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
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ABSTRACT |
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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.
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INTRODUCTION |
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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 MHCIMHCIIa
MHCIId(x)
MHCIIb. 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.
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MATERIALS AND METHODS |
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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 atTroponin 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 -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.
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.
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RESULTS |
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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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