Structural characterization of the human fast skeletal muscle troponin I gene (TNNI2)

Three troponin I genes have been identified in vertebrates that encode the isoforms expressed in adult cardiac muscle (TNNI3), slow skeletal muscle (TNNI1) and fast skeletal muscle (TNNI2), respectively. While the organization and regulation of human cardiac and slow skeletal muscle genes have been investigated in detail, the fast skeletal troponin I gene has to date only been examined in birds. Here, we describe the structure and complete sequence of the human fast skeletal muscle troponin I gene (TNNI2) and identify putative regulatory elements within both the 5' flanking region and the first intron. In particular, a region containing MEF-2, E-box, CCAC and CAGG elements was identified in intron 1 that closely resembles the fast internal regulatory element (FIRE) of the quail intronic enhancer. We have previously shown that the fast skeletal muscle troponin I gene is located at 11p15.5 and noted potential close linkage with the fast skeletal muscle troponin T gene (TNNT3). Here, we have isolated two independent human PAC genomic clones that contain either TNNI2 or TNNT3 and demonstrate by interphase FISH mapping that they are less than 100 kb apart in the genome. The results demonstrate that the human TNNI2 gene is closely related to its avian counterparts with conserved elements within both the putative promoter and first intron. Our data further confirm close physical linkage of TNNI2 and TNNI3 on 11p15.5.     

Isolation and cloning by a polymerase chain reaction of a genomic DNA fragment of the human slow skeletal troponin (TNNT1) gene

The genomic 3′ structure of the gene coding for the human slow skeletal troponin T (TNNT1) gene, is reported. An intron of 912 nucleotides containing an Alu-element has been identified and characterized. The complexity of the sequenced region suggests an alternative exon use. The present results may be valuable for further studies on the gene structure of TNNT1 and the related troponin gene family.     

Sequences of complete cDNAs encoding four variants of chicken skeletal muscle troponin T

cDNAs containing the complete coding sequences of four isoforms of troponin T derived from 1-week-old chick skeletal muscle have been isolated and sequenced. While the 5' and 3' untranslated regions and most of the coding sequence were identical for each, dramatic differences were observed in the NH2-terminal region corresponding to amino acid residues 10-37 of rabbit skeletal troponin T. These sequence differences correspond to the alternatively spliced but not mutually exclusive exons 4 to 8 of the rat skeletal muscle troponin T gene. In addition, we observe a sequence corresponding to an extra exon or exons (between 5 and 6) present in the chicken skeletal muscle gene and not previously detected in the rat skeletal or chicken cardiac genes. This sequence of 63 nucleotides consists of an almost perfect repeat of 30 and 33 nucleotides and has previously been shown to be represented as a protein variant in chicken skeletal muscle. A difference is also present in one cDNA clone corresponding to the alternatively spliced (mutually exclusive) exons 16 and 17 of the rat gene. In the protein, this corresponds to a region implicated in the interaction of troponin T with troponin C, tropomyosin, and perhaps troponin I and F-actin.     

Assignment of the Human Fast Skeletal Troponin T Gene (TNNT3) to Chromosome 11p15.5: Evidence for the Presence of 11pter in a Monochromosome 9 Somatic Cell Hybrid in NIGMS Mapping Panel 2

Human fast skeletal troponin T (TnT_f), the tropomyosin binding component of the multisubunit troponin complex, plays an important role in the Ca²⁺regulation of striated muscle contraction. Specific primers designed from the 3′ end of human TnT_fcDNA were used to amplify an intronic region by polymerase chain reaction (PCR). This TnT_f-specific PCR product was detected from two somatic cell hybrids containing human chromosomes 9 and 11, respectively, in NIGMS mapping panel 2. However, further studies with other somatic hybrid cell lines (Bios Laboratory) localized the TnT_fgene (HGMW-approved symbol TNNT3) only to chromosome 11. This observation was further confirmed by fluorescencein situhybridization with a 12-kb TnT_fgenomic probe generated by extended PCR, showing the sublocalization of the gene to band p15.5 on chromosome 11. This locus is of specific interest, as Beckwith–Wiedemann syndrome and various childhood and adult tumor-related abnormalities have been mapped to this region. The study also indicates the presence of an 11pter region in the NIGMS cell hybrid GM10611, which has previously been reported to contain only human chromosome 9.     

A novel nemaline myopathy in the Amish caused by a mutation in troponin T1

The nemaline myopathies are characterized by weakness and eosinophilic, rodlike (nemaline) inclusions in muscle fibers. Amish nemaline myopathy is a form of nemaline myopathy common among the Old Order Amish. In the first months of life, affected infants have tremors with hypotonia and mild contractures of the shoulders and hips. Progressive worsening of the proximal contractures, weakness, and a pectus carinatum deformity develop before the children die of respiratory insufficiency, usually in the second year. The disorder has an incidence of approximately 1 in 500 among the Amish, and it is inherited in an autosomal recessive pattern. Using a genealogy database, automated pedigree software, and linkage analysis of DNA samples from four sibships, we identified an approximately 2-cM interval on chromosome 19q13.4 that was homozygous in all affected individuals. The gene for the sarcomeric thin-filament protein, slow skeletal muscle troponin T (TNNT1), maps to this interval and was sequenced. We identified a stop codon in exon 11, predicted to truncate the protein at amino acid 179, which segregates with the disease. We conclude that Amish nemaline myopathy is a distinct, heritable, myopathic disorder caused by a mutation in TNNT1.     

Assessment of a creatine kinase isoform M defect as a cause of myotonic dystrophy and the characterization of two novel CKMM polymorphisms

Summary Recent studies have shown the gene encoding creatine kinase isoform M (CKMM) to be very closely linked to the myotonic dystrophy (DM) locus on the long arm of chromosome 19. Given this close linkage to DM and the postulated role of CKMM in skeletal muscle contraction, the possibility of a defect in CKMM causing DM was investigated. CKMM cDNA was isolated from the skeletal muscle of an individual with DM. Sequencing of the CKMM cDNA from the DM chromosome 19 revealed two novel polymorphisms but no translationally significant mutation. This work rules out a defect in the coding segment of CKMM as a cause of DM in this family and, in light of genetic homogeneity shown to date for DM, probably in all cases of DM.     

cDNA sequence, tissue-specific expression, and chromosomal mapping of the human slow-twitch skeletal muscle isoform of troponin I

Troponin I (TnI) is a myofibrillar protein involved in the calcium-mediated regulation of striated muscle contraction. Three isoforms of TnI are known and each is expressed in a muscle fiber-type-specific manner. TnI-fast and TnI-slow are expressed exclusively in fast-twitch and slow-twitch skeletal muscle myofibers, respectively, while a third isoform, TnI-card, is expressed in both the atrium and the ventricle of the heart. An explanation of the myofiber-type-restricted expression of the troponin I multigene family will further aid in understanding how various types of striated muscle fibers are established. To initiate the study of TnI isoform gene expression, we have isolated a full-length cDNA representing the human slow-twitch skeletal muscle isoform of troponin I. Sequence comparisons demonstrate that the TnI-slow protein is highly conserved between species. Therefore, the cDNA was used as a probe to investigate the tissue-specific and developmental regulation of the TnI-slow gene in both rodent and human myogenic cells. TnI-slow message appears to be restricted to muscle tissue containing slow-twitch skeletal muscle myofibers. TnI-slow gene expression is induced in differentiated cultures of primary human muscle cells and several (but not all) myogenic cell lines. In addition, a human-specific probe prepared from the 3' untranslated region of the cDNA has been used to probe a panel of human/mouse somatic cell hybrid lines, resulting in the assignment of the human TnI-slow gene to the q12----qter region of chromosome 1. The locus is designated TNNI1.     

Close physical linkage of human troponin genes: organization, sequence, and expression of the locus encoding cardiac troponin I and slow skeletal troponin T

Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.     

Polymorphic forms of troponin T and troponin C and their localization in striated muscle cell types

1. 1. Immunochemical studies have shown that the major forms of troponin T present in fast skeletal, slow skeletal and cardiac muscles are different proteins.

2. 2. Similar studies indicate that the major form of troponin C present in fast skeletal muscles differs from troponin C present in slow skeletal and cardiac muscle cells. The forms of troponin C present in slow skeletal and cardiac muscles are immunochemically very similar.

3. 3. The antibodies to the polymorphic forms of troponin T and troponin C are specific for the muscle type, except in the case of the slow skeletal and cardiac muscle forms of troponin C.

4. 4. By the immunoperoxidase technique, it has been shown that the fast skeletal muscle troponin T is localized in type II cells and slow skeletal muscle troponin T in type I cells.

5. 5. Fast skeletal muscle troponin C is present in type II cells and a different troponin C, identified by its reaction with the antibody against cardiac troponin C, is present in type I cells.

6. 6. It is concluded that in normal adult skeletal muscle, fast muscle forms of troponin I, troponin T and troponin C are present together as a homocomplex in type II cells and the slow muscle forms exist as an analagous homocomplex in type I cells.

     

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.     

Troponin from the myocardium and skeletal muscles: structure and properties

The literary and experimental data on the structure and properties of cardiac and skeletal muscle troponin are reviewed. The cation--binding sites of cardiac and skeletal muscle troponin C are distinguished by specificity; the sites localized in the C-terminal part of the protein molecule can bind both Ca2+ and Mg2+, whereas the sites localized at the N-end specifically bind Ca2+. The use of bifunctional reagents revealed a number of helical sites within the structure of cardiac troponin C (residues 84-92 and 150-158) and of skeletal muscle troponin C (residues 90-98 and 125-136). A comparison of experimental data with the results of an X-ray analysis testifies to the presence in the central part of the troponin C molecule of a long alpha-helical sequence responsible for troponin C interaction with the inhibiting peptide of troponin I. The efficiency of interaction of troponin components depends on Ca2+ concentration; the integrity of the overall troponin complex is mainly provided for by troponin C interaction with troponin I and by troponin I interaction with troponin T. The interaction between troponins T and C is relatively weak, especially in the case of cardiac troponin components. Both skeletal and cardiac muscles synthesize several troponin T isoforms differing in length and amino acid composition of N-terminal 40-60 member peptides. Troponin T isoforms can undergo phosphorylation by several protein kinases. The single site of troponin T which exists in a phosphorylated state in vivo (residue Ser-1) undergoes phosphorylation by specific protein kinase (troponin T kinase) related to casein kinases II. It was assumed that the phosphorylation of Ser-1 residue of troponin T as well as the synthesis of troponin T isoforms differing in the structure of the N-terminal peptide, provides for the regulation of interaction between two neighbouring tropomyosin molecules.     

Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart

We have isolated and sequenced a cDNA encoding rat cardiac troponin I. The predicted amino acid sequence was highly identical with previously reported chemically derived amino acid sequences for rabbit and bovine cardiac troponin I. Clones for slow skeletal muscle troponin I were also obtained from neonatal rat cardiac ventricle by the polymerase chain reaction. The nucleotide sequences of these clones were determined to be more than 99% identical with a previously reported rat slow skeletal troponin I cDNA [Koppe et al. (1989) J. Biol. Chem. 264, 14327-14333]. The troponin I clones hybridized to RNA from the appropriate muscle from adult animals. However, RNA from fetal and neonatal rat heart also hybridized with the slow skeletal troponin I cDNA, demonstrating its expression in fetal and neonatal rat heart. Slow skeletal troponin I steady-state mRNA levels decreased with increasing age, but cardiac troponin I mRNA levels increased through fetal and early neonatal cardiac development. Thus, during fetal and neonatal development, slow skeletal and cardiac troponin I isoforms are coexpressed in the rat heart and regulated in opposite directions. The degree of primary sequence differences in these isoforms, especially at phosphorylation sites, may result in important functional differences in the neonatal myocardium.     

Phosphorylation of isolated components of the troponin complex of skeletal and cardiac muscle phosphorylase kinase from bird skeletal muscles

Pigeon and chicken skeletal muscle phosphorylase kinase purified to a nearly homogeneous state is able to phosphorylate both cardiac and skeletal troponin I and T. After 1-hr incubation, the enzyme transfers up to 0.35 mole of phosphorus per mole of skeletal troponin I, up to 0.5 mole of cardiac troponin I and up to 0.1 mole of cardiac and skeletal troponin T. Avian muscle phosphorylase kinase does not phosphorylate the first serine residue of cardiac and skeletal troponin T, but catalyzes the phosphate incorporation into the site(s) of troponin T located in the central or C-terminal parts of the protein molecule. The rate of troponin phosphorylation by pigeon muscle phosphorylase kinase is pH-dependent: the 6.8/8.2 ratio for troponin I is close to 0,2, whereas that with troponin T varies in the range of 0.5-0.7. Troponin phosphorylation by avian phosphorylase kinase depends on the presence of Ca2+ in the incubation mixture. In the presence of 3 mM EGTA troponin I phosphorylation is inhibited by 70-90%, whereas that of troponin T--by 50%. The experimental results indicate that the phosphorylation of troponin I and T is catalyzed either by two different active centers or by different conformations of the single center of avian phosphorylase kinase.