Cardiac troponin T in developing, regenerating and denervated rat skeletal muscle

Troponin T (TnT) is the tropomyosin-binding subunit of the troponin complex which is involved in the calcium-dependent regulation of striated muscle contraction (Ebashi et al. 1973). Cardiac and skeletal muscles contain multiple molecular isoforms of TnT (Dhoot et al. 1979) derived from different genes and from differential RNA splicing (Breibart et al. 1985; Cooper and Ordahl, 1985; Gahlmann et al. 1987). In both mammalian and avian muscles, alternative pre-mRNA splicing of exons in the 5’ and 3’ regions of the fast skeletal TnT transcripts generates a large number of mRNAs, which are differentially expressed in embryonic and adult skeletal muscles (Breibart et al. 1985; Bucher et al. 1989). In the chicken heart, a single gene generates two developmentally regulated mRNAs that code for an embryonic and an adult isoform of TnT (Cooper and Ordahl, 1985). The embryonic cardiac isoform is also transiently expressed during development in chicken skeletal muscle (Toyota and Shimada, 1981; Cooper and Ordahl, 1985; Ogasawara etal. 1987). Two distinct cardiac TnT isoforms, presumably derived from a single gene by alternative RNA splicing (Jin and Lin, 1989) are expressed in the fetal and adult rat heart (Jin and Lin, 1988; Dhoot, 1988; Saggin et al. 1988).

In the present work, we show that cardiac TnT is present in fetal and neonatal rat skeletal muscle and is re-expressed in regenerating and denervated muscle. We also show that the expression of cardiac TnT is not coordinated with the expression of the embryonic skeletal myosin heavy chain (MHC).

Heart and skeletal muscles from normal and experimentally treated Wistar rats of different ages were used in this study. Muscle regeneration was induced by a cold injury as previously described (Sartore et al. 1982). In brief, 3-month-old rats were anesthetized with ether, the tibialis anterior and the soleus muscles were exposed and a copper rod, precooled in liquid nitrogen, was applied to the muscle surface for 5 s. Specimens from injured muscles were removed at different time intervals (3, 7, 11 days) and frozen in liquid nitrogen. Denervation experiments were performed in 3-month-old rats by cutting out a segment of the right sciatic nerve in the thigh under ether anesthesia. The soleus and tibialis anterior muscle from the operated and controlatéral side were removed after 7 and 15 days and frozen in liquid nitrogen.

Two anti-TnT and one anti-MHC monoclonal antibodies (mAbs) were used in this study. RV-C2 is a cardiac TnT-specific mAb obtained from mice immunized with myofibrillar proteins from hypothyroid rabbit (Saggin er al. 1988). T-59, which reacts with both cardiac and skeletal TnT isoforms, was obtained from mice immunized with a myofibrillar preparation from bovine heart, as previously described (Saggin et al. 1988 and 1989). BF-G6 is an anti-embryonic MHC obtained from mice immunized with bovine fetal skeletal myosin (Schiaffino et al. 1986; Gorza et al. 1988; Schiaffino et al. 1988).

Skeletal and cardiac muscles from fetuses, newborns and adult rats were directly homogenized in Laemmli’s buffer (Laemmli, 1970) to minimize proteolytic degradation. Proteins were separated by SDS-PAGE using 8 % gels (Anderson and Oakeley, 1989) and stained with Coomassie blue. Duplicate gels were electrophoretically transferred to nitro-cellulose sheets essentially as described by Piperno and Fuller (1985). By this method, we observed a complete elution of all the proteins having an apparent relative molecular mass less than 100x10³ without a significant transfer to a second sheet of nitrocellulose. The transfer was monitored by Coomassie blue staining of the gel and Ponceau Red staining of the nitrocellulose. The blots were first incubated with anti-TnT mAbs and then with peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dakopatts) and developed with 3,3’-diaminobenzidine (Sigma)’ in the presence of imidazole, as described elsewhere (Saggin et al. 1988).

Immunoperoxidase staining was performed using previously described procedures (Gorza et al. 1988). Briefly, cryosections of unfixed tissues were incubated with appropriate dilutions of anti-TnT or anti-MHC mAbs for 30 min at 37°C. Unbound antibodies were removed by repeated rinses in phosphate-buffered saline (PBS). Sections were then incubated with appropriate dilutions of anti-mouse immunoglobulins conjugated with peroxidase (Dakopatts) for 30 min at 37 °C. After several rinses in PBS, the sections were incubated in 1 mg ml^-1 of p-phenylendiamine-pyrocathecol (Polysciences) in Tris-HCl 0.05 M, pH 7.6 in the presence of 0.01% hydrogen peroxide, to reveal peroxidase activity. Correlation of TnT and MHC expression was studied using double immunofluorescence. Sections were first stained for TnT using unlabelled RV-C2 followed by rhodamine-conjugated anti-mouse immunoglobulins (Dakopatts), then treated with non-immune mouse immunoglobulins to block anti-mouse immunoglobulins free binding sites and subsequently stained for MHC using mAb BF-G6 directly conjugated with fluorescein isothiocyanate (Sigma) as previously described (Azzarello et al. 1987).

Immunoblotting analysis of mAbs RV-C2 and T-59 with skeletal and cardiac muscle is illustrated in Fig. 1. We have previously reported that RV-C2 reacts specifically with two TnT isoforms expressed in fetal and adult rat heart, the adult isoform showing a slightly higher electrophoretic mobility (Saggin et al. 1988). As shown in Fig. 1A, the two isoforms are better resolved using the SDS-PAGE procedure described by Anderson and Oakeley (1989). In addition, this procedure reveals another band with intermediate mobility, which is absent in fetal heart and progressively increases from neonatal to adult stages, and a higher mobility band, whose nature remains to be determined.

Adult skeletal muscle is completely unreactive with RV-C2; by contrast, skeletal muscle from 18-day-old fetus shows a reactive band having the same electrophoretic mobility as the major embryonic cardiac TnT isoform. Both the embryonic and the major adult cardiac TnT isoforms are present in skeletal muscle from 1- and 7-day-old rats, while only a trace amount of the adult form is barely detectable by 15 days.

To determine whether other TnT species are present in developing skeletal muscle, we used a mAb (T-59) that reacts with both cardiac and skeletal muscle TnTs in rat tissue homogenates (Fig. IB). Immunoblotting analysis shows that TnT switching in developing rat skeletal muscle involves the sequential expression of distinct fetal, neonatal and adult TnT isoforms. In fetal muscle, only the embryonic cardiac TnT isoform is identified. In 1- and 7-day-old rats, T-59 stains two major neonatal-specific bands, in addition to the two cardiac TnTs: neonatal bands have higher electrophoretic mobility than cardiac TnTs but lower mobility than the TnT species present in fast and slow muscles from adult rats. A complex TnT profile is apparent by 15 days: in addition to the two neonatal bands, T-59 stains a slower migrating component and faster migrating bands similar to those seen in adult muscle.

Muscle fibers in fetal and neonatal rat skeletal muscle are labelled by anti-cardiac TnT, whereas adult muscle fibers are completely unreactive (Fig. 2). The distribution of cardiac TnT in developing skeletal muscle was compared to that of embryonic MHC using immuno-peroxidase and double immunofluorescence procedures. In 11-day embryos, numerous cells in the myotomal region of the somites are labelled by both antibodies; at this stage, as well as in 13.5-day embryos, only rare fibers stain selectively with either one or the other antibody (Fig. 3). In the late fetal and neonatal stages, the reactivity for cardiac TnT varies in different muscles without any obvious relation to specific fiber types: thus a stronger reaction is seen in soleus and tibialis anterior compared to gastrocnemius muscles. In contrast, reactivity for embryonic MHC is homo-geneously strong in all hindlimb muscles (Fig. 4). During postnatal development, the reactivity for cardiac TnT is progressively and homogeneously reduced in the different fiber types in soleus and tibialis muscles, while embryonic MHC persists for several weeks in type 2A fibers (Fig. 5).

The intrafusal fibers of muscle spindles are the only fibers reacting for cardiac TnT and MHC in adult hindlimb muscles: however, as shown in Fig. 6, the bag fibers are more reactive for cardiac TnT, whereas the chain fibers are more reactive for embryonic MHC (see also Rowlerson et al. 1985). Embryonic MHC is known to be expressed also in extraocular muscles of adult rats (Wieczorek et al. 1985; Sartore et al. 1987); in contrast, no reactivity for cardiac TnT is observed in extraocular muscles (data not shown).

3 to 5 days following a cold injury numerous regenerating fibers in the injured area were found to stain for cardiac TnT (Fig. 7A). Reactivity persists in subsequent days and begin to disappear 11 days after the lesion (Fig. 7B). Double-labelling experiments showed that most regenerating fibers reacting for cardiac TnT are also reactive for embryonic MHC. However, the relative intensity of staining with the two antibodies is not closely correlated in the different fibers and some fibers stain only with one or the other antibody (Fig. 8).

Cardiac TnT was also detected in a number of denervated muscle fibers like embryonic MHC (Schiaffino et al. 1988). As shown in Fig. 9, many fibers staining for cardiac TnT are often unreactive for embryonic MHC and the opposite is also true. We have previously reported that re-expression of embryonic MHC after denervation is confined to a specific fiber type, the type 2A fibers (Schiaffino et al. 1988). In contrast, as determined by analysis of serial sections with fiber type-specific anti-MHC mAbs (Schiaffino et al. 1989), cardiac TnT immunoreactivity can be detected in different fiber types (not shown).

This study shows that in the rat, like in the chicken (Toyota and Shimada, 1981; Cooper and Ordhal, 1985; Ogasawara et al. 1987), two TnT isoforms antigenically and electrophoretically similar to those expressed in the embryonic and adult heart are transiently expressed in fetal and neonatal skeletal muscle. This is a further example of developmentally regulated sarcomeric protein isoforms, like embryonic myosin light chain (Whalen et al. 1978), embryonic and neonatal MHCs (Whalen et al. 1981), and cr-cardiac actin (Minty et al. 1982), which are transiently expressed in developing skeletal muscle. Embryonic and adult cardiac TnT are sequentially expressed during skeletal muscle development: the embryonic isoform is predominantly expressed in fetal stages while both isoforms are present in 1-day and 7-day newborns and the adult form is the only detectable TnT species by 15 days. These findings suggest that in the rat, like in the chicken (Cooper and Ordhal, 1985), a similar switch in splicing of cardiac TnT pre-mRNA takes place during development in cardiac and skeletal muscle. However, the level of expression of the cardiac TnT gene in developing skeletal muscle appears to vary according to species. In the chicken, the cardiac TnT gene is expressed at similar levels in embryonic skeletal and cardiac muscle (Cooper and Ordhal, 1985). In the rat, the pattern of accumulation of cardiac TnT closely parallels that of the corresponding mRNA, which is present in much lower amounts in fetal skeletal compared to fetal cardiac muscle (Ausoni et al. in preparation). These findings suggest that the level of cardiac TnT protein in developing skeletal muscle is regulated by changes in the level of the corresponding mRNA.

Immunoblotting analysis with a mAb that reacts with both cardiac and skeletal muscle TnT species reveals a distinct TnT profile in neonatal skeletal muscle. In addition to the cardiac TnT isoforms, hindlimb muscles from 1- and 7-day-old rats contain two other TnT bands that migrate ahead of cardiac TnTs but have lower mobility than the TnT species found in fast and slow skeletal muscles from adult animals. An additional lower mobility component is seen in muscles from 15-day-old animals. The nature of these neonatal-specific TnT variants remains to be determined. Previous genomic and cDNA analyses have shown that alternative splicing of the fast skeletal muscle TnT gene generates multiple mRNAs, which are differentially expressed in developing and adult rat skeletal muscle (Medford et al. 1984; Breitbart et al. 1985). It is therefore likely that the neonatal-specific TnT forms are the product of mRNAs derived from alternative splicing of the fast skeletal muscle TnT gene. Multiple TnT variants, presumably derived by developmentally regulated pre-mRNA splicing of the fast skeletal TnT gene (Bucher et al. 1989), have been detected in neonatal chicken breast muscle (Abe et al. 1986).

Cardiac TnT is re-expressed during muscle regeneration, like embryonic and neonatal MHCs (Sartore et al. 1982) and embryonic myosin light chain (Carraro et al. 1983), and can thus represent a useful marker for regenerating fibers. Cardiac TnT is also re-expressed in a number of muscle fibers after denervation, as previously described for embryonic and neonatal MHCs (Schiaffino et al. 1988). A reversion to an embryonic pattern of troponin T synthesis has also been described in regenerating and denervated chicken muscle, where the leg-type TnT typical of the embryonic stages is re-expressed in those muscles that contain the breast-type TnT as the major adult isoform (Obinata et al. 1984; Matsuda et al. 1984; Shimizu and Shimada, 1985).

In this study, we have used an immunocytochemical approach to investigate at the single fiber level the expression of two developmentally regulated sarcomeric proteins, cardiac TnT and embryonic MHC, during normal development, regeneration and denervation. Our findings show that there is no close correlation between the expression of the two proteins. Non-coordinated regulation for the synthesis of myofibrillar proteins has previously been observed in regenerating and denervated chicken skeletal muscle (Matsuda et al. 1984; Saad et al. 1988). The difference in the pattern of disappearance of cardiac TnT and embryonic MHC during postnatal development is particularly striking: whereas the level of cardiac TnT declines progressively and homogeneously in all fiber types, embryonic MHC rapidly disappears after birth in all fiber types except in type 2A fibers where it persists for several weeks (see also Schiaffino et al. 1988). In developing chicken skeletal muscle repression of the cardiac TnT gene is controlled at the level of transcriptional initiation (Long and Ordhal, 1988), and is probably nerve-dependent (Toyota and Shimada, 1983). It is not known whether transcriptional and/or post-transcriptional mechanisms operate to repress the embryonic MHC gene, but our results suggest that this process is modulated by fiber-type-specific factors that may also be implicated in the selective re-appearance of embryonic MHC immunoreactivity in type 2A fibers after denervation. In contrast, fiber-type-specific factors do not appear to be involved in the regulation of the cardiac TnT gene.

This work was supported in part by grants from Ministero della Pubblica Istruzione and from Consiglio Nazionale delle Ricerche (Special Project FATMA). We thank Massimo Fabbri and Maurizio Moretto for technical assistance.