Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat

Laurence Stevens1, Carole Firinga1, Bärbel Gohlsch2, Bruno Bastide1, Yvonne Mounier1, and Dirk Pette2

1 Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France; and 2 Faculty of Biology, University of Constance, D-78457 Constance, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the plasticity of slow and fast muscles undergoing slow-to-fast transition, rat soleus (SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscles were exposed for 14 days to 1) unweighting by hindlimb suspension (HU), or 2) treatment with the beta 2-adrenergic agonist clenbuterol (CB), or 3) a combination of both (HU-CB). In general, HU elicited atrophy, CB induced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoforms revealed HU- and CB-induced slow-to-fast transitions in SOL (increases of MHCIIa with small amounts of MHCIId and MHCIIb) and the upregulation of the slow MHCIa isoform. The HU- and CB-induced changes in GAS consisted of increases in MHCIId and MHCIIb ("fast-to-faster transitions"). Changes in the MLC composition of SOL and GAS consisted of slow-to-fast transitions and mainly encompassed an exchange of MLC1s with MLC1f. In addition, MLC3f was elevated whenever MHCIId and MHCIIb isoforms were increased. Because the EDL is predominantly composed of type IID and IIB fibers, HU, CB, and HU-CB had no significant effect on the MHC and MLC patterns.

extensor digitorum longus; gastrocnemius; hindlimb suspension; myosin; slow-to-fast transition; soleus


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE HAS THE CAPACITY to change its phenotype, not only in response to altered functional demands, but also under the influence of specific growth factors and hormones. As shown in numerous studies, increased neuromuscular activity and loading both elicit fast-to-slow transitions and decreased neuromuscular activity, and unloading causes transitions in the reverse direction (22). Recently, the effects of mechanical unloading by hindlimb unweighting (HU) on the expression of myosin heavy chain (MHC) isoforms were investigated in rat soleus (SOL) muscle (30). In agreement with previous studies (e.g., 5, 8, 31), pronounced atrophy and slow-to-fast transitions in the MHC isoform pattern were observed. These transitions in MHC protein isoforms were preceded by corresponding changes at the mRNA level (28).

Clenbuterol (CB), a beta 2-adrenergic agonist, is known to induce muscle hypertrophy (9, 15, 33) and has been shown to counteract unloading-induced atrophy (1, 23, 33). Moreover, several studies have established that CB additionally induces slow-to-fast transitions in rat SOL muscle (7, 19, 23). Taking into account these effects, it could be expected that CB not only counteracts atrophy under conditions of unweighting but also enhances slow-to-fast transitions under the same conditions.

The validity of these assumptions was investigated in the present study on the effects of unweighting, CB treatment, and a combination of both on three different rat muscles, SOL, extensor digitorum longus (EDL), and the red portion of the gastrocnemius (GAS) muscle. It was of interest to determine the extent of the adaptive responses of these muscles because of their different fiber type composition and their different functions. The slow SOL as well as the fast GAS are antigravity muscles. The EDL is also fast, but is a nonpostural muscle.

Adult male rats were exposed to one of the following conditions: CB treatment for 14 days, unweighting by hindlimb suspension for 14 days, and CB treatment combined HU for 14 days. Control and experimental muscles were investigated for changes in weight and myosin composition. The analysis of myosin composition encompassed electrophoretic studies of both the MHC and myosin light chain (MLC) isoforms.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and muscles. Adult male Wistar rats (initial body wt ~260 g) were randomly divided into four groups: control, CB-treated, hindlimb unweighted (HU), and CB combined with hindlimb unweighting (HU-CB). The animal experiments, as well as the animal maintenance conditions, were approved by the French Ministries of Agriculture and Education (veterinary service of health and animal protection, authorization no. 03805). HU for 14 days was performed as previously described (29). CB (Sigma, St. Louis, MO) was administered via the drinking water (30 mg/l) for 14 days (23). It was freshly prepared every day. At the end of treatment, the animals were anesthetized by intraperitoneal injection of ethylcarbamate, killed by exsanguination, and EDL, GAS (red portion), and SOL muscles were dissected, blotted, and weighed (Table 1). Thereafter, the muscles were frozen in liquid N2 and stored at -70°C until analyzed.

                              
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Table 1.   Comparison of body and muscle weights among the different groups for soleus, gastrocnemius, and extensor digitorum longus muscles

MHC and MLC electrophoresis. Frozen muscle tissue was pulverized under liquid N2 in a small steel mortar (21). MLC isoforms were separated by one-dimensional electrophoresis according to Laemmli (16) using the protocol of Salviati et al. (25). The positions of the fast and slow MLC isoforms were identified on the gels by their apparent molecular masses. Immunoblotting with an antibody that recognized slow and fast troponin C isoforms (17) was used to delineate the separate bands of MLC2f and troponin Cslow (Fig. 1).


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Fig. 1.   One-dimensional separation of myofibrillar proteins from rat soleus (SOL) and gastrocnemius (GAS) muscles in a 10-20% SDS polyacrylamide gradient gel (silver stained). The position of the slow (MLC1s, MLC2s) and the fast (MLC1f, MLC2f, MLC3f) myosin light chains is indicated. The identity of bands representing fast and slow troponin C subunits TnCf and TnCs was proved by immunoblotting.

MHC isoforms were analyzed on a glycerol-containing 7% SDS polyacrylamide gel as previously described (13). The gels were silver stained (20) and evaluated by integrating densitometry. At least two measurements were performed on each sample.

Statistical analyses. Data are presented as means ± SD. After one-way analysis of variance (ANOVA), Student's t-test was used as a post hoc test to establish intergroup comparisons. The acceptable level of significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in body and muscle mass. CB did not affect body mass, however, rats exposed to HU and HU-CB lost weight (Table 1). As judged from absolute and relative muscle masses, CB induced hypertrophy, especially in the two fast muscles (EDL, GAS), and to a lesser extent in slow SOL muscle. In contrast, HU induced atrophy, which was greatest in SOL muscle, and less in the two fast muscles (Table 1). CB treatment during HU alleviated the atrophic response, thus partially counteracting the HU-induced atrophy. This effect was greatest in the EDL, whereas compared with the control, muscle mass was maintained. SOL muscle, with an almost 60% weight loss after 14 days of HU, exhibited a smaller loss in mass. There was ~30% more muscle mass following HU-CB compared with HU alone.

Effects on MHC isoforms. CB treatment, HU, and HU-CB elicited marked changes in the MHC isoform pattern of SOL muscle (Fig. 2, Table 2). Generally, these changes consisted of slow-to-fast transitions increasing in the order CB < HU < HU-CB. CB markedly elevated the relative concentration of MHCIIa at the expense of MHCI and, in addition, induced low amounts of MHCIId and MHCIIb. Under the HU condition, the relative concentration of MHCIIa was less elevated than with CB, whereas MHCIId attained higher levels. This shift toward the faster isoform indicated a more extensive slow-to-fast transition by unweighting than by CB. The HU-CB condition was characterized by a stronger shift toward the faster isoforms, especially in view of the additional increases in MHCIId and MHCIIb. The three fast isoforms together accounted for ~35% of the total MHC complement, reaching relative concentrations of ~14%, ~12%, and ~7%, for MHCIIa, MHCIId, and MHCIIb, respectively. An additional effect of HU and HU-CB on SOL was the appearance of small amounts of the previously characterized slow MHCIa isoform (11) (see Fig. 2). MHCIa was not detected in control and CB SOL muscles but attained relative concentrations of ~5% and ~1.5% under conditions of HU and HU-CB, respectively.


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Fig. 2.   Electrophoretic separation of slow and fast myosin heavy chain (MHC) isoforms in extracts from rat SOL (top) and GAS (bottom) muscles exposed to different experimental conditions: Cont, control; CB, clenbuterol; HU, hindlimb unweighting; HU-CB, hindlimb unweighting combined with CB treatment. The electrophoretic separations of SOL and GAS muscle extracts were performed on same gels. The order of the lanes at bottom was changed to match the order of the lanes at top.


                              
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Table 2.   Effects of CB, HU, and HU-CB on percentage distribution of MHC isoforms in rat soleus muscle

Except for slight decreases in the relative concentration of MHCI, CB, HU, and HU-CB remained without effects on the MHC complement of EDL muscle (Table 3). This contrasted with the conspicuous changes seen in the red GAS (Table 4). Its predominantly fast fiber population responded with shifts toward faster MHC isoforms to the various experimental conditions. These changes were less pronounced than in SOL muscle. CB tended to increase MHCIIb, most likely at the expense of MHCIIa, as this isoform was reduced, and MHCIId was unaltered. Similar changes were observed after HU where, in addition to the MHCIIa to MHCIIb shift, MHCI tended to decrease. HU-CB did not induce additional changes.

                              
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Table 3.   Effects of CB, HU, and HU-CB on percentage distribution of MHC isoforms in rat extensor digitorum longus muscle


                              
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Table 4.   Effects of CB, HU, and HU-CB on percentage distribution of MHC isoforms in rat gastrocnemius muscle (red portion)

Effects on MLCs. Similar to the effects on the MHC complement, the changes in MLCs reflected slow-to-fast transitions and were greater in the slow compared with the fast muscles (Table 5). In SOL, CB induced decreases in the slow isoforms of the alkali (MLC1s) and regulatory (MLC2s) light chains with concomitant increases in the relative concentrations of the corresponding fast isoforms, MLC1f/MLC3f and MLC2f. HU had a slightly different effect with a reduction in the relative concentration in MLC1s and an increase in MLC1f and MLC3f, but little change in the regulatory light chains. The changes in MLC composition of SOL by HU-CB were restricted to a partial exchange of MLC1s with MLC1f.

                              
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Table 5.   Effects of CB, HU, and HU-CB on percentage distribution of MLC isoforms in rat soleus muscle

Effects of CB and HU on the light chain pattern of fast muscle were only observed in the deep GAS (Table 6). On the whole, they also consisted of slow-to-fast transitions. CB treatment decreased MLC1s and increased MLC1f. As for the regulatory light chain, this transition did not reach statistical significance. A similar exchange was observed in the unweighted GAS where, in addition to the elevation of MLC1f, an increase was also evident for MLC3f. GAS muscle exposed to CB and HU was characterized by a partial exchange of MLC1s with MLC1f and a pronounced increase in MLC3f.

                              
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Table 6.   Effects of CB, HU, and HU-CB on percentage distribution of MLC isoforms in rat gastrocnemius muscle (red portion)


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results on the effects of CB with regard to muscle hypertrophy and a tempering of HU-induced muscle atrophy are in agreement with similar data in the literature (1). We also confirm that differences exist between the adaptive responses of fast and slow muscles following unweighting and CB treatment. In addition, our results show different responses of the two fast-twitch muscles under study. According to the present investigation, CB induces a greater hypertrophic effect in GAS than in EDL. Moreover, although both muscles respond to unweighting with similar losses in mass (~30%), CB treatment counteracts the HU-induced atrophy more efficiently in EDL than in GAS. This difference may be related to the higher content of type I fibers in GAS than in EDL (see differences in MHC isoform composition in Tables 3 and 4). According to the literature (7, 15) and the present results, CB appears to have a greater hypertrophic effect on type II fibers than on type I fibers. It is not unexpected, therefore, that CB counteracts the HU-induced atrophy more efficiently in the EDL compared with the GAS.

As judged from the MHC analyses, CB elicits exchanges of slow with faster isoforms in both slow and fast muscles. These findings add to our understanding of muscle plasticity. In SOL muscle, the slow-to-fast transition in MHC isoforms occurs mainly in the direction of MHCI to MHCIIa, with the induction of small amounts of MHCIId and MHCIIb. In the fast GAS muscle, the changes in MHC isoforms may be described as "fast-to-faster" transitions, consisting mainly of a decrease in MHCIIa accompanied by an increase in MHCIIb. The relative concentration of MHCIId was unaltered under these conditions. This could point to a direct transition from MHCIIa to MHCIIb, but might also result from a two-step transition, i.e., MHCIIa right-arrow MHCIId right-arrow MHCIIb, with MHCIId as an intermediate between MHCIIa and MHCIIb. In this case, the relative concentration of MHCIId would appear unaltered. A direct MHCIIa-to-MHCIIb transition would result in hybrid fibers displaying coexistence of MHCIIa with MHCIIb without MHCIId. Although no single fiber studies were performed in the present study, we favor the two-step transition model and based our assumption on single fiber analyses performed on unweighted, slow-to-fast transforming rat SOL muscle. In these muscles, fibers with coexisting MHCIIa and MHCIIb were not detected, but fibers with the following MHC combinations could be delineated: MHCI + MHCIIa, MHCI + MHCIIa + MHCIId, as well as MHCI + MHCIIa + MHCIId + MHCIIb (28).

The changes in MHC isoforms in unweighted muscles are qualitatively similar and in the same direction as those elicited by CB. However, HU-induced slow-to-fast transitions appear to be more extensive than those induced by CB treatment alone. Thus HU causes transitions that extend to MHCIId and also to MHCIIb. The combination of HU and CB further enhances the slow-to-fast transition with even higher increases in MHCIId and MHCIIb compared with HU alone.

Interestingly, SOL muscle expresses small amounts of MHCIa, an additional slow isoform (10), under the conditions of HU and HU-CB. We assume that MHCIalpha , another slow isoform, is also elevated under the conditions of HU and of HU-CB, because mRNA and immunohistochemical studies have recently demonstrated its upregulation in unweighted rat SOL muscle (30). Because of the comigration of MHCIalpha and MHCI, electrophoretic separation of these two isoforms is difficult. As such, elevations in MHCIalpha may therefore be hidden within the bulky MHCI band.

The changes in the MHC isoform pattern of unweighted GAS muscle also appear to fit this general scheme of slow-to-fast transitions. According to the predominantly fast phenotype of this muscle, slow-to-fast transitions are, for the most part, confined to the fast fiber types and consist mainly of IIA right-arrow IID right-arrow IIB (i.e., fast-to-faster transitions). In fact, the MHC isoform pattern of GAS exposed to HU displays increases in the relative concentration of MHCIIb, and similar changes occur under the influence of CB. The similar effects induced by CB and HU in the MHC isoform pattern of GAS muscle suggest that these changes represent maximally attainable responses under these conditions. It is not unexpected, therefore, that the combined actions of HU and CB do not lead to additional fast-to-faster transitions, i.e., greater amounts of MHCIId and MHCIIb.

In contrast to reports in the literature (9, 14, 24), we noted significant changes in MLC expression under the conditions of HU, CB, and HU-CB. In general, these alterations amounted to partial exchanges of the slow MLC isoforms with their fast counterparts. These slow-to-fast transitions appear to be greater for essential (alkali) light chains compared with regulatory light chains. Disproportionate slow-to-fast transitions of essential and regulatory light chains undoubtedly lead to the appearance of hybrid myosins (e.g., both fast and slow MLC isoforms in combination with fast MHC isoforms) (2, 26, 27). Such hybrid fibers have been previously observed in muscles undergoing fast-to-slow transitions (18). Higher apparent affinities of the fast MLC3f for MHCIId and MHCIIb instead of MHCIIa (32) may explain why increases in MLC3f are observed in the present study under conditions that lead to significant increases in MHCIId and MHCIIb.

In summary, SOL muscle is capable of changing its phenotype from slow to fast. HU, CB treatment, and the combination of both (HU-CB) all cause the upregulation of the fast MHCIIa and, in addition, the induction of considerable amounts of the faster MHCIId and fastest MHCIIb isoforms. These data lend support to the concept of muscle plasticity spanning from one end to the other along the spectrum of fiber types. According to the previously established time courses of induction and increases in fast MHC isoforms in unweighted SOL muscle (30), it seems that the changes observed in the present study could occur as progressive slow-to-fast transitions in the order of MHCI right-arrow MHCIIa right-arrow MHCIId right-arrow MHCIIb. The changes in MHC isoform expression observed under the same conditions in the GAS muscle might be interpreted as fast-to-faster transitions, probably in the order of MHCIIa right-arrow MHCIId right-arrow MHCIIb. These changes in MHC composition are accompanied in both muscles by similar transitions in MLC expression. Together, these slow-to-fast changes occur in the same order established in independent studies on MHC-based fiber types for contractile properties, myosin ATPase activity, tension cost, and ATP phosphorylation potential (3, 4, 6, 12).


    ACKNOWLEDGEMENTS

This study was supported by Association Française contre les Myopathies Grant 7109, Fonds Européen de Développement Régional Grant F007, and Deutsche Forschungsgemeinschaft Grant Pe 62/25-3.


    FOOTNOTES

Addresses for reprint requests and other correspondence: L. Stevens, Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d' Ascq, France (E-mail: laurence.stevens{at}univ-lille1.fr); and D. Pette, Fachbereich Biologie, Universität Konstanz, D-78547 Konstanz, Germany (E-mail: dirk.pette{at}uni-konstanz.de).

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. Section 1734 solely to indicate this fact.

Received 2 February 2000; accepted in final form 13 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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