1Laboratoire de Biologie Moléculaire et Génie Génétique, Université de Liège, Campus du Sart-Tilman, Liege, Belgium; 2Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, Institut Fédératif de Recherche 118, Unité Propre de Recherches de lEnseignement Supérieur Équipe dAccueil 1032, Villeneuve dAscq Cedex, France; and 3Section of Molecular Cardiology, Evanston Hospital, Evanston, Illinois
Submitted 28 July 2004 ; accepted in final form 21 March 2005
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ABSTRACT |
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skinned fibers; skeletal muscle; troponin subunit exchange; hindlimb unloading; atrophy
Here we show that the diversity of sTnT isoforms is higher than previously thought, because a fourth sTnT isoform was found in rat muscles. Our goal was to investigate the mRNA and protein expression levels of the different sTnT isoforms in the slow rat soleus muscle as well as in a model of functional atrophy allowing a slow-to-fast phenotype transition, namely, hindlimb unloading (HU). In addition, we studied sTnT expression levels in two fast muscles as well as the functional properties of the different sTnT isoforms by substituting slow troponin complexes for fast troponin subunits in fast skinned muscle fibers.
Our data show a tissue-specific expression pattern of sTnT isoforms that is not modified by HU. Moreover, the four sTnT isoforms can be separated into two classes according to their ability to modulate Ca2+ activation characteristics.
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EXPERIMENTAL PROCEDURES |
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The experiments as well as the maintenance conditions of the animals received authorization from the Ministry of Agriculture and the Ministry of Education (veterinary service of health and animal protection, authorization no. 03805). The experiments were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.
Experiments were carried out on two groups of adult male Wistar rats (300 g) anesthetized with pentobarbital sodium (3 mg/kg). The first group (n = 3) was composed of control animals. The second group (n = 3) was composed of animals submitted to 15 days of HU, an animal model for functional atrophy (22). In the HU experiment, the rats were suspended by the tail to induce hindlimb muscle unloading and atrophy. The two groups of animals were matched for age and weight. The slow soleus muscle and the fast extensor digitorum longus (EDL) and tibialis anterior (TA) muscles were used for sTnT expression.
Analysis of sTnT mRNAs and Cloning of sTnT cDNAs
Isolation of mRNA was performed on control muscles with a MicroPolyA Pure kit (Ambion). sTnT cDNAs were then amplified by RT-PCR (Titan One Step RT-PCR kit, Roche). The primer design was based on sTnT sequences available for the mouse (15), assuming a high degree of sequence conservation between the two rodent species. Two different pairs of primers were used. One pair of forward CCAGGATGTCAGACACCGA and reverse GTTCTGAAGCGCTGTTGCTC primers was designed to amplify the 5' alternatively spliced region for subsequent separation of the putative isoforms by gel electrophoresis. The second pair of synthetic oligonucleotides was designed to amplify the whole sTnT coding sequence from the ATG start codon to the TGA stop codon (forward primer: GGGAATTCCATATGTCAGACACCGAAGAACA, reverse primer: CATGCCATGGTCACTTCCAGCGGCCTCCAAC). The PCR products obtained with the second primer pair were cloned with a Topo TA cloning kit (Invitrogen). Sequencing of the cDNAs was performed on a 310 Applied Biosystems sequencing unit.
Recombinant Protein Expression
sTnT.
The cDNAs coding for the four different rat sTnT isoforms were transferred into a pET30a vector. Because sTnT cDNA contains many arginine codons, synthesis of sTnT was performed in BL21(DE3) CodonPlus RIL cells (Stratagene). Cells transformed with the pET expression vectors were grown overnight at 37°C. This culture was diluted 1:100 and induced at an optical density at 600 nm of 0.8 with 1 mM isopropyl
-D-1-thiogalactopyranoside for 45 h. Bacterial pellets from 600-ml sTnT cultures were resuspended in 30 ml of homogenization buffer (50 mM Tris·HCl pH 8.5 and a cocktail of protease inhibitors from Sigma). The cells were lysed with a French cell press, and the lysate was centrifuged at 25,000 g for 30 min at 4°C. The supernatant was then loaded on an anion exchange column (HiTrap Q HP-Sepharose, Amersham Biosciences). TnT was eluted with an increasing gradient from 0 to 50% buffer containing 1 M NaCl.
Skeletal slow troponin C and troponin I. Rat skeletal slow troponin C (sTnC) and troponin I (sTnI) were cloned and expressed as described for sTnT, except that sTnI was loaded on a cation exchange column.
Troponin complex. Equimolar quantities of each subunit of the troponin complex were mixed in 6 M urea, 1 M NaCl, 50 mM Tris pH 8.0, 3 mM CaCl2, 2.5 mM MgCl2, and 1 mM DTT. After 1 h, the complex was successively dialyzed against decreased urea concentrations (4, 2, and 0 M) and then against decreased NaCl concentrations (0.8, 0.6, 0.3, and 0.1 M). The complex was then loaded on a HiTrap Q HP and eluted with an increasing gradient from 0.1 to 0.3 M NaCl. Only fractions containing equimolar quantities (1:1:1) of purified ternary complexes were pooled and dialyzed against (in mM) 20 MOPS pH 7.0, 180 NaCl, 3 CaCl2, 5 MgCl2, and 0.1 DTT.
Calcium Activation Characteristics of Single Skinned Skeletal Fibers
Experimental setup.
The experiments were carried out in a thermostatically controlled room (19 ± 1°C) as described previously (17). Briefly, the fibers were randomly removed from EDL muscles, and fibers of 5 mm were sectioned into two segments. The first segment was dissolved and stored to serve as control for endogenous TnT expression analysis by electrophoresis. The second 2- to 2.5-mm fiber segment was connected to a force transducer (Fort 10, WPI) in an experimental chamber under constant stirring and submitted to TnT exchange.
Solutions. All solutions were described previously (16), and pCa values were calculated with the Fabiato (8) computer program. The calculation was performed with the stability constants listed for Ca2+ (24).
Determination of Ca2+ activation characteristics. The tension (T)-pCa curves were obtained as previously described (16). The steepness of the T-pCa curve was determined by the Hill coefficients nH, either n1 or n2, according to the following equation (4): P/P0 = {([Ca2+]/K)nH/[1 + ([Ca2+]/K)nH]}, where P/P0 is the normalized tension, [Ca2+] is Ca2+ concentration, and K is the apparent dissociation constant (pK = log K = pCa50); n1 corresponds to P/P0 > 50% and n2 to P/P0 < 50% (23).
Exchange of troponin complexes. After T-pCa relationship determination in the presence of their endogenous troponin complexes, the fibers were put into rigor and were bathed with an excess of 1:1:1 recombinant complexes (sTnT-sTnC-sTnI) as previously described (19), with sTnT1, sTnT2, sTnTx, and sTnT3, at an average concentration of 1.5 mg/ml. Incubation time was 2 h, with slow stirring to ensure optimal displacement of endogenous troponin complexes. After T-pCa relationships were obtained, all fibers were dissolved in SDS sample buffer, heated at 90°C for 3 min, and stored at 80°C until electrophoretic analysis.
Biochemical Analysis at Protein Level
For each technique, at least three experiments were carried out to ensure the reproducibility of the results.
SDS-PAGE. Separation of TnT isoforms of whole muscles, single fibers (intact segments and matching reconstituted segments), or recombinant proteins was performed by SDS-PAGE using 1020% linear gradient gels (32). Proteins were detected by immunoblotting. To assess whether sTnTs were phosphorylated or not, alkaline phosphatase treatment was applied as previously described (13).
Immunoblotting. Electrotransfer to a 0.2-µm-pore size nitrocellulose sheet (Advantec MFS) was performed. The sTnT isoforms were detected with a highly specific monoclonal antibody (CT3-mAb) directed against sTnT (2, 15). Detection of fTnT was performed with a monoclonal antibody (JLT12, Sigma). TnT antibodies were revealed by an extravidin-biotin peroxidase staining kit (Sigma), and TnT isoforms were visualized by chemiluminescence (ECL, Amersham). Signal intensities were measured with a Multi Analyst system (Bio-Rad).
Statistical analysis. All data are reported as means ± SE. The statistical significance of the difference between means was determined with Students t-test or paired t-test when data were obtained from the same fiber under different experimental conditions. Differences at or above the 95% confidence level were considered significant.
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RESULTS |
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Using mRNA from rat soleus muscle, we cloned the cDNAs encoding sTnT isoforms, encompassing the complete coding region from the translational start (ATG) to the stop codons (TGA), with primers corresponding to the conserved sequences in the mouse sTnT sequences. In view of the high abundance of clones similar to mouse sTnT1 and sTnT2 (15 of 16), we took advantage of an AvaI restriction site, present in sTnT1 and sTnT2 (position 7479, see Fig. 1B), but not in mouse sTnT3. We cloned the undigested 800-bp cDNAs and found clones similar to mouse sTnT3, as well as clones containing three additional base pairs that had no matching equivalent in the mouse. This isoform is therefore referred to as sTnTx. The sequences of sTnT cDNAs were deposited under GenBank accession numbers AY334079, AY334080, AY334081, and AY334082 for sTnT1, sTnT2, sTnTx, and sTnT3, respectively. Protein sequence alignment of the three mouse sTnT isoforms along with the four predicted rat sTnT isoforms is shown in Fig. 1A. The differential splicing occurring in the rat led to four isoforms. sTnT1 is a 786-bp isoform. sTnT2 has only a 3-bp deletion relative to sTnT1 (GAA, encoding glutamic acid) and is thus 783 bp long. sTnTx and sTnT3 cDNAs lack 33 pb relative to sTnT1 and sTnT2 and are constituted of 753 and 750 bp, respectively. sTnTx vs. sTnT3 has the same codon difference as sTnT1 vs. sTnT2 (Fig. 1B).
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Recombinant sTnTs. The sTnT isoforms were bacterially expressed and purified to near homogeneity, and equal amounts of each recombinant sTnT were analyzed by Western blotting with the mouse monoclonal antibody CT3-mAb (15). As illustrated in Fig. 3A (lanes 14), an immunoreactive protein of the expected size was detected in each preparation. Interestingly, a distinct migration was observed between the sTnT1 and sTnT2 isoforms and between sTnTx and sTnT3, although these proteins differ by only a single glutamic acid. The signal intensity was equivalent for each recombinant sTnT, indicating that CT3-mAb recognized the four isoforms equally well.
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Ca2+ Activation Characteristics of Control and Reconstituted EDL Fibers
To investigate the functional properties of the various sTnT isoforms in a physiologically relevant setup, the endogenous fast troponin complexes of skinned EDL fibers were replaced with complexes made of the three slow troponin subunits sTnT, sTnC, and sTnI. EDL fibers were chosen because 1) replacement of the endogenous fast troponin complex with a slow troponin complex was thought to induce noticeable changes in the T-pCa relationship and 2) it is easy to evaluate the disappearance of fTnT and the appearance of sTnT by immunoblotting after displacement of troponin complexes. As illustrated in the inset of Fig. 4, sTnT was not detected in the control EDL fibers (lane 1) and appeared only after the exchange period (lane 2). Conversely, fTnT was only clearly detected in the control segment (lane 3). The band that is observed after replacement (lane 4) is not a residual fTnT but the substituted sTnT migrating at the same level as fTnT2. Indeed, the membrane was not stripped after sTnT detection but directly reprobed with fTnT.
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DISCUSSION |
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sTnTx increases to four the number of sTnT isoforms described so far. Previously, the presence of only one sTnT isoform (27) or three sTnT isoforms (2, 30) was described in the soleus muscle of adult rats. In other rodents, two sTnT isoforms were found in guinea pigs (13) and three sTnT isoforms in mice (15). Some studies also suggested a higher diversity for sTnT in adult human muscle (27, 28). Rat sTnT1, sTnT2, and sTnT3 cDNAs showed 96% sequence similarity to the corresponding mouse sTnT isoform cDNAs. In fact, the only "real" difference is a GAG codon missing in rat isoforms at positions 6770, and thus a deleted glutamic acid in all the rat protein sequences. All the other nucleotide sequence differences are located in the third "wobble" position of the codons, thus encoding identical amino acids (see Fig. 1A). In mouse (14), sTnT1 mRNA results from splicing exon 5 to exon 6' (Fig. 1B), sTnT2 is generated by alternatively splicing exon 5 to exon 6, and sTnT3 results from splicing exon 4 to exon 6. No equivalent to rat sTnTx, corresponding to the insertion of an AAG codon between exon 4 and exon 6 in sTnT3, has been described previously. However, in the mouse genomic sTnT sequence, intron 5 ends with a putative alternative AG splice acceptor site followed by the AAG codon corresponding to exon 6' in mouse or rat sTnT1. This alternative splice site seems to be conserved in the two rodent species. Thus a hypothesis can be put forward for the generation of rat sTnTx. We suggest that alternative splicing of exon 4 to exon 6' or exon 6 generates sTnTx or sTnT3, respectively, whereas splicing of exon 4 to exon 5 followed by splicing of exon 5 to exon 6' or 6 generates sTnT1 or sTnT2 (Fig. 1B). The alternative splicing of exon 6' is therefore responsible for the single amino acid difference between sTnT1 and sTnT2 and between sTnTx and sTnT3. In a similar way, Yonemura et al. (33) found variants of chicken sTnT cDNA differing by only one codon.
Comparison of the deduced amino acid sequences for the different isoforms revealed one very striking feature: the different number of glutamic acids in their amino-terminal region. Indeed, sTnT1 contains 20 glutamic acid residues, whereas sTnT3 is composed of "only" 15 glutamates out of the first 40 amino acids. Very few positively charged amino acids (basic Lys and Arg) are present in the sTnT amino-terminal domain. Thus the density of negatively charged amino acids is very high in the amino-terminal region of the sTnTs. This observation suggests that the number of glutamic acid residues in different sTnTs might play an important physiological role in their Ca2+ activation properties. Our results are in good agreement with this, because differences in Ca2+ affinity were revealed between high- and low-molecular-weight isoforms. The most striking difference observed between the two sTnT classes was that the higher-molecular-weight sTnT isoforms conferred a higher Ca2+ affinity to the skinned skeletal fibers (Fig. 4). Consistent with our results, Gomes et al. (11) demonstrated that the variable amino-terminal region of the four human cTnT isoforms contributed to the determination of the calcium sensitivity of force development in a charge-dependent manner. Although we failed to detect any functional difference, with our functional assay between isoforms differing by only a single amino acid (sTnT1 vs. sTnT2 and sTnTx vs. sTnT3), we cannot rule out that this single amino acid can affect muscle function. Indeed, it is clear from the data gathered on familial hypertrophic cardiomyopathy mutations that even a single amino acid difference can alter muscle functional properties (12). This hypothesis makes sense if we consider that the amino acid involved is a glutamate. Our data showing specific expression in different muscle types provide some additional support for such a functional role. Indeed, we show the presence of almost only high-molecular-weight sTnT in slow muscles, in agreement with the usually higher Ca2+ affinity of these muscle fibers. On the other side, the expression of low-molecular-weight isoforms in the fast EDL and TA muscles, with sTnTx being the main sTnT isoform, is consistent with the typically lower Ca2+ affinity of these muscle fibers.
Moreover, our observations raise a very important issue: we clearly demonstrate that adult fast muscles express sTnT isoforms, although it was previously reported that sTnT expression was restricted to slow muscle fiber-forming regions after late fetal development (18). To further investigate sTnT expression in fast muscles, we analyzed the TnT isoform pattern of soleus muscle in a HU model. Exposure to HU is known to trigger functional atrophy and transformations leading slow postural muscles toward a fast phenotype (9). These transformations are thought to be triggered by alterations of the neuronal firing pattern (3). Because we discovered in the present study that high-molecular-weight sTnTs are specific to slow postural muscles, whereas low-molecular-weight sTnTs are specific to fast muscles, we expected the expression of sTnT isoforms in the soleus muscle to be switched to a faster phenotype. Although an increase in fTnT expression and a slight decrease of total sTnT expression was observed after HU (2, 30), it appears that the sTnT expression pattern is not influenced by 15 days of HU, suggesting that this pattern is not altered by changes in neuromuscular activity, in contrast to other contractile proteins. Nevertheless, the specific expression of one of the two possible exon 6 variants in slow or fast muscles suggests a functional role for the two alternative splice variants. HU does not alter the pattern of sTnT isoforms in soleus muscle. Thus their role might be a more structural one, possibly effective mainly during embryogenesis. At present, it is difficult to attribute a specific function to these specific sTnT isoforms in fast muscles.
To conclude, sTnTx increases the diversity of rat sTnT isoforms described so far. TnT expression has been shown to be highly sensitive to muscle functional demand, probably to ensure a rapid adaptation of the muscle to new conditions (2, 13, 30). The new, low-molecular-weight isoform is predominantly expressed in fast muscles, and both small isoforms conferred lower Ca2+ affinity than the higher-molecular-weight isoforms. The observed heterogeneity in the expression patterns of rat sTnT isoforms between slow and fast muscle fibers may contribute to a fine contractile adaptation to various physiological, structural, or developmental conditions. We show here that the sTnT expression pattern does not switch from slow to fast type in soleus muscle under strong environmental conditions. Further work is required to understand the role of sTnT isoforms in fast muscle fibers and the significance of the predominant expression of specific splicing variants.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* P. Kischel and B. Bastide contributed equally to this work.
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