal antibodies and an
anti-B-crystallin antibody in rat skeletal muscle. We also examined
the relationship between B-crystallin and muscle fiber type after
fiber transformation with T3 and propylthiouracil (PTU)
treatments, which induce shifts in the expression of MHC isoforms (17,
26). Here, we show that the expression of B-crystallin significantly
decreased after T3 treatment in both soleus and EDL muscles
and significantly increased after PTU treatment in EDL muscle. The
intensity of B-crystallin for each fiber in EDL muscle is
systematically related to fiber type, and the relationship is better
maintained under the influence of thyroid hormone.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals.
The same experiments were performed twice for biochemical
(experiment 1) and immunohistochemical (experiment 2)
analyses of B-crystallin expression. Adult male Wistar rats weighing
201-229 g (n = 18 × 2) were randomly assigned to one
of three groups designated control, hyperthyroid (T3), and
hypothyroid (PTU). All animals were provided with standard rat chow ad
libitum and were housed at room temperature with a 12:12-h light-dark
cycle. The rats assigned to the T3 group were injected
subcutaneously with 300 µg/kg body wt of T3 (sodium salt)
every other day. The rats in the PTU group were supplemented with 0.1%
PTU in drinking water throughout the 8-wk experimental period. After 8 wk, the rats were anesthetized with diethyl ether, and then the EDL and
soleus muscles were dissected. The conditions of these two experiments were the same, except that the animals in experiment 1 were
older than those in experiment 2 by 1 wk.
Purification of B-crystallin.
For measuring the protein content of B-crystallin in muscle
homogenate, B-crystallin was purified from bovine lens, which is
detailed in a previous study (1).
Preparation of antibodies.
Antibody C1 was raised against COOH-terminal (SH)KPAVTAAPKK peptides of
rat B-crystallin (synthesized and purified by Dr. S. Aimoto,
Research Center for Protein Engineering, Institute for Protein
Research, Osaka University, Osaka, Japan) conjugated to BSA with
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce Chemical, Rockford, IL) (35). IgG, precipitated by 50% NH4SO4 and dialyzed against PBS, was purified
by using COOH-terminal peptide-conjugated affinity chromatography using
MBS linker and EAH Sepharose 4B (Pharmacia Biotech, Shinagawa, Japan).
Preparation of muscle samples for SDS-PAGE and immunoblot analyses.
The procedure followed Atomi et al. (4, 5) with a slight modification.
Muscles were dissected out, immediately frozen in liquid nitrogen, and
stored at 
70°C until use. Muscle samples were crushed in
liquid nitrogen and were directly solubilized in low-salt buffer
containing 20 mM KCl, 2 mM sodium phosphate, pH 6.8, 2 mM EGTA, and 0.1 mM diisopropyl fluorophosphate. Protein concentration was determined
with bicinchoninic acid protein assay kit using BSA as standard. Total
muscle homogenates and supernatant solutions after centrifugation
(soluble fractions) were solubilized with an equal volume of 2×
SDS sample buffer (27) and heated for 2 min in boiling water. Transfer
of proteins to nitrocellulose membrane and immunoblotting were
performed in accordance with the method of Towbin et al. (43) with a
slight modification (5). An anti-B-crystallin antibody was generally
used at 1:5,000 dilution. Immunoblots were visualized by the use of an
enhanced chemiluminescence kit (Amersham International, Bucks, UK).
Measurement of B-crystallin protein contents of
experimental muscles.
The protein contents of B-crystallin in total muscle homogenates in
control, T3, and PTU groups (experiment 1) were
calculated from the measurement of an area of the band corresponding to
B-crystallin in the gel of SDS-PAGE stained with Coomassie brilliant
blue (CBB), and a calibration curve was obtained by using four to five
different quantities of purified B-crystallin (from 0 to 0.54 µg/lane) per each gel. The relation between CBB-stained area
[obtained by National Institutes of Health (NIH) Image] and the
concentration of B-crystallin was approximated with a quadratic
equation (r2 = 0.99). The gradient gels (at
5-20% acrylamide concentration) were used for the measurement of
B-crystallin protein content. Twenty micrograms of total muscle
homogenate were applied to each lane. The band concerned was clearly
separated in the gradient gel and identified to be B-crystallin with
Western blotting against anti-B-crystallin antibody.
Immunohistochemistry.
For immunohistochemical analysis, small pieces from the midsection of
the muscles were then frozen in melting isopentane and stored at
70°C. Cross sections were cut at 10 µm thickness on a
cryostat microtome at 25°C. Muscle sections were
sequentially incubated in PBS for 10 min, 1.5% goat serum in PBS for
30 min, anti-B-crystallin Ig for 1 h, PBS for 10 min, FITC-labeled
goat anti-rabbit IgG (TAGO, Burlingame, CA) at room temperature for 1 h, and PBS for 10 min. The sections were mounted in
glycerol-para-phenylenediamine and were examined with
epifluorescent illumination with a Zeiss Axioplan photomicroscope.
Controls using preimmune rabbit serum did not show any significant
staining. For analysis of immunohistochemical staining, sections were
visualized with a video camera attached to a microscope, and images
were processed with a microcomputer-based image analysis system (IBAS,
Carl Zeiss, Oberkochen, Germany). More than 250 muscle fibers were
digitized per muscle using a computer that calculated the average gray
level of pixels in a circle drawn within each fiber. The gray level of
each fiber was measured by pixel at 0-256 graduation. This
graduation of gray level was determined relative to the sections with
the highest and the lowest pixilation. Type I fibers demonstrated the
highest level, and the lowest level was observed mostly in type IIb fibers.
Skeletal muscle fiber-type classification.
To compare the staining pattern of B-crystallin in a muscle composed
of different fiber types, 10-µm sections were consecutively cut and
mounted on slides. These consecutive sections were then incubated in
the five different antibodies. The specific activities of the five
antibodies [BF-G6 (39), BF-35, SC-71 (40), BA-F8, and BF-13
(8)] are shown in Table 1. Four fiber
types were designated as type I, type IIa, type IIx, and type IIb.
These monoclonal antibodies were a generous gift from Dr. S. Schiaffino, Department of Biomedical Science, University of Padova
(Padova, Italy). Bound antibodies were revealed by FITC-labeled goat
anti-mouse IgG.
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Statistical analysis. Data are given as means ± SD of number of fibers (n). Statistical analysis of data was performed according to ANOVA, Student's unpaired t-test, or its modified method [by Welch and Aspin (see Ref. 41)]. Differences at P < 0.05 were regarded as significantly different.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Antibody characteristics.
The polyclonal antibody against COOH-terminal peptide of
B-crystallin purified by peptide affinity chromatography
specifically reacted to a 22-kDa B-crystallin in the homogenates of
rat soleus and EDL muscles (Fig. 1).
Criteria to determine fiber type with monoclonal antibodies against
MHCs produced by Dr. Schiaffino were well ascertained in this study for
the skeletal muscles of Wistar rats (Table 1).
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Quantification of B-crystallin in soleus and EDL
muscles.
In experiment 1, the B-crystallin content in soleus and EDL
muscles was determined by the intensity stained with CBB and calculated
using NIH Image software. B-crystallin content of EDL
muscles was 0.8% of that of soleus muscles and roughly agreed with the
ratio by immunoassay (22).
Physical characteristics of rats after EDL and soleus muscles were
treated with T3 and PTU.
Body weights after the 8-wk experiment were significantly lower in
T3- and PTU-treated animals than in the control rats (Table 2). The soleus muscle weight significantly
decreased in PTU-treated animals, whereas the EDL muscle weight
significantly decreased in both T3 and PTU groups. This
finding was consistent with previous studies of thyroid hormone-treated
rats (17).
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Specific changes of B-crystallin after
T3 and PTU treatment in soleus and EDL muscles.
The B-crystallin protein content in the total muscle homogenate in
the experimental groups was calculated from the areas in SDS-PAGE
stained with CBB (Fig. 2,
Aa and Ab). The approximation with a quadratic
equation used for this calculation was obtained from the areas for five
different contents of purified B-crystallin. The relation between
them became linear after logarithm exchange. Typical examples of
B-crystallin in total muscle homogenate of soleus and EDL muscles in
control, T3, and PTU groups subjected to SDS-PAGE and
Western blotting are shown in Fig. 2B. The B-crystallin content in total muscle homogenate, identified by Western blotting and
estimated using the standard calibration curve for B-crystallin with
Western blotting, was similar to the mean values obtained by CBB
staining. The mean B-crystallin protein content was significantly lower in both T3-treated soleus (17%) and EDL (55%)
muscles than in those of the control group (Fig. 2C). Although
mean B-crystallin protein content after PTU treatment did not
significantly change in soleus muscle, it significantly increased over
twofold in EDL muscle (150%).
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Fiber-type frequencies of the EDL and soleus muscles in control and
in T3- and PTU-treated rats.
About 10% of 250 fibers examined in the experimental groups reacted to
antibodies against more than two MHC isoforms. These fibers were not
included in the fiber-type distribution analysis in this study.
Fiber-type frequencies of EDL and soleus muscles in control,
T3, and PTU groups are shown in Table
3. After T3 treatment, fiber
frequencies of type I and type IIx significantly decreased in EDL
muscle. In soleus muscle, fiber frequencies of type I significantly
decreased to a level that was similar to that found in EDL muscle,
whereas type IIa levels increased after hyperthyroidism was induced. On
the other hand, significant increases in type I and IIa and significant
decreases in type IIx and IIb were observed in hypothyroid EDL muscle.
Significantly decreased type IIa fiber frequencies were observed in
PTU-treated soleus muscles. These muscle-type-specific changes of fiber
type after T3 and PTU treatments are consistent with the
results obtained in thyroid hormone-treated rats (17).
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Serial relation of B-crystallin expression to fiber
type in control soleus and EDL muscles.
The relationship between the expression of B-crystallin and MHC
isotypes was examined in control soleus and EDL muscles (Fig. 3). Because the staining intensity against
anti-B-crystallin antibody was different between fibers, the
relationships between the reactivity to anti-B-crystallin antibody
and the reactivity to anti-MHC antibodies were examined. Type I fibers
showed a stronger reactivity to anti-B-crystallin IgG than did type
II fibers in both soleus and EDL muscles. The differences in the
reactivity to anti-B-crystallin IgG among the fast subtypes were
faint in soleus muscle and considerable in EDL muscle. In soleus
muscle, the staining intensities of type IIa and IIx fibers were almost identical but that of type IIx was slightly higher than that of type
IIa (Fig. 3A, top). In EDL muscle, type IIa, IIx, and IIb fibers showed moderate, weak, and faint immunoreactivity, respectively (Fig. 3A, bottom). The distributions of the gray levels
of B-crystallin per fiber type in EDL muscle (Fig. 3A,
bottom) reinforced the above results. From these observations, it
was suggested that the expression of B-crystallin in rat skeletal
muscles varies systematically with fiber type; however, this variation
in expression might be muscle specific.
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Maintained fiber-type-specific B-crystallin
expression in hyperthyroidism.
Typical sections of soleus and EDL muscles from control and
experimental groups stained with anti-B-crystallin antibody are shown in Fig. 4A. Most of the
fibers of soleus muscles both in control and T3 groups
(except for an unknown few in control soleus muscles) were classified
into only two groups of type I and type IIa. The gray levels of type I
fibers in soleus muscle were consistently higher than those of type IIa
fibers in other areas in the muscle sections of the T3
group (data not shown). Few fibers expressed type IIx, although the
number of fibers expressing both type IIa and type IIx increased after
T3 treatment. Although the difference in the
intensities of the gray level between type IIa and IIx was very small,
any intensity of the gray level of five type IIx fibers observed in the
areas where type IIx and IIa fibers were closely located was slightly
higher than that of type IIa. Because the differences among the gray
levels of type I, IIa, and IIx fibers and the numbers of type IIx were
too small, especially in the soleus muscle treated with T3
or PTU, statistical analysis was not performed for the data. From these
observations for both control and T3 groups, the
intensities of anti-B-crystallin antibody in soleus muscles were
greater in the order of type I 
type IIx type IIa. The relative
percentages of the mean gray values of these five type IIx fibers and
four type IIa fibers found near areas of T3 soleus were
~75% and 62% respectively, compared with the mean gray values for
nine type I fibers (100%).
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B-crystallin
expression, of the different fiber-type groups for EDL muscles in the
all control, T3, and PTU groups were significant (P < 0.001, Table 4). This means that the
averages of the gray levels among three type II fiber groups were
statistically different from each other. After T3
treatment, the mean gray level of B-crystallin was significantly
less in the type IIa group and was significantly larger in the type IIb
group compared with results shown in controls. The relation between the
B-crystallin expression and fiber type observed in control EDL
muscles was consistently maintained in T3 EDL muscles.
Distributions of the gray levels after T3 treatment were
almost the same as those in the control group for EDL (Fig. 4, A-C,
right). The orders of intensities of the gray levels in control
groups with reference to fiber type did not change in both soleus and
EDL muscles.
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Hypothyroidism condition depressed B-crystallin
expression levels in type IIa and IIx in EDL muscle.
Twenty percent of fibers were uncharacterized in PTU-treated soleus
muscle. Most of the fibers in soleus muscles were included and
classified into two groups of type I and type IIa as shown in Table
3. The gray levels of type I fibers in PTU-treated fibers of soleus muscle were consistently higher than those of type IIa fibers
in these groups. More precise examination was not performed for soleus
muscle. In EDL muscle, the mean B-crystallin levels of type IIa and
IIx were significantly less (45% and 57% for control) in the PTU than
in the control group (Table 4, Fig. 4). This means that
fiber-type-specific expression of B-crystallin is maintained only
under the influence of thyroid hormone in both soleus and EDL muscles.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Immunohistochemical fiber typing. Both immunohistochemical and electrophoretical analyses have revealed that most of the fibers express only a single type of MHC and can therefore be classified as type I, IIa, IIb, and IIx fibers. Only a few contain detectable levels of two MHC isoforms (11, 16, 38, 42). Monoclonal antibodies used in this study could not detect the coexistence of type IIa with type IIx or type IIx with type IIb MHC because none of them did specifically stain type IIx MHC. Type IIx MHC was identified by negative staining with the other antibodies used. Some fibers identified as type IIa or type IIb might contain various amounts of type IIx MHC isoform coexisting with a predominant one. It is not known whether anti-MHC antibodies used in this experiment react to embryonic and neonatal MHC isoforms. Therefore, a possible induction of these nonadult myosin isoforms under hypothyroidism (a reduction in circulating T3 level) (26) could not be detected in this experiment.
Evaluation of B-crystallin expression by
immunoreactivity of each fiber.
Although qualitative classification with immunocytochemical and
histochemical techniques is available for fiber typing in skeletal
muscle, it is difficult to quantitatively evaluate protein content per
fiber with reference to fiber type by MHC expression. Possible reasons
for this are as follows. 1) The thickness of muscle sections is
not perfectly constant. 2) The reactivity of the protein in the
muscle sections, therefore, is not necessarily constant. 3)
Even if they are constant, there is some technical difficulty in
staining the fibers equally. 4) Distribution of muscle fibers
in a muscle section is usually mosaic and variable, especially under
various physiological conditions. All these could induce variations in
the reaction to antibody IgG, even if a given protein content per
muscle fiber is reacted to a given IgG content. For these reasons, in
the present study, we selected muscle fibers in the areas where there
was relatively constant staining, to quantify B-crystallin from the
gray level of each muscle section, and we classified them with respect
to fiber type in each experimental muscle.
Effects of thyroid state on MHC gene expression. It is useful to examine the changes of protein expression relating to muscle fiber type under the various thyroid hormone levels, which directly and/or indirectly control expressions of muscle-specific genes. Hypothyroidism induces alterations in MHC phenotypes consisting of an increased expression of the slow type I MHC isoform, coupled with a decreased expression of the fast type IIa and IIx MHC. These alterations vary depending on muscle type (26). In this study, the changes of fiber type were evaluated by anti-isomyosin antibodies with T3 and PTU treatments for 8 wk. In slow soleus muscle, PTU-induced hypothyroidism caused a slight increase in the expression of type I MHC and a decrease in the expression of type IIa/IIx MHCs. In fast-twitch EDL muscle, hypothyroidism caused increases in type I and IIa MHC expression coupled with a decrease in type IIx and IIb MHC expression.
These results were consistent with previous results in rat soleus muscle (26) and in the fast-twitch plantaris muscle (17). In contrast, hyperthyroidism of hindlimb skeletal muscles induces the upregulation of type IIa/IIx MHC expression of the fibers in the soleus muscle and induces the downregulation of both type I and IIa/IIx MHC isoforms as well as a concomitant increase in the expression of type IIb MHC isoform in plantaris muscles (17). These findings about changes after thyroid hormone treatment agreed with the previous results (17, 26). The results in the present study also roughly agreed with the assumption that the expression ofB-crystallin in skeletal muscle
might be dependent on the similar mechanism to the changes of
fiber-type, from the response to T3/PTU treatment.
The thyroid hormone can alter the transcriptional rate of a gene by
direct interaction with a thyroid hormone receptor-thyroid hormone
responsive element (TR-TRE) of the target gene promoter. TR-TRE has
been found in the promoter regulatory regions of several genes
expressed in skeletal muscle including those genes for type I MHC (14),
-actin (10), sarcoplasmic reticulum Ca2+-ATPase pump
(19), and myogenic factors of MyoD and myogenin (12). MyoD mRNA and
myogenin mRNA preferentially accumulate in fast- and slow-twitch
muscles, respectively (21).
Effects of thyroid hormone on the expression of
B-crystallin in soleus and EDL muscles.
Biochemical analysis in the present study showed that 8-wk
T3 treatment significantly decreased the expression of
B-crystallin in both soleus and EDL muscles. Further PTU treatment
increased it over twofold in EDL muscle, although significant change
was not observed in soleus muscle. The difference of reactivity in muscles may be related to other factors, such as Ca2+
sensitivity (9), myogenic factor (21), and possibly the mechanical
environment, which may regulate myosin expression.
T3-regulated fiber-type transformation and
B-crystallin expression.
Interestingly, a complete serial order in the quantity of
B-crystallin expression in EDL muscle, that is, I > IIa > IIx > IIb, matches the sequence of fiber-type transition inferred from studies of MHC expression in normal and electrically stimulated muscles
(16, 38). Furthermore, this relationship received some modifications
after the withdrawal of thyroid hormone in PTU treatment but almost
unchanged under T3. This means that the relation between
B-crystallin and fiber type is well maintained under physiological
influence of thyroid hormone. There is at least one E box in MRE, the
muscle-specific regulatory region, in the upstream of B-crystallin
gene (15), but no TRE was found there. Therefore, possible indirect
regulation for B-crystallin expression by thyroid hormone might be related.
Possible physiological significances of strict relation between
sHSP, B-crystallin, and fiber type.
Previously, we have found that a 22-kDa protein that specifically
decreases in slow-twitch muscle atrophy is B-crystallin. B-crystallin does not decrease by hindlimb suspension with passive stretch (4). B-crystallin localizes at Z disks of glycerinated myofibrils (5). Although the function of B-crystallin in skeletal muscle is still unknown, a recent study (20) showed the role of
B-crystallin as a molecular chaperone. One possible
function of B-crystallin in slow-twitch muscle is a chaperone for
proteins that are easily denatured. Because slow-twitch muscles have
higher metabolic and protein turnover rates compared with fast-twitch muscles (34), it seems that slow-twitch muscles may have an essential
need for a chaperone system.
B-crystallin completely
associates with tubulin/microtubule and intermediate filament in L6
myoblast cells (unpublished data) and that B-crystallin binds
temperature dependently to the tubulin molecule by immunoprecipitation with anti-B-crystallin antibody and can suppress tubulin aggregation by complex formation (1). It is well known that tubulin is a labile
protein that is easily denatured even in physiological condition.
Together, the strict correlation between the expression of
B-crystallin and fiber type in the present study suggests that the
function of B-crystallin keeps dynamic stability and/or metabolic
activity in skeletal muscle, particularly in slow-twitch muscle.
The amino acid sequence of B-crystallin has a striking similarity to
the sequence of sHSPs, and increasing evidence indicates that
B-crystallin has a function similar to sHSPs. The expression of
B-crystallin as well as sHSPs is induced by various stresses (37).
Recently it was found that overexpression of sHSPs, including B-crystallin, can prevent cells from dying by reactive oxygen species (ROS) induced by tumor necrosis factor- and stimuli of ROS-inducing reagents (32). From these observations and from the fact
that slow-twitch soleus muscle is continuously subjected to much more
stress, including high temperature, oxidant injury, a higher rate of
protein turnover, and continuous contraction with passive stretching
compared with the other skeletal muscles, it seems reasonable to assume
that the constitutive expressions of B-crystallin/sHSP and other
HSPs (described below) are required in slow-twitch muscle.
Other stress proteins in muscle.
Although many stressors can induce HSP synthesis in various cultured
cells from bacteria to humans, only a few studies have demonstrated the
function of mammalian HSPs in vivo. Studies on diseases causing the
abnormal expression of HSPs have been carried out in recent years (6).
Increased expression of HSPs has been demonstrated in vivo by heat
shock in anesthetized animals (18, 36). Physiological stress during
treadmill running to exhaustion induces syntheses of HSP72 and HSP90 in
rat peripheral lymphocytes, spleen cells, and soleus muscles (29). In
addition, HSP70 is constitutively expressed in soleus muscle (30) and
shows type I MHC-dependent expression (31). Such expression patterns
and the localization and their changes of stress protein, including HSP70 and B-crystallin in the previous (2, 4), and the present
results suggest the existence of unknown characteristics that could
explain different phenotypes of skeletal muscle fibers.
B-crystallin expression was muscle
specific and fiber-type specific in both soleus and EDL muscles from
the histochemical analysis using specific antibodies for myosins and
B-crystallin and was significantly lower under T3 treatment in both muscles. This relation was well maintained in physiological conditions under thyroid hormone influence.
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ACKNOWLEDGEMENTS |
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We sincerely thank Dr. Stefano Schiaffino for his generous gift of antibodies against MHCs and for critical reading of the manuscript.
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FOOTNOTES |
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This study was supported in part by a Grant-in-Aid of Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, by a Fund for Basic Experiments Oriented to Space Station Utilization from Institute of Space Astronomical Science, and by a Fund for Scientific Experiments Oriented to Mechanical Stimulus in Biology from Japan Proportion and Beauty Science Institute.
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: Y. Atomi, Dept. of Life Sciences, Graduate School of Arts and Sciences, The Univ. of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902 Japan (E-mail: atomi{at}idaten.c.u-tokyo.ac.jp).
Received 7 July 1998; accepted in final form 14 January 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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