Time-dependent changes in expression of troponin subunit isoforms in unloaded rat soleus muscle

Laurence Stevens1, Bruno Bastide1, Philippe Kischel1, Dirk Pette2, and Yvonne Mounier1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study focuses on the effects of mechanical unloading of rat soleus muscle on the isoform patterns of the three troponin (Tn) subunits: troponin T (TnT), troponin I (TnI), and troponin C (TnC). Mechanical unloading was achieved by hindlimb unloading (HU) for time periods of 7, 15, and 28 days. Relative concentrations of slow and fast TnT, TnI, and TnC isoforms were assessed by electrophoretic and immunoblot analyses. HU induced profound slow-to-fast isoform transitions of all Tn subunits, although to different extents and with different time courses. The effectiveness of the isoform transitions was higher for TnT than for TnI and TnC. Indeed, TnI and TnC encompassed minor partial exchanges of slow isoforms with their fast counterparts, whereas the expression pattern of fast TnT isoforms (TnTf) was largely increased after HU. Moreover, slow and fast isoforms of the different Tn were not affected in the same manner by HU. This suggests that the slow and fast counterparts of the Tn subunit isoforms are regulated independently in response to HU. The changes in TnTf composition occurred in parallel with previously demonstrated transitions within the pattern of the fast myosin heavy chains in the same muscles.

hindlimb suspension; isoform transition; troponin T, I, and C.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADULT FAST- AND SLOW-TWITCH MUSCLE FIBERS have the capacity to change their phenotypes in response to altered functional demands (20). Thus mechanical unloading of rat soleus muscle by hindlimb suspension results in atrophy and in pronounced slow-to-fast transitions of myosin heavy chain (MHC) and myosin light chain (MLC) isoforms (22, 26). In a previous paper (25), we showed that hindlimb unloading (HU) of rat soleus muscle for different time periods generally elicited orchestrated slow-to-fast transitions in MHC protein and mRNA isoform levels that occurred in the order MHCIright-arrowMHCIIaright-arrowMHCIId(x)right-arrowMHCIIb. It has also previously been demonstrated (13) that changes in troponin C (TnC), the Ca2+ sensor of the troponin (Tn) complex, alter the Ca2+-activated properties (tension-pCa relationships) of 14-day unloaded rat soleus muscle, although to a lesser extent than the changes in MHC and MLC complement. In addition, Campione et al. (2) noticed the appearance of fast-type troponin T (TnT) and troponin I (TnI) isoforms in unloaded rat soleus muscle, but this study was restricted to a 21-day HU period. Therefore, these data emphasized the necessity to perform additional studies to investigate the effects of mechanical unloading on Tn subunit composition in more detail, especially with regard to the extent and time course of HU-induced slow-to-fast transitions in the isoform patterns of TnT, TnI, and TnC.

TnC, the Ca2+-binding subunit, exists in skeletal muscle as fast (TnCf) and slow (TnCs) isoforms, both encoded by separate genes (6). TnT, the subunit interacting with tropomyosin, exists as multiple fast and slow isoforms originating from different genes and alternative splicing (19). Five major fast TnT isoforms (TnT1f, TnT2f, TnT3f, TnT4f, and TnT5f) have been identified at the protein level in fast-twitch muscles, whereas two major slow protein isoforms, TnT1s and TnT2s, exist in slow-twitch muscles (19). TnI, the inhibitory subunit of the Tn complex, exists as three isoforms that are encoded by specific genes. The cardiac TnI isoform (TnIc) is restricted to adult cardiac muscle, whereas slow TnI (TnIs) and fast TnI (TnIf) isoforms are expressed in slow and fast skeletal muscles, respectively.

The present study was undertaken to investigate the time course of changes in the isoform patterns of the three Tn subunits in rat soleus muscle exposed to different time periods (7, 15, and 28 days) of mechanical unloading by HU. Changes in Tn subunit isoforms were assessed by electrophoretic and immunoblot analyses by using specific antibodies to identify slow and fast TnI, TnT, and TnC isoforms.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and muscles. The animal maintenance conditions, as well as the HU experiments, were approved by the French Ministries of Agriculture and Education (veterinary service of health and animal protection, authorization no. 03805). Adult male Wistar rats (n = 5, initial body weight ~280 g) were hindlimb unloaded as previously described (24) for either 7, 15, or 28 days. For the control rats, preliminary experiments on MHC isoforms (24) were done on rats covering the time period parallel to 4- to 28-day HU: no difference appeared between control rats age-matched for 4-, 7-, 15-, and 28-day HU. Thus, in this study, five rats age-matched for 15-day HU were used.

At the end of the treatment, all the rats were killed by intraperitoneal injection of an overdose of ethylcarbamate, and soleus (soleus, n = 5) and tibialis anterior (TA, n = 5) muscles were dissected and weighed. TA muscle samples were used as a reference for fast troponin band migration. The muscles were frozen in liquid N2 and stored at -80°C until analyzed. The muscles investigated in the present study were the same as those previously analyzed for time-dependent changes in myosin isoform composition (23, 25).

Troponin subunit analysis. Frozen muscle tissue was pulverized under liquid N2 in a small steel mortar (17). The muscle powder was suspended 1:10 (mass/vol) in ice-cold preextraction medium (100 mM KCl, 20 mM Tris · HCl, and 2 mM dithiothreitol, pH 7.5) and treated with a Polytron homogenizer (Kinematica, Lucerne, Switzerland) at intense cooling. The pellet obtained after 5-min centrifugation at 10,000 g (4°C) was resuspended 1:5 (mass/vol) in 30 mM KCl, 2 mM dithiothreitol, and 20 mM Tris · HCl (pH 7.5) and subjected to homogenization as described above. Protein concentration of this myofibrillar homogenate was determined according to Lowry et al. (16).

TnT, TnI, and TnC isoforms were separated by one-dimensional 10-20% gradient gel electrophoresis of the myofibrillar homogenate according to Laemmli (15) as previously described (21). Fast and slow troponin subunits, as well as actin, were identified by immunoblotting. The actin amount, which proved to be unaltered during the period of HU studied (see Immunoblot analyses), served as a control for constant sample loading.

Immunoblot analyses. After electrophoretic separation, proteins were electrotransferred to a 0.2-µm nitrocellulose sheet (Advantec MFS, Pleasanton, CA). The membranes were blocked with phosphate-buffered saline (PBS, pH 7.4) containing 5% nonfat dry milk and 0.2% sodium azide. The following mouse monoclonal antibodies were from Sigma: clones no. JLT-12 (specific to fast TnT isoforms) and no. 5C5 (specific to alpha -sarcomeric actin). Previously characterized polyclonal antibodies from guinea pig (9, 10, 17) were used to identify slow isoforms of TnT, as well as slow and fast isoforms of TnC and TnI.

The primary antigen/antibody complexes were detected by a peroxidase staining kit (Sigma) consisting of extravidin peroxidase and biotinylated goat conjugate antibodies against mouse or guinea pig IgGs. The specific signals for actin and the various Tn subunit isoforms were visualized by an enhanced chemiluminescence (ECL) kit and hyperfilm ECL (Amersham International, Little Chalfont, UK). Signal intensities were evaluated by integrating densitometry using a GS-700 Imaging Densitometer (Biorad, Ivry sur Seine, France). The efficiency of the electrotransfer was checked by reversibly staining the gels immediately after the transfer and before immunoblot with SYPRO Orange dye (Molecular Probes, Eugene, OR), revealed under a 312-nm ultraviolet transilluminator (13). The measurements of signal intensities of both SYPRO Orange-stained gels and ECL films after immunoblots proved that the actin signal was not significantly altered during HU up to 28 days [percentage of control soleus signal (n = 5): 91 ± 5% (7-day HU); 90 ± 5% (15-day HU); 99 ± 1% (28-day HU)] and thus could be used as a reference for estimating relative expression levels of Tn subunit isoforms. To ensure that results were obtained in the linear range of the optical scanner densities, a range of various dilutions was used for each sample and the different ratios of Tn to actin were determined and compared.

Statistical analyses. Data are presented as means ± SD. All data were analyzed by using Student's t-test to determine differences between values from control and experimental muscles. The acceptable level of significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HU-induced changes in the TnI isoform pattern. The actin signal, which proved to be unaltered during HU up to 28 days, was used as a reference for estimating relative expression levels of slow (TnIs) and fast (TnIf) TnI isoforms (Fig. 1B). As shown in Figs. 1 and 2, HU induced pronounced changes in the levels of TnIs and TnIf. Compared with control (0-day HU), TnIs was moderately reduced after 7 days of HU and was greatly decreased after 15 days (~50%) and 28 days (60%). Conversely, TnIf was ~4.5-fold elevated after 7 and 15 days of HU and continued to increase with prolonged HU. Its relative concentration was ~8-fold elevated over control in 28-day unweighted soleus muscle (Fig. 2).


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Fig. 1.   A: changes in the isoform pattern of slow troponin I (TnIs) in rat soleus muscle. Relative amounts of TnIs isoforms (related to actin signal) were densitometrically evaluated from 1-dimensional immunoblots. Values are means ± SD, n = 5 for each time point. Muscles were investigated after 0, 7, 15, and 28 days of hindlimb unloading (HU). B: immunoblot illustration of HU-induced effects on the isoform pattern of TnIs in soleus muscle. Note that TnIs is not expressed in tibialis anterior (TA) muscle sample. aSignificantly different from control soleus muscles (0-day HU). bSignificantly different from 7-day HU soleus. Error bars are not mentioned when they merge with the data points.



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Fig. 2.   A: changes in the isoform pattern of fast TnI (TnIf, open circle ) in rat soleus muscle. Relative amounts of TnIf isoforms (related to actin) were densitometrically evaluated from 1-dimensional immunoblots. Values are means ± SD, n = 5 for each time point. Muscles were investigated after 0, 7, 15, and 28 days of HU. B: immunoblot illustration of HU-induced effects on the isoform pattern of TnIf in soleus muscle. Note that TnIf signal is the highest in TA muscle sample. aSignificantly different from control soleus muscles (0-day HU). bSignificantly different from 7-day HU soleus. cSignificantly different from 15-day HU soleus.

HU-induced changes in the TnT isoform pattern. The same procedure used for TnI was used to estimate changes in relative concentrations of slow (TnTs) and fast (TnTf) TnT isoforms. An unexpected finding was that the polyclonal antibody detected three slow TnT isoforms in soleus muscle, namely, two major bands previously identified in rabbit muscle as TnT1s and TnT2s (6, 10, 19) and a third faster-migrating band (Fig. 3B). The mobility of this band was clearly distinct from any of the fast TnT isoforms. We therefore assumed that this band corresponded to the third slow TnT isoform, TnT3s, previously detected by Jin et al. (11) in mouse skeletal muscle. TnT1s, TnT2s, and TnT3s represented ~44%, ~39%, and ~17%, respectively, of the total slow TnT isoforms in control soleus muscle. As documented by the data in Fig. 3A, HU for up to 28 days did not affect the relative concentrations of the three slow TnT isoforms.


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Fig. 3.   A: changes in the isoform pattern of slow TnT in rat soleus muscle. Relative amounts of each slow TnT isoform (TnT1s, ; TnT2s, ; TnT3s, black-down-triangle ) were densitometrically evaluated from 1-dimensional immunoblots and related to the total amount of TnTs. Values are means ± SD, n = 5 for each time point. Muscles were investigated after 0, 7, 15, and 28 days of HU. B: immunoblot illustration of HU-induced effects on the isoform pattern of TnTs in soleus muscle.

In contrast, HU markedly affected the fast TnT isoform pattern (Fig. 4). TnT3f, the predominant fast TnT isoform in normal soleus muscle (~61% of total TnT), decreased to relative concentrations of ~45%, ~35%, and ~32% after 7, 15, and 28 days of HU, respectively. Conversely, relative concentrations of TnT2f, TnT1f, and TnT4f increased during HU. TnT1f, present at only 2.5% in control soleus, increased ~5-fold after 7 days, and its relative concentration reached a level of ~28% after 28 days. TnT4f, which was undetectable in normal rat SOL muscle, was induced by HU, reaching ~5% at 7 and 15 days and ~10% after 28 days of HU. The amount of TnT2f, the second major isoform in control soleus (~30%), increased and reached a level similar to that of TnT3f (~40%) after 15 and 28 days of HU.


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Fig. 4.   A: changes in the isoform pattern of fast TnT in rat soleus muscle. Relative amounts of each fast TnT isoform (TnT1f, open circle ; TnT2f, ; TnT3f, diamond ; TnT4f, down-triangle) were densitometrically evaluated from 1-dimensional immunoblots and related to the total amount of TnTf. Values are means ± SD, n = 5 for each time point. Muscles were investigated after 0, 7, 15, and 28 days of HU. B: immunoblot illustration of HU-induced effects on the isoform pattern of TnTf in soleus muscle. Note that the 4 TnTf bands are also observed in TA muscle sample. aSignificantly different from control soleus muscles (0-day HU). bSignificantly different from 7-day HU soleus. cSignificantly different from 15-day HU soleus.

The total amounts of TnTs (1s + 2s + 3s) and TnTf (1f + 2f + 3f + 4f) were related to their respective actin signal and are represented in Fig. 5, A and B. Both isoforms could not be studied on the same gel, so we could not establish their relative proportions vs. a total amount of Tn. Thus they were expressed as percentages of their respective control values (equivalent to 100%). The results show that the whole signal of TnTs presented a tendency (not significant) to decrease from 7 days of HU and then remain stable until 28 days. The signal of TnTf started to increase significantly at 7 days and progressively rose up until 28 days.


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Fig. 5.   A: changes in the pattern of the total slow TnT isoforms (1s + 2s + 3s) in rat soleus muscle. The signals of the TnTs isoforms (related to actin) after HU were densitometrically evaluated from 1-dimensional immunoblots and are expressed as percentages of the control. Values are means ± SD, n = 5 for each time point. Muscles were investigated after 0, 7, 15, and 28 days of HU. B: changes in the pattern of the total fast TnT isoforms (1f, 2f, 3f and/or 4f) in rat soleus muscle. The signals of the TnTf isoforms (related to actin) were densitometrically evaluated from 1-dimensional immunoblots and are expressed as percentages of the control. Values are means ± SD, n = 5 for each time point. Muscles were investigated after 0, 7, 15, and 28 days of HU. aSignificantly different from control soleus muscles (0-day HU). bSignificantly different from 7-day HU soleus. cSignificantly different from 15-day HU soleus.

HU-induced changes in the TnC isoform pattern. Figure 6 illustrates the changes in the slow (TnCs) and fast (TnCf) isoforms of TnC after 7, 15, and 28 days of HU. TnCs and TnCf were both recognized by the polyclonal antibody. Both isoforms could thus be studied on the same gels, and their proportion could be determined as percentages of total TnC. TnCs, the predominant isoform, represented ~96% of total TnC in control soleus muscle. It was partially replaced by TnCf during HU. First changes became detectable after 15 days and increased with prolonged HU. However, the increase in TnCf remained moderate such that TnCs remained the predominant isoform (~80%) also in 28-day unloaded soleus muscle.


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Fig. 6.   A: changes in the isoform pattern of slow (TnCs, ) and fast (TnCf, open circle ) TnC in rat soleus muscle. Relative amounts of TnCs and TnCf isoforms were densitometrically evaluated from 1-dimensional immunoblots. Values are means ± SD, n = 5 for each time point. Muscles were investigated after 0, 7, 15, and 28 days of HU. B: immunoblot illustration of HU-induced effects on the isoform pattern of TnC in soleus muscle. aSignificantly different from control soleus muscles (0-day HU). b Significantly different from 7-day HU soleus. cSignificantly different from 15-day HU soleus.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study reveal that mechanical unloading has a profound effect on the composition of troponin, the major regulatory protein complex of the thin filament. Alterations in TnI expression at both mRNA and protein levels have previously been observed in response to reduced neuromuscular activity (2, 4, 5). Here we show that reduced neuromuscular activity by mechanical unloading affects all three subunits of the Tn complex in rat SOL muscle. Collectively, the HU-induced changes encompass slow-to-fast transitions in the isoform patterns of TnT, as well as those of TnI and TnC. Time courses and degrees of these transitions, however, differ between the three subunits.

The changes in TnT isoforms are focused on great slow-to-fast rearrangements in the pattern of the fast isoforms: increases in TnT1f, concomitant with a decrease in TnT3f, and induction of the TnT4f isoform that is not detectable in normal SOL muscle. These changes agree with the notion that the expression of fast TnT and MHC isoforms occurs in a coordinated manner (7, 17, 18). As derived from single-fiber studies, the following preferential coexpression patterns of fast MHC and TnT isoforms revealed in rat muscle are MHCIIa-TnT3f, MHCIId-TnT1f, and MHCIIb-TnT4f (7). It is not surprising, therefore, that the induction of TnT4f occurs in parallel with the previously demonstrated upregulation of MHCIIb in the same muscles investigated in the present study (25). Similarly, as demonstrated in the same muscles, the increase of TnT1f fits the upregulation of MHCIId, whereas the decrease of TnT3f follows the decay of MHCIIa (25).

An interesting finding of the present study was the detection of a third slow TnT isoform, identified by its electrophoretic mobility and immunoblotting in control and unloaded soleus muscles. We suggest that this compound corresponds to the TnT3s isoform previously identified in mouse muscle by Jin et al. (11). According to these authors, three slow TnT isoforms (TnT1s, TnT2s, and TnT3s) are generated in mouse muscle by alternative splicing from the primary transcript of a common slow TnT gene. Interestingly, the proportions of the three TnTs isoforms in SOL muscle remain unaffected by mechanical unloading. This finding is in line with similar observations on unaltered proportions of TnT1s and TnT2s in both denervated developing and denervated adult soleus muscles of rabbit (17). It appears thus that slow TnT isoforms are under the control of an intrinsic program and are less affected by neural input and neuromuscular activity than TnTf isoforms.

TnI also exhibits important changes as reflected by a decrease of its slow isoform to ~40% of its normal level after 28 days of HU. Interestingly, the moderate decay of TnIs during the initial 7-day period of HU coincides with a steep rise in TnIf. This discrepancy probably indicates that downregulation of TnIs and upregulation of TnIf are independently regulated. Possibly, the steep rise of TnIf is neurally induced and coincides with a change in electromyographic activity, which has been reported to turn into a more phasic pattern after 7 days of HU (1, 3).

The slow-to-fast transition in TnC expression is in accord with our previous observations on the effects of 14 days of HU, namely an increase in TnCf from ~10% in control soleus to ~20% in the unloaded muscle (12, 13, 14). Using a polyclonal antibody that recognizes fast and slow TnC isoforms, we show here that the decrease in TnCs is accompanied by a similar increase in TnCf. However, compared with the fast TnT and the TnI isoforms, the slow-to-fast transition in TnC is small and occurs only after longer periods of HU. The relatively small changes of TnC isoforms at the protein level are in agreement with reports in the literature that fast-to-slow transitions of TnC are less pronounced at the protein level than at the mRNA level (5, 10).

The functional significance of these changes in Tn subunit isoform expression could be discussed in terms of Ca2+-activated properties. Indeed, we showed (24) in accordance with other studies (8, 27) that, after HU or spaceflight missions, rat soleus fibers displayed modifications in the different parameters derived from the tension-pCa relationships [i.e., a higher threshold for Ca2+ activation, a rightward shift of the tension-pCa curve, and an increased Hill coefficient nH (or slope of the curve)]. The Hill coefficient parameter, functionally corresponding to the cooperativity along the thin filament, is generally related to the type of TnT and tropomyosin isoform present in the muscle fibers, whereas TnC isoforms mainly dictate the threshold and pCa50 parameter values (14).

Another physiological explanation could be linked to the level of phosphorylation states of the different Tn subunits according to their various isoforms (10). For instance, the fact that TnIf increased very steeply and faster than TnIs could be linked to the presence of phosphorylation sites on rabbit fast TnI isoforms (10) and the failure to detect charge variants of slow TnI (and thus the absence of phosphorylation sites on this isoform). In addition, a possible explanation for the differential response of slow and fast TnT isoforms to unloading might be related to their phosphorylated, partially phosphorylated, or unphosphorylated states (9, 10).

Taken together, the slow-to-fast transitions in mechanically unloaded rat soleus muscle encompass changes in the isoform patterns of all three Tn subunits. These changes, however, differ with regard to their temporal patterns and the extent of the transitions. The asynchronic and quantitatively different slow-to-fast transitions of the three subunits lead to the coexistence of slow and fast TnI, TnT, and TnC isoforms at various ratios in individual fibers. Hybrid myosin fibers of unloaded soleus muscles have been described at both the MHC protein and mRNA levels (23, 25). Assuming that the stoichiometry of the Tn complex is maintained during the transformation process, this necessarily leads to the existence of hybrid and, most likely, functional Tn complexes composed of slow and fast subunit isoforms.


    ACKNOWLEDGEMENTS

This study was supported by Centre National d'Etudes Spatiales Grant 2000/3027, Fond Européen de Développement Régional Grant F007, and the Conseil Régional du Nord Pas-de-Calais.


    FOOTNOTES

Address 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).

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.

First published January 9, 2002;10.1152/ajpcell.00252.2001

Received 6 June 2001; accepted in final form 20 December 2001.


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MATERIALS AND METHODS
RESULTS
DISCUSSION
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