Fast Skeletal Muscle Troponin Research Articles

Assignment of the human fast skeletal muscle troponin C gene (TNNC2) between D20S721 and GCT10F11 on chromosome 20 by somatic cell hybrid analysis

The chromosomal location of the human fast skeletal muscle troponin C (TNNC2) gene was determined using somatic cell hybrids. PCR-based analysis of a 'monochromosomal' hybrid panel identified the presence of the TNNC2 gene on human chromosome 20 and subsequent analysis of the Genebridge4 radiation hybrid panel located the gene between D20S721 and GCT10F11 with a lod score of > 3.

Characterization of the Single Tyrosine Containing Troponin C from Lungfish White Muscle. Comparison with Several Fast Skeletal Muscle Troponin C's from Fish Species

Troponin C molecules from fast skeletal muscle of the following fish species (trout, whiting, lungfish, tilapia, and cod) have been purified to homogeneity. Upon binding of Ca2+ or Mg2+, lungfish troponin C is the only troponin C from fish white muscle to show the typical increase of tyrosine fluorescence emission quantum yield reported for rabbit fast skeletal muscle troponin C. The increase of tyrosine fluorescence signal occurring upon Ca2+ and Mg2+ titration of lungfish troponin C has been used to determine the corresponding affinity constants. With K(Ca) = 7.0 107 M−1 and K(Mg) = 3.6 103 M−1, the sites probed by the tyrosine residue of lungfish troponin C are typical of the COOH-terminal domain of fast skeletal troponin C's. The amino acid sequencing of the tyrosine containing tryptic peptides has allowed us to position the single tyrosine residue at position 7 in the Ca2+ binding loop of the third site, in identical position to Tyr109 of troponin C from rabbit fast skeletal muscle. Metal ion binding studies followed by intrinsic fluorescence or Tb3+ luminescence indicate that the conformation of the structural domain of lungfish troponin C with one metal ion bound is close to the physiological conformation of this domain.

Primary structure and developmental acidic to basic transition of 13 alternatively spliced mouse fast skeletal muscle troponin T isoforms

Large samples of original cDNAs encoding neonatal and adult mouse fast skeletal muscle troponin T (fTnT) have been isolated and characterized. The results demonstrate expression relationships of 8 alternatively spliced exons of the fTnT gene and reveal the primary structure of as many as 13 fTnT isoforms that diverge into acidic and basic classes due to differential mRNA splicing in the N-terminal variable region. In the C-terminal variable region encoded by the mutually exclusive exons 16 and 17, the splicing pathway and structure of exon 16 appears to be adult fTnT-specific, suggesting an adaptation to the functional demands of mature fast skeletal muscle. The cloned cDNAs were expressed in E. coli as standards to identify a high M r to low M r, acidic to basic fTnT isoform transition in postnatal developing skeletal muscles. Different from the developmental cardiac TnT switch generated by alternative splicing of a single exon, the fTnT isoform transition is an additive effect of alternative splicing of multiple N-terminal-coding exons, especially exons 4, 8 and fetal that are expressed at higher frequencies in the neonatal than in the adult muscle. The developmental fTnT isoform primary structure transition in both N- and C-terminal variable regions suggest a physiological importance of the apparently complex TnT isoform expression.

Cardiac and skeletal muscle troponin I isoforms are encoded by a dispersed gene family on mouse chromosomes 1 and 7.

We mapped the locations of the genes encoding the slow skeletal muscle, fast skeletal muscle, and cardiac isoforms of troponin I (Tnni) in the mouse genome by interspecific hybrid backcross analysis of species-specific (C57BL/6 vs Mus spretus) restriction fragment length polymorphisms (RFLPs). The slow skeletal muscle troponin I locus (Tnni1) mapped to Chromosome (Chr) 1. The fast skeletal muscle troponin I locus (Tnni2), mapped to Chr 7, approximately 70 cM from the centromere. The cardiac troponin I locus (Tnni3) also mapped to Chr 7, approximately 5-10 cM from the centromere and unlinked to the fast skeletal muscle troponin I locus. Thus, the troponin I gene family is dispersed in the mouse genome.

Pretreatment of myoblast cultures with basic fibroblast growth factor increases the efficacy of their transplantation in mdx mice.

The effect of pretreatment of cultures with basic fibroblast growth factor (bFGF) on myoblast allotransplantation to C57BL/10ScSn mdx/mdx mouse (mdx mouse) muscles not previously damaged and not irradiated was studied. Transgenic CD1 mice which have a beta-galactosidase gene under the control of the promoter of the quail fast skeletal muscle troponin I gene, were used as donors. The myoblasts were grown with 100 ng/mL bFGF during the last 2 days before injecting them in the left tibialis anterior (TA) muscles of mdx mice. Myoblasts from the same primary cultures were also grown without bFGF and injected in the right TA muscles as control. The recipient mice were immunosuppressed with FK 506. Twenty-eight days after myoblast transplantation, the percentage of beta-galactosidase-positive fibers was significantly higher (more than fourfold) following culture with bFGF than without bFGF. Almost all beta-galactosidase-positive fibers were also dystrophin positive. Direct intramuscular injections of bFGF or of Hank's balanced salt solution (HBSS) at the time of myoblast transplantation and at several intervals afterwards were also investigated. The percentage of beta-galactosidase-positive fibers did not differ significantly following intramuscular injection of bFGF from controls injected with HBSS. In vitro, this high concentration of bFGF significantly reduced the formation of myotubes, and the percentage of mononuclear cells which were myoblasts was significantly increased by 34%. These observations alone do not account for the fourfold increase in transplantation success. The presence of bFGF in the culture did not significantly increase the cell survival 3 days after their transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Utilization of myoblasts from transgenic mice to evaluate the efficacy of myoblast transplantation

A possible treatment for Duchenne muscular dystrophy is the injection of normal myoblasts into dystrophic muscles to induce the formation of new, healthy, and dystrophin-positive muscle fibers. To develop this therapy, it is important to identify the muscle fibers formed by the injected myoblasts in the host muscles. In this study, we used myoblasts from transgenic mice which have a gene expressing beta-galactosidase under the control of the promoter of quail fast skeletal muscle troponin I. This transgene is expressed in myotubes and muscle fibers, but not in myoblasts. Twenty-eight days after myoblast transplantation in nude and in mdx mice, muscle fibers containing of beta-galactosidase were identified by x-gal staining. In mdx mice, most of the beta-galactosidase-positive muscle fibers resulting from the myoblast transplantation were also dystrophin positive. This technique could make it possible to follow the success of myoblast transplantation even in mice that are not depleted of dystrophin.

Expression of troponin C genes during development in the chicken.

Expression of cardiac troponin C (C/STnC) and fast skeletal muscle troponin C (FTnC) genes during development was analyzed by in situ hybridization and Northern blot. At Hamburger-Hamilton stage 14, FTnC mRNA was detected in somites. At stage 20, FTnC mRNA was identified in truncus arteriosus. FTnC mRNA was undetectable in embryonic ventricle at the other developmental stages. This suggested that transcription of FTnC mRNA at stage 20 was stage and region-specific. At early stage 10, C/STnC mRNA was detected in truncus arteriosus, ventricle and vitelline veins at the proximal region where right and left veins bifurcate from ventricle. In the proximal region of vitelline vein, C/STnC mRNA was transcribed in both the rostral and caudal walls of the veins. The area of C/STnC gene expression in vitelline vein is wider than those of alpha-cardiac and smooth muscle actin genes (Ruzicka and Schwartz, J. Cell Biol. 107: 2575-2586, 1988). It is suggested that C/STnC plays an important role relating embryonic excitation-contraction-coupling in the wider area including vitelline vein. C/STnC mRNA was identified also in somites, embryonic breast muscle and adult breast muscle. Since C/STnC protein was undetectable in adult breast muscle by immunofluorescence microscopy (Toyota and Shimada, J. Cell Biol. 91: 497-504, 1981), the imbalance of C/STnC mRNA and protein levels suggested the operation of specific mechanisms that separately regulated the accumulation of C/STnC mRNA and protein.

Novel developmentally regulated exon identified in the rat fast skeletal muscle troponin T gene

In theory, the rat fast skeletal muscle troponin T gene can generate 64 different isoforms. Here we report the identification of a novel alternative exon (exon y) that increases the potential isoform variation to 128. The inclusion of exon y in fast skeletal muscle troponin T mRNA occurs in perinatal, but not adult, skeletal muscle. Exon y is located between exons 8 and 9. This is the first time that a developmentally regulated exon located amongst a set of alternatively spliced exons has been described. Exon y is included in two mRNA isoforms. The proteins that these mRNAs would encode have molecular masses greater than that of the largest fast skeletal muscle troponin T isoform lacking exon y. These two proteins correlate well in both size and pattern of expression with the two fast skeletal muscle troponin T isoforms expressed in perinatal skeletal muscle. These results indicate that there is coordinated regulation of the splicing of exon y with other alternative exons.

Complex fiber-type-specific expression of fast skeletal muscle troponin I gene constructs in transgenic mice

We analyzed, in transgenic mice, the cellular expression pattern of the quail fast skeletal muscle troponin I (TnIfast) gene and of a chimeric reporter construct in which quail TnIfast DNA sequences drive expression of E. coli beta-galactosidase (beta-gal). Both constructs were actively expressed in skeletal muscle and specifically in fast, as opposed to slow, muscle fibers. Unexpectedly, both constructs showed a marked differential expression among the adult fast fiber subtypes according to the pattern IIB > IIX > IIA. This expression pattern was consistent in multiple lines and differed from the endogenous mouse TnIfast pattern, which shows approximately equal expression in all fast fibers. These observations indicate that distinct regulatory mechanisms contribute to high-level expression of TnIfast in the various fast fiber subtypes and suggest that the outwardly simple pattern of equal expression in all fast fiber types shown by the endogenous mouse TnIfast gene is based on an intricate system of counterbalancing mechanisms. The adult expression pattern of the TnIfast/beta-gal construct emerged in a two-stage developmental process. Differential expression in fast versus slow fibers was evident in neonatal animals, although expression in fast fibers was relatively weak and homogeneous. During the first two weeks of postnatal life, expression in maturing IIB fibers was greatly increased whereas that in IIA/IIX fibers remained weak, giving rise to marked differential expression among fast fiber types. Thus at least two serially acting (pre- and post-natal) fiber-type-specific regulatory mechanisms contribute to high-level gene expression in adult fast muscle fibers. Unexpected similarities between TnIfast transgene expression and that of the myosin heavy chain gene family (which includes differentially expressed IIB-, IIX- and IIA-specific members) suggest that similar mechanisms may regulate adult fast muscle gene expression in a variety of unrelated muscle gene families.

Gene structure and expression of an unusual protein kinase from Plasmodium falciparum homologous at its carboxyl terminus with the EF hand calcium-binding proteins.

An unusual protein kinase gene, termed PfCPK, was isolated from Plasmodium falciparum. The gene, which contains five exons and four introns, encodes a product with a predicted length of 524 amino acids. The amino-terminal segment of the predicted protein contains all of the conserved sequences characteristic of a protein kinase catalytic domain and has a high homology to several protein serine-threonine kinase subfamilies (30-41% amino acid identities). These subfamilies include calcium/calmodulin-dependent protein kinases, calcium-dependent protein kinase, ribosomal S6 protein kinase, cyclic nucleotide-dependent protein kinases, protein kinase C, and the yeast SNF1 subfamily. All of these protein kinases are relatively close in the phylogeny tree and within the kinase catalytic domains have about 35% amino acid identities to each other, suggesting that PfCPK is also in this region of the phylogeny tree. An unusual feature of PfCPK is that its carboxyl-terminal segment displays homology to the EF hand calcium-binding proteins, for example 34% amino acid identity to chicken fast skeletal muscle troponin C and 35% amino acid identity to human calmodulin. Like troponin Cs and calmodulins, PfCPK also contains four EF hand calcium-binding motifs. Furthermore, the four introns in the PfCPK gene are all located in the carboxyl-terminal putative EF hand calcium-binding region (EF hand calcium-binding proteins from higher eukaryotes generally contain multiple introns). This combination of a protein kinase and an EF hand calcium-binding protein in a single polypeptide implies that PfCPK may be directly activated by calcium. Constructs containing the full-length PfCPK cDNA have been expressed in Escherichia coli at a high level to generate a 60-kDa recombinant protein. Compared with similar fractions from control cells, the fraction containing PfCPK recombinant protein exhibited an elevated protein kinase activity which was Ca(2+)-dependent.

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.

Effect of Myosin Cross-Bridge Interaction with Actin on the Ca2+-Binding Properties of Troponin C in Fast Skeletal Myofibrils1

Ca2+ binding to fast skeletal muscle troponin C reincorporated into troponin C-depleted (CDTA-treated) myofibrils has been measured directly by using 45Ca and indirectly by using a fluorescent probe. Direct Ca2(+)-binding measurements have shown that the Ca2+ affinity of the low-affinity sites is enhanced in the absence of ATP and conversely reduced when myosin is selectively extracted from myofibrils, compared to the Ca2+ affinity in the presence of ATP. Fluorescence intensity changes of a dansylaziridine label at the Met-25 residue of troponin C have shown the same Ca2(+)-sensitivity whether or not ATP is present, while much lower Ca2(+)-sensitivity is seen in the myosin-extracted myofibrils. Since the Met-25 residue is in the amino terminal side alpha-helix of Ca2(+)-binding site I and far from Ca2(+)-binding site II in the primary structure, Ca2+ binding to site II has been evaluated by assuming that the fluorescence change monitors Ca2+ binding to site I alone. Ca2+ binding to site II thus estimated has shown high positive cooperativity only in the presence of ATP and has been found to be nearly proportional to the activation of myofibrillar ATPase, suggesting that Ca2(+)-binding site II is directly involved in the activation of myofibrillar ATPase activity. On the other hand, Ca2(+)-binding site I has been suggested to regulate the interaction of weakly binding cross-bridges with the thin filament, since the fluorescence change in the presence of ATP is saturated at the free Ca2+ concentration required for the activation of myofibrillar ATPase.

Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity.

To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.

The structure and regulation of expression of the murine fast skeletal troponin C gene. Identification of a developmentally regulated, muscle-specific transcriptional enhancer.

Fast skeletal muscle troponin C (sTnC) is the calcium-binding subunit of the myofibrillar thin filament that regulates excitation-contraction coupling. Utilizing a polymerase chain reaction cloning strategy, we have isolated cDNA clones encoding murine sTnC. The 160-amino acid sTnC protein shares 70% amino acid sequence identity with the slow/cardiac isoform of troponin C (cTnC). However, three areas of significant sequence divergence were identified. Southern blot analyses demonstrated that murine sTnC is encoded by a single copy gene that is distinct from that which encodes cTnC. Northern blot analyses showed that the sTnC gene is expressed exclusively in skeletal muscle (extensor digitorum and anterior tibialis) and not in neonatal or adult heart, brain, kidney, liver, lung, or testes. Studies of the murine C2C12 myoblast cell line demonstrated that sTnC gene expression is developmentally regulated during the differentiation of these myoblasts into myotubes. A full-length murine sTnC genomic clone was isolated and characterized by DNA sequence, primer extension, and S1 nuclease protection analyses. The sTnC gene is composed of six exons spanning 2.6 kilobase pairs of genomic DNA. Although the introns do not divide the gene into functional domains, the intron-exon borders are nearly identical to those of the other members of the troponin C multigene family. Transient transfection assays using chloramphenicol acetyltransferase reporter plasmids demonstrated that the sTnC promoter alone is relatively inactive in muscle cells and that high level sTnC gene expression in these cells is controlled by a potent transcriptional enhancer element located within the first intron of the gene. In additional transfection experiments, the sTnC enhancer was shown to display three important biological activities. (i) It was required for high level transcription from the sTnC promoter in muscle cells; (ii) its activity was muscle cell specific; and (iii) its activity was developmentally regulated during the differentiation of C2C12 myoblasts to myotubes. Taken together, these data define the sTnC gene as an excellent model system for studies of developmentally regulated gene expression in skeletal muscle.

Cloning, structural analysis, and expression of the human fast twitch skeletal muscle troponin C gene.

The gene encoding human fast skeletal muscle troponin C (TnC) was cloned, mapped, and sequenced. The locations of intron positions in this gene were compared to those in the related genes for mouse slow skeletal TnC and vertebrate and nonvertebrate calmodulins. We detected strikingly similar purine-rich DNA sequences on the coding strand in the basal promoter of the genes for fast and slow troponin C and chicken calmodulin II which may represent conserved regulatory elements in genes of the vertebrate troponin C/calmodulin gene family. We mapped the transcriptional start site of the gene and analyzed the expression of TnC test genes in the myogenic cell lines C2, L8, and H9c2(2-1) and in the human fibroblast line HuT12. Constructs comprising 4.7 or 6.2 kilobase pairs of 5'-flanking sequence (including the genuine transcriptional start site) upstream of the chloramphenicol acetyltransferase gene as reporter expressed the hybrid gene in C2 cells but not in nonmuscle cells. Surprisingly, no expression was found in cell lines L8 and H9c2(2-1) despite the fact that all three muscle cell lines vigorously express the endogenous TnC fast mRNA after differentiation. The discrepancy between the expression of endogenous genes and the test gene in these cell lines indicates different requirements for regulatory elements in different myogenic cells.

CDNA Clone and Expression Analysis of Rodent Fast and Slow Skeletal Muscle Troponin I mRNAs

We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.

Developmental and Muscle-specific Regulation of Avian Fast Skeletal Troponin T Isoform Expression by mRNA Splicing

We have investigated the developmental regulation of the avian fast skeletal muscle troponin T (TnTf) gene of the Japanese quail. Sequence analysis of troponin T mRNA, cDNA clones, and a genomic DNA segment demonstrate that the avian, fast skeletal TnTf protein isoforms are produced from a single gene. This TnTf gene is expressed in skeletal muscle, but not in adult cardiac muscles or in non-muscle tissues. In addition to known TnT isoforms, three new isoforms of TnT are described. These isoforms arise by regulated alternative RNA splicing of exons in the 5' and 3' regions of TnTf transcripts. Alternative splicing of the 5' TnTf exons involves splicing of multiple exons in different combinations (i.e. not mutually exclusive), whereas 3' alternative splicing involves mutually exclusive splice choices between two exons (alpha or beta exons). S1 nuclease protection and primer extension analyses show that alternative splicing of both 5' and 3' exons is precisely regulated and coordinated in physiologically different striated muscles, which express distinct, restricted combinations of 5' and 3' alternatively spliced exons in mRNA transcripts. In contrast, different embryonic muscles and clonal embryonic myoblast cultures coexpress the 3' alternative splice choices. This indicates that alternative splicing of TnTf mRNAs is controlled in different adult muscles by specific trans factors, and not by the restricted expression of different spliced forms in different embryonic myoblast lineages. Comparison of TnTf isoform expression in quail and chicken flight muscle (Wilkinson, J. M., Moir, A. J., and Waterfield, M. D. (1984) Eur. J. Biochem. 143, 47-56) to TnTf isoforms of the rat (Breitbart, R. E., and Nadal-Ginard, B. (1986) J. Mol. Biol. 188, 313-324), and rabbit (Pearlstone, J. R., Carpenter, M. R., and Smillie, M. B. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1902-1906) indicates that the avian gene contains an additional exon(s) not present in mammalian genes. The alternative exon sequences TnTf mRNAs expressed in anatomically distinct quail muscles can be correlated with sequences in TnTf protein isoforms in these chicken muscles. Thus, the regulated splicing of alternative exons in TnT transcripts, and not selective translation of stochastically spliced TnT mRNAs, regulates TnTf isoform expression in specific muscles.

N-terminal amino acid sequences of three functionally different troponin T isoforms from rabbit fast skeletal muscle

The different isoforms of fast skeletal muscle troponin T (TnT) are generated by alternative splicing of several 5′ exons in the fast TnT gene. In rabbit skeletal muscle this process results in three major fast TnT species, TnT 1f, TnT 2f and TnT 3f, that differ in a region of 30 to 40 amino acid residues near the N terminus. Differential expression of these three isoforms modulates the activation of the thin filament by calcium. To establish a basis for further structure-function studies, we have sequenced the N-terminal region of these proteins. TnT 2f is the fast TnT sequenced by Pearlstone et al.. The larger species TnT 1f contains six additional amino acid residues identical in sequence and position to those encoded by exon 4 in the rat fast skeletal muscle TnT gene. TnT 3f also contains that sequence but lacks 17 amino acid residues spanning the region encoded by exons 6 and 7 of the rat gene. These three TnTs appear to be generated by discrete alternative splicing pathways, each differing by a single event. Comparison of these TnT sequences with those from chicken fast skeletal muscle and bovine heart shows that the splicing pattern resulting in the excision of exon 4 is evolutionarily conserved and leads to a more calcium-sensitive thin filament.

Differential expression of slow and fast skeletal muscle troponin C: Slow skeletal muscle troponin C is expressed in human fibroblasts

We have isolated and sequenced the cDNAs for human slow and fast skeletal muscle troponin C (TnC). Each cDNA is encoded by one of the two TnC genes in the human genome. The fast skeletal muscle TnC gene appears to be expressed exclusively in skeletal muscle. Only the slow TnC gene is expressed in human cardiac ventricle. The slow skeletal TnC gene is also expressed in skeletal muscle and, surprisingly, in several human fibroblast cell lines. Thus, at least one of the three proteins of the troponin complex appears to be expressed in non-muscle cells of higher vertebrates. The relative steady-state amounts of the slow and fast skeletal TnC mRNAs in various adult and embryonic striated muscles are similar to the expected amounts of the corresponding protein, suggesting that the expression of TnC genes is controlled predominantly by the production or accumulation of mRNA rather than by translational or post-translational mechanisms.

Isolation and sequence of a cDNA clone for rabbit fast skeletal muscle troponin C. Homology with calmodulin and parvalbumin.

The binding of Ca2+ to troponin C (TnC) regulates skeletal muscle contraction. We have isolated a full-length cDNA clone for fast skeletal muscle TnC from a neonatal rabbit skeletal muscle library and determined its nucleic acid sequence. The amino acid sequence deduced from this clone matches the previously reported amino acid sequence (Collins, J. H., Greaser, M. L., Potter, J. D., and Horn, M. J. (1977) J. Biol. Chem. 252, 6356-6362) except at the amino terminus. According to the nucleotide sequence, the first 2 residues of TnC are threonine-aspartic acid, which is the reverse of the order reported previously. The isolation of the adult form of TnC from a neonatal library suggests that there may be no developmental isoforms of fast TnC. The protein coding region of the fast TnC clone has 67% homology with the reported nucleotide sequence for chicken slow TnC (Putkey, J. A., Carroll, S. L., and Means, A. R. (1987) Mol. Cell. Biol. 7, 549-1553). The homologies between the nucleotide sequences of TnC, calmodulin, and parvalbumin provide evidence that all three proteins were derived from a common precursor molecule which had four Ca2+-binding sites.