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J Appl Physiol 88: 1355-1364, 2000;
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Vol. 88, Issue 4, 1355-1364, April 2000

Fiber-type-specific alpha B-crystallin distribution and its shifts with T3 and PTU treatments in rat hindlimb muscles

Yoriko Atomi, Kyoko Toro, Tsuyoshi Masuda, and Hideo Hatta

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-8902 Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in alpha 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 alpha 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-alpha B-crystallin antibody. alpha 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. alpha 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 alpha 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 alpha 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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). alpha B-crystallin is one of the small HSPs (sHSPs) and can function as a molecular chaperone (20). alpha 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 alpha 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, alpha 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 alpha B-crystallin specifically decreases in atrophied soleus muscle, but the expression of alpha B-crystallin can be sustained if the muscle is passively stretched (3, 4). Recently, it was reported that the expression of alpha B-crystallin is related to maintaining the stability of the cytoskeleton (2, 23, 33). alpha 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 alpha 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 alpha 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-alpha B-crystallin antibody in rat skeletal muscle. We also examined the relationship between alpha 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 alpha 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 alpha 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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. The same experiments were performed twice for biochemical (experiment 1) and immunohistochemical (experiment 2) analyses of alpha 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 alpha B-crystallin. For measuring the protein content of alpha B-crystallin in muscle homogenate, alpha 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 alpha 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-alpha 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 alpha B-crystallin protein contents of experimental muscles. The protein contents of alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha B-crystallin with Western blotting against anti-alpha 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-alpha 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 alpha 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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  		Table 1.   Staining intensities in rat muscle reacted to antibodies against MHC isoforms

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Antibody characteristics. The polyclonal antibody against COOH-terminal peptide of alpha B-crystallin purified by peptide affinity chromatography specifically reacted to a 22-kDa alpha 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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  		Fig. 1.   Characteristics of anti-alpha B-crystallin COOH-terminal peptide antibody. Rat total proteins of soleus and extensor digitorum longus (EDL) muscle homogenates (lanes 1 and 2) were subjected to SDS-PAGE and followed by staining with Coomassie brilliant blue (CBB) (A, lanes 1 and 2; 7.5 µg per each lane) or immunoblotted with anti-alpha B-crystallin COOH-terminal peptide antibody (B, lanes 3 and 4; 1 µg per each lane). Molecular mass markers (lane M) are phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and alpha -lactoalbumin (144 kDa).

Quantification of alpha B-crystallin in soleus and EDL muscles. In experiment 1, the alpha B-crystallin content in soleus and EDL muscles was determined by the intensity stained with CBB and calculated using NIH Image software. alpha 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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  		Table 2.   Mean body weight and muscle mass of rats in control, T3, and PTU groups for biochemical (experiment 1) and immunohistochemical (experiment 2) analyses

Specific changes of alpha B-crystallin after T3 and PTU treatment in soleus and EDL muscles. The alpha 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 alpha B-crystallin. The relation between them became linear after logarithm exchange. Typical examples of alpha 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 alpha B-crystallin content in total muscle homogenate, identified by Western blotting and estimated using the standard calibration curve for alpha B-crystallin with Western blotting, was similar to the mean values obtained by CBB staining. The mean alpha 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 alpha 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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  		Fig. 2.   Quantification of alpha B-crystallin content in total muscle homogenate and effects of thyroid hormone 3,5,3'- triiodothyronine (T3) and propylthiouracil (PTU) treatments on alpha B-crystallin content of soleus and EDL muscles. A: sample used to make a standard curve to measure alpha B-crystallin content in hindlimb muscles. a: Five levels of content of purified alpha B-crystallin (lanes 1-5; 0.068, 0.136, 0.272, 0.408, and 0.544 µg per each lane) in SDS-PAGE stained by CBB. b: Approximation with a quadratic equation obtained from 5 different contents of purified alpha B-crystallin shown in a. c: Regression equation exchanged from a quadratic equation shown in b. B: rat total proteins of soleus (lanes 1-3 and 7-9; 2 µg per each lane) and EDL (lanes 4-6 and 10-12; 2 µg per each lane) muscle homogenates of control (lanes 1 and 4), T3 (lanes 2 and 5), and PTU (lanes 3 and 6) treatments were subjected to SDS-PAGE (lanes 1-6) and followed by immunoblotting with anti-alpha B-crystallin COOH-terminal peptide antibody (lanes 7-12). Lane A shows alpha B-crystallin (137 ng) in SDS-PAGE (left) and in Western blotting (right). Molecular mass markers (lane M) are myosin (203 kDa), beta -galactosidase phosphorylase (123 kDa), BSA (83 kDa), ovalbumin (50.7 kDa), carbonic anhydrase (35.7 kDa), soybean trypsin inhibitor (29.6 kDa), and lysozyme (21.9 kDa). C: mean alpha B-crystallin protein content of soleus (a) and EDL (b) muscles measured by CBB staining were compared among control (lane 1), T3 (lane 2), and PTU (lane 3) treatments. Graphs show means and SD of alpha B-crystallin content of each experimental group. Significant differences at ** P < 0.01 and *** P < 0.001 for control groups.

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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  		Table 3.   Fiber-type frequencies after thyroid T3 and PTU treatments

Serial relation of alpha B-crystallin expression to fiber type in control soleus and EDL muscles. The relationship between the expression of alpha B-crystallin and MHC isotypes was examined in control soleus and EDL muscles (Fig. 3). Because the staining intensity against anti-alpha B-crystallin antibody was different between fibers, the relationships between the reactivity to anti-alpha B-crystallin antibody and the reactivity to anti-MHC antibodies were examined. Type I fibers showed a stronger reactivity to anti-alpha B-crystallin IgG than did type II fibers in both soleus and EDL muscles. The differences in the reactivity to anti-alpha 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 alpha 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 alpha B-crystallin in rat skeletal muscles varies systematically with fiber type; however, this variation in expression might be muscle specific.


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  		Fig. 3.   Comparisons of reactivities to anti-alpha B-crystallin and myosin heavy chain (MHC) isoform-specific monoclonal antibodies in serial sections of rat soleus and EDL muscles with immunohistochemical staining. Top: photographs of soleus muscle stained with the anti-alpha B-crystallin COOH-terminal peptide antibody (A) and monoclonal antibodies against MHC isoforms: F8 (B), 13 (C), 71 (D), G6 (E), and 35 (F). Numbers in photographs of soleus muscle show MHC isoforms: 1 = type I, 2 = type IIa, and 3 = type IIx. Most of the fibers in the soleus muscle were included into type I and type IIa isoforms, but only a few fibers showing as 3 could be assigned as type IIx. Bottom: photographs of EDL muscle stained with the anti-alpha B-crystallin COOH-terminal peptide antibody (A) and monoclonal antibodies against MHC isoforms: F8 (B), 13 (C), 71 (D), G6 (E), and 35 (F). Numbers in photographs show MHC isoforms: 1 = type I, 2 = type IIa, 3 = type IIx, and 4 = type IIb. Fibers were classified by the criteria compiled in Table 1. FITC-stained interstitial areas in photographs (C and E) are derived from artifacts due to the weakness of antibodies (BF-13, BF-G6). Scale bar, 50 µm.

Maintained fiber-type-specific alpha B-crystallin expression in hyperthyroidism. Typical sections of soleus and EDL muscles from control and experimental groups stained with anti-alpha 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-alpha 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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  		Fig. 4.   Effects of thyroid hormone T3 and PTU treatments on the reactivities to the anti-alpha B-crystallin COOH-terminal peptide antibody. Left: immunohistochemically stained rat soleus (A, C, E) and EDL (B, D, F) muscles with the anti-alpha B-crystallin antibody from control (A and B), T3 (C and D) and PTU (E and F) groups. Scale bar, 50 µm. Right: histograms showing the distribution of gray level of immunohistochemical staining intensity, as a typical representative of 6 rat EDL muscles showing same staining pattern, for alpha B-crystallin among 250 fibers. A, B, and C: histograms for control, T3, and PTU experimental groups, respectively. Staining intensity in individual muscle fibers correlates with fiber types. x-axis, fiber type; y-axis, average gray levels on a scale where 0 = black and 255 = white; z-axis, number of fibers.

The mean gray levels, showing the intensities of alpha 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 alpha 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 alpha 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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  		Table 4.   Mean gray level of the reactivity to anti-alpha B-crystallin antibody in control, T3, and PTU groups in EDL muscles

Hypothyroidism condition depressed alpha 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 alpha 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 alpha B-crystallin is maintained only under the influence of thyroid hormone in both soleus and EDL muscles.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha B-crystallin from the gray level of each muscle section, and we classified them with respect to fiber type in each experimental muscle.

The analysis using BF-35 antibody, which detects type IIx by negative reactivity to type IIx, was available for EDL muscle but not for soleus muscle. The reason for the difference in the reactivity between soleus and EDL muscles is unknown at present. Therefore, the classification of type IIx in soleus muscle was performed by the comparison of reactivity to the other four anti-myosin antibodies. From the difficulty to quantify the relation in soleus muscle, the above-mentioned results of the relation observed for soleus muscles were described only in the text.

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 of alpha B-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), alpha -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 alpha B-crystallin in soleus and EDL muscles. Biochemical analysis in the present study showed that 8-wk T3 treatment significantly decreased the expression of alpha 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 alpha B-crystallin expression. Interestingly, a complete serial order in the quantity of alpha 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 alpha 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 alpha B-crystallin gene (15), but no TRE was found there. Therefore, possible indirect regulation for alpha B-crystallin expression by thyroid hormone might be related.

Possible physiological significances of strict relation between sHSP, alpha B-crystallin, and fiber type. Previously, we have found that a 22-kDa protein that specifically decreases in slow-twitch muscle atrophy is alpha B-crystallin. alpha B-crystallin does not decrease by hindlimb suspension with passive stretch (4). alpha B-crystallin localizes at Z disks of glycerinated myofibrils (5). Although the function of alpha B-crystallin in skeletal muscle is still unknown, a recent study (20) showed the role of alpha B-crystallin as a molecular chaperone. One possible function of alpha 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.

It is found in our recent study that alpha B-crystallin completely associates with tubulin/microtubule and intermediate filament in L6 myoblast cells (unpublished data) and that alpha B-crystallin binds temperature dependently to the tubulin molecule by immunoprecipitation with anti-alpha 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 alpha B-crystallin and fiber type in the present study suggests that the function of alpha B-crystallin keeps dynamic stability and/or metabolic activity in skeletal muscle, particularly in slow-twitch muscle.

The amino acid sequence of alpha B-crystallin has a striking similarity to the sequence of sHSPs, and increasing evidence indicates that alpha B-crystallin has a function similar to sHSPs. The expression of alpha B-crystallin as well as sHSPs is induced by various stresses (37). Recently it was found that overexpression of sHSPs, including alpha B-crystallin, can prevent cells from dying by reactive oxygen species (ROS) induced by tumor necrosis factor-alpha 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 alpha 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 alpha 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.

In summary, stress protein alpha 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 alpha B-crystallin and was significantly lower under T3 treatment in both muscles. This relation was well maintained in physiological conditions under thyroid hormone influence.


    ACKNOWLEDGEMENTS

We sincerely thank Dr. Stefano Schiaffino for his generous gift of antibodies against MHCs and for critical reading of the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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