Troponin T Gene Research Articles

Early Divergence of the C-Terminal Variable Region of Troponin T Via a Pair of Mutually Exclusive Alternatively Spliced Exons Followed by a Selective Fixation in Vertebrate Heart.

Troponin T (TnT) is the thin filament anchoring subunit of troponin complex and plays an organizer role in the Ca2+-regulation of striated muscle contraction. From an ancestral gene emerged ~ 700 million years ago in Bilateria, three homologous genes have evolved in vertebrates to encode muscle type-specific isoforms of TnT. Alternative splicing variants of TnT are present in vertebrate and invertebrate muscles to add functional diversity. While the C-terminal region of TnT is largely conserved, it contains an alternatively spliced segment emerged early in C. elegans, which has evolved into a pair of mutually exclusive exons in arthropods (10A and 10B of Drosophila TpnT gene) and vertebrates (16 and 17 of fast skeletal muscle Tnnt3 gene). The C-terminal alternatively spliced segment of TnT interfaces with the other two subunits of troponin with functional significance. The vertebrate cardiac TnT gene that emerged from duplication of the fast TnT gene has eliminated this alternative splicing by the fixation of an exon 17-like constitutive exon, indicating a functional value in slower and rhythmic contractions. The vertebrate slow skeletal muscle TnT gene that emerged from duplication of the cardiac TnT gene has the exon 17-like structure conserved, indicating its further function in sustained and fatigue resistant contractions. This functionality-based evolution is consistent with the finding that exon 10B-encoded segment of Drosophila TnT homologous to the exon 17-encoded segment of vertebrate fast TnT is selectively expressed in insect heart and leg muscles. The evolution of the C-terminal variable region of TnT demonstrates a submolecular mechanism in modifying striated muscle contractility and for the treatment of muscle and heart diseases.

Familial Hypertrophic Cardiomyopathy - Identification of cause and risk stratification through exome sequencing

BackgroundHypertrophic Cardiomyopathy (HCM) with variable clinical presentations and heterogeneity is the common cause of sudden cardiac death. Genetic diagnosis is challenging in these complex diseases but exome sequencing as a genetic diagnostic tool provides explainable results. MethodsIn a familial Hypertrophic Cardiomyopathy with multigenerational inheritance with apparent phenotype, had a history of sudden death and severe arrhythmia followed by implantation of Implantable cardioverter defibrillator (ICD). Exome sequencing (100×) trailed by effective filtering steps for exome variants on the basis of different parameters, segregated variants are prioritized for the disease and further clinical relevance are evaluated for the variants. ResultsA rare causal variant in troponin-T gene (TNNT2, NM_000364.3;c.274C > T;p.Arg92Trp) is identified, shared by only affected members, absent in unaffected members and also in 200 unrelated control chromosomes. TNNT2 mutation act as a driver mutation but mutations in other disease-related genes, KCNMB1, LPL, APOE and other biochemical factors provides risk stratification within affected family members. ConclusionThis study contributes to the role of “rare variants” in complex disease phenotypes and heterogeneity within family and the necessity of whole exome targeted approaches in complex cardiomyopathy, which are known to harbor private mutations.

Hypertrophic Cardiomyopathy. A Study of the Troponin-T Gene in 127 Spanish Families

The information available on the correlation between genotype and phenotype and the prognostic implications of different troponin-T gene mutations is sparse and, at times, contradictory. We studied the TNNT2 gene in 127 patients with hypertrophic cardiomyopathy and identified three mutations in patients from four families (3.1%): the Phe87Leu mutation, which has not been previously reported, the Arg278Cys mutation (two families) and the Asp271Ile mutation. Seven carriers of the Phe87Leu mutation (aged 29 to 52 years) were found to have mild hypertrophy (i.e., a wall thickness <16 mm). There were 11 deaths associated with the condition (seven sudden deaths), and four of those who died were aged between 14 and 16 years. No sudden deaths occurred in the other three families. In conclusion, troponin-T mutations were responsible for 3% of the hypertrophic cardiomyopathy cases in our study population. The Phe87Leu mutation was associated with only mild hypertrophy but with a high risk of sudden death.

Deletion of a Genomic Segment Containing the Cardiac Troponin I Gene Knocks Down Expression of the Slow Troponin T Gene and Impairs Fatigue Tolerance of Diaphragm Muscle

The loss of slow skeletal muscle troponin T (TnT) results in a recessive nemaline myopathy in the Amish featured with lethal respiratory failure. The genes encoding slow TnT and cardiac troponin I (TnI) are closely linked. Ex vivo promoter analysis suggested that the 5'-enhancer region of the slow TnT gene overlaps with the structure of the upstream cardiac TnI gene. Using transgenic expression of exogenous cardiac TnI to rescue the postnatal lethality of a mouse line in which the entire cardiac TnI gene was deleted, we investigated the effect of enhancer deletion on slow TnT gene expression in vivo and functional consequences. The levels of slow TnT mRNA and protein were significantly reduced in the diaphragm muscle of adult double transgenic mice. The slow TnT-deficient (ssTnT-KD) diaphragm muscle exhibited atrophy and decreased ratios of slow versus fast isoforms of TnT, TnI, and myosin. Consistent with the changes toward more fast myofilament contents, ssTnT-KD diaphragm muscle required stimulation at higher frequency for optimal tetanic force production. The ssTnT-KD diaphragm muscle also exhibited significantly reduced fatigue tolerance, showing faster and more declines of force with slower and less recovery from fatigue as compared with the wild type controls. The natural switch to more slow fiber contents during aging was partially blunted in the ssTnT-KD skeletal muscle. The data demonstrated a critical role of slow TnT in diaphragm function and in the pathogenesis and pathophysiology of Amish nemaline myopathy.

The role of Akt/GSK-3β signaling in familial hypertrophic cardiomyopathy

Mutations in cardiac troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). Transgenic mice expressing a missense mutation (R92Q) or a splice site donor mutation (Trunc) in the cardiac TnT gene have mutation-specific phenotypes but mice of both models have smaller hearts compared to wild type and exhibit hemodynamic dysfunction. Because growth-related signaling pathways in the hearts of mice expressing TnT mutations are not known, we evaluated the impact of increased Akt or glycogen synthase kinase-3β (GSK-3β) activity in both mutant TnT mice; molecules that increase heart size via physiologic pathways and block pathologic growth, respectively. Expression of activated Akt dramatically augments heart size in both R92Q and Trunc mice; however, this increase in heart size is not beneficial, since Akt also increases fibrosis in both TnT mutants and causes some pathologic gene expression shifts in the R92Q mice. Activated GSK-3β results in further decreases in left ventricular size in both R92Q and Trunc hearts, but this decrease is associated with significant mutation-specific phenotypes. Among many pathologic consequences, activating GSK-3β in R92Q hearts decreases phosphorylation of troponin I and results in early mortality. In contrast, increased GSK-3β activity in Trunc hearts does not significantly impact cardiac phenotypes. These findings demonstrate that increased Akt and its downstream target, GSK-3β can impact both cardiac size and phenotype in a mutation-specific manner. Moreover, increased activity of these molecules implicated in beneficial cardiac phenotypes exacerbates the progression of disease in the R92Q TnT mutant.

Decreased Fatigue Tolerance In Diaphragm Muscle Of Slow Troponin T Knockdown Mice

The loss of slow skeletal muscle troponin T (TnT) results in a severe type of nemaline myopathy in the Amish (ANM). The genes encoding TnT and troponin I (TnI) are closely linked in pairs in which the 5′-enhancer region of the slow TnT gene overlaps with the cardiac TnI gene. In a mouse line with the entire cardiac TnI gene deleted, a partial destruction of the slow TnT gene promoter produces a knockdown effect. By crossing with transgenic mouse lines that over-express a core structure of cardiac TnI (cTnI-ND) under the control of cloned alpha-MHC promoter, we rescued the postnatal lethality of the cardiac TnI gene-deleted mice with no detrimental cardiac phenotypes or leaking expression in non-cardiac tissues. The double transgenic mice exhibited decreased expression of slow TnT mRNA and protein in adult diaphragm muscle. Functional analysis of isolated muscle strips showed that the slow TnT deficient (sTnT-KD) diaphragm had significantly decreased fatigue tolerance evident by the faster decrease in force and slower rate of recovery as compared with that in wild type controls. As a consequence of slow TnT deficiency, the sTnT-KD diaphragm muscle contained a higher proportion of fast TnT, decreased slow TnI with increased fast TnI, and decreased type I myosin with increased type II myosin. Consistent with the switch toward fast myofilament contents, the sTnT-KD diaphragm muscle produced higher specific tension in twitch and tetanic contractions as well as shorter time to develop peak tension in twitch contractions. The decreased fatigue tolerance of sTnT-KD diaphragm muscle explains the terminal respiratory failure seen in virtually all ANM patients and this double transgenic mouse model provides a useful experimental system to study the pathogenesis and treatment of ANM.

Cloning and characterization of a cDNA clone encoding troponin T from tick Haemaphysalis qinghaiensis (Acari: Ixodidae)

Troponin T (TnT) is a key protein for Ca 2+-sensitive molecular switching of muscle contraction. The entire cDNA sequence of Haemaphysalis qinghaiensis troponin T gene ( HqTnT) from the hard tick was cloned here. The cDNA sequence of HqTnT possesses an ORF of 1170 bp coding for a mature protein with 389 amino acid residues and a molecular mass of 46.5 kDa. Search of the cloned sequence in GenBank revealed that HqTnT gene shared some homology with TnT genes of other organisms. By sequence comparison, two conserved domains were identified in the middle (residues 54–78) and close to the carboxyl-terminal end (residues 181–229) of the amino acid sequence of HqTnT. These conserved domains might be responsible for the interaction with tick tropomyosin and troponin I subunit, respectively. Reverse transcription-polymerase chain reaction showed a ubiquitous expression of HqTnT gene at different developmental stages and in different tissues of the tick. The HqTnT was expressed as glutathione S-transferase fused protein in a prokaryotic system. Though rHqTnT could induce specific antibodies in sheep, the antibodies could not protect sheep from the infestation of ticks. Therefore, recombinantly produced HqTnT could not be a candidate antigen for developing anti-tick vaccine. To our knowledge, this is the first report of tick TnT gene.

Aberrant Splicing of an Alternative Exon in the Drosophila Troponin-T Gene Affects Flight Muscle Development

During myofibrillogenesis, many muscle structural proteins assemble to form the highly ordered contractile sarcomere. Mutations in these proteins can lead to dysfunctional muscle and various myopathies. We have analyzed the Drosophila melanogaster troponin T (TnT) up1 mutant that specifically affects the indirect flight muscles (IFM) to explore troponin function during myofibrillogenesis. The up1 muscles lack normal sarcomeres and contain "zebra bodies," a phenotypic feature of human nemaline myopathies. We show that the up(1) mutation causes defective splicing of a newly identified alternative TnT exon (10a) that encodes part of the TnT C terminus. This exon is used to generate a TnT isoform specific to the IFM and jump muscles, which during IFM development replaces the exon 10b isoform. Functional differences between the 10a and 10b TnT isoforms may be due to different potential phosphorylation sites, none of which correspond to known phosphorylation sites in human cardiac TnT. The absence of TnT mRNA in up1 IFM reduces mRNA levels of an IFM-specific troponin I (TnI) isoform, but not actin, tropomyosin, or troponin C, suggesting a mechanism controlling expression of TnT and TnI genes may exist that must be examined in the context of human myopathies caused by mutations of these thin filament proteins.

Troponin T isoform expression is modulated during Atlantic halibut metamorphosis.

BackgroundFlatfish metamorphosis is a thyroid hormone (TH) driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT), a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied.ResultsIn the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT) gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT) expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature.ConclusionMuscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

Differential regulation of myofilament protein isoforms underlying the contractility changes in skeletal muscle unloading

Weight-bearing skeletal muscles change phenotype in response to unloading. Using the hindlimb suspension rat model, we investigated the regulation of myofilament protein isoforms in correlation to contractility. Four weeks of continuous hindlimb unloading produced progressive atrophy and contractility changes in soleus but not extensor digitorum longus muscle. The unloaded soleus muscle also had decreased fatigue resistance. Along with the decrease of myosin heavy chain isoform I and IIa and increase of IIb and IIx, coordinated regulation of thin filament regulatory protein isoforms were observed: gamma- and beta-tropomyosin decreased and alpha-tropomyosin increased, resulting in an alpha/beta ratio similar to that in normal fast twitch skeletal muscle; troponin I and troponin T (TnT) both showed decrease in the slow isoform and increases in the fast isoform. The TnT isoform switching began after 7 days of unloading and TnI isoform showed detectable changes at 14 days while other protein isoform changes were not significant until 28 days of treatment. Correlating to the early changes in contractility, especially the resistance to fatigue, the early response of TnT isoform regulation may play a unique role in the adaptation of skeletal muscle to unloading. When the fast TnT gene expression was upregulated in the unloaded soleus muscle, alternative RNA splicing switched to produce more high molecular weight acidic isoforms, reflecting a potential compensation for the decrease of slow TnT that is critical to skeletal muscle function. The results demonstrate that differential regulation of TnT isoforms is a sensitive mechanism in muscle adaptation to functional demands.

Evidence of post‐transcriptional regulation in the maintenance of a partial muscle phenotype by electrogenic cells of S.macrurus

Electrocytes, the current-producing cells of electric organs (EOs) in electric fish, are unique in that they derive from striated muscle and they possess biochemical characteristics of both muscle and non-muscle cells. In the freshwater teleost Sternopygus macrurus, electrocytes are multinucleated cells that do not contract yet retain expression of some proteins common to skeletal muscle cells. Given the role that transcriptional regulation plays in the activation of the myogenic program in vertebrates, we examined the expression patterns of several genes associated with multiple functions of skeletal muscle in mature electrocytes of S. macrurus. Our expression analyses detected transcripts for alpha-actin, alpha-acetylcholine (ACh) receptor (alpha-AChR), desmin, muscle creatine kinase (MCK), myosin heavy chain (MHC) isoforms, titin, tropomyosin, and troponin-T genes in the EO. However, immunolabeling studies revealed that electrocytes do not contain MCK, MHCs, or tropomyosin or troponin-T proteins. These results underscore the contribution of gene regulatory mechanisms in the maintenance of the muscle-like phenotype of EO that may be transcriptional-independent. We also report the classification and frequency of distinct transcripts from a random selection of 420 clones from an EO cDNA library. This is the first characterization of expressed genes in an EO, and it is an important step toward identifying mechanisms that affect different muscle protein systems for the evolution of highly specialized noncontractile tissues. Evidence of post-transcriptional regulation in the maintenance of a partial muscle phenotype by electrogenic cells of S. macrurus.

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.

Cellular Fate of Truncated Slow Skeletal Muscle Troponin T Produced by Glu180 Nonsense Mutation in Amish Nemaline Myopathy

A nonsense mutation at codon Glu180 in exon 11 of slow skeletal muscle troponin T (TnT) gene (TNNT1) causes an autosomal-recessive inherited nemaline myopathy. We previously reported the absence of intact or prematurely terminated slow TnT polypeptide in Amish nemaline myopathy (ANM) patient muscle. The present study further investigates the expression and fate of mutant slow TnT in muscle cells. Intact slow TnT mRNA was readily detected in patient muscle, indicating unaffected transcription and RNA splicing. Sequence of the cloned cDNAs revealed the single nucleotide mutation in two alternatively spliced isoforms of slow TnT mRNA. Mutant TNNT1 cDNA is translationally active in Escherichia coli and non-muscle eukaryotic cells, producing the expected truncated slow TnT protein. The mutant mRNA was expressed at significant levels in differentiated C2C12 myotubes, but unlike intact exogenous TnT, truncated slow TnT protein was not detected. Transfective expression in undifferentiated myoblasts produced slow TnT mRNA but not a detectable amount of truncated or intact slow TnT proteins, indicating a muscle cell-specific proteolysis of TnT when it is not integrated into myofilaments. The slow TnT-(1-179) fragment has substantially lower affinity for binding to tropomyosin, in keeping with the loss of one of two tropomyosin-binding sites. Our findings suggest that inefficient incorporation into myofilament is responsible for the instability of mutant slow TnT in ANM muscle. Rapid degradation of the truncated slow TnT protein, rather than instability of the nonsense mRNA, provides the protective mechanism against the potential dominant negative effect of the mutant TnT fragment.

Troponin T isoforms alter the tolerance of transgenic mouse cardiac muscle to acidosis

Troponin T (TnT) is an essential protein in the Ca 2+ regulatory system of striated of muscle. Three fiber type-specific TnT genes have evolved in higher vertebrates to encode cardiac, slow and fast skeletal muscle TnT isoforms. To understand the functional significance of TnT isoforms, we studied the effects of acidosis on the contractility of transgenic mouse cardiac muscle that expresses fast skeletal muscle TnT. Contractility analysis of intact cardiac muscle strips showed that while no differences were detected at physiological pH, the transgenic cardiac muscle had significantly greater decreases in +d F/d t max at acidic pH than that of the wild-type control. Contractility of skinned cardiac muscles demonstrated that the presence of fast TnT resulted in significantly larger decreases in force and Ca 2+ sensitivity at acidic pH than that of the wild-type control. The effect of TnT isoforms on the tolerance of muscle to acidosis may explain the higher tolerance of embryonic versus adult cardiac muscles. The results are consistent with the hypothesis that charge differences in TnT isoforms contribute to the contractility of muscle. The data further support a hypothesis that slow TnT is similar to the cardiac, but not fast, and TnT may contribute to the higher tolerance of slow muscles to stress conditions. Therefore, TnT isoform diversity may contribute to the compatibility of muscle thin filaments to cellular environments in different fiber types, during development and functional adaptation.

Role of troponin T in disease

Several striated muscle myopathies have been directly linked to mutations in contractile and associated proteins. Troponin T (TnT) is one of the three subunits that form troponin (Tn) which together with tropomyosin is responsible for the regulation of striated muscle contraction. All three subunits of cardiac Tn as well as tropomyosin have been associated with hypertrophic cardiomyopathy (HCM). However, TnT accounts for most of the mutations that cause HCM in these regulatory proteins. To date 30 mutations have been identified in the cardiac TnT (CTnT) gene that results in familial HCM (FHC). The CTnT gene has also been associated with familial dilated cardiomyopathy (DCM). CTnT deficiency is lethal due to impaired cardiac development. A recessive nonsense mutation in the gene encoding slow skeletal TnT has been associated with an unusual, severe form of nemaline myopathy among the Old Order Amish. How each mutation leads to the diverse clinical symptoms associated with FHC, DCM or nemaline myopathy is unclear. However, the use of animal model systems, in particular transgenic mice, has significantly increased our knowledge of normal and myopathic muscle physiology. In this review, we focus on the role of TnT in muscle physiology and disease.

Genomic structure of the chicken slow skeletal muscle troponin T gene

Troponin T (TnT) is a key protein for Ca 2+-sensitive molecular switching of muscle contraction. In vertebrates, three TnT genes have been identified, which produce isoforms characteristic of cardiac, fast skeletal, and slow skeletal muscles through alternative splicing in a tissue-specific and developmentally regulated manner. The diversification of myofibers into forms with specific metabolic and contractile characteristics is thought to be closely associated with the differential expression of these TnT isoforms. Herein, we determined the nucleotide sequence of the chicken slow skeletal muscle TnT gene and its upstream region. The gene was simpler in structure than the two other chicken genes. The transcription initiation site was positioned 183 bp upstream of the 3′ end of exon 1. Alternative splicing of exon 5 using an internal acceptor site generated two distinct slow skeletal muscle troponin T (sTnT) transcripts. We identified possible regulatory elements, M-CAT-like, CACC-box, and E-box (E-box1 to E-box3) motifs in the upstream region and an E-box motif (E-box4) in exon 1.

Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster.

The indirect flight muscles (IFM) of Drosophila melanogaster provide a good genetic system with which to investigate muscle function. Flight muscle contraction is regulated by both stretch and Ca(2+)-induced thin filament (actin + tropomyosin + troponin complex) activation. Some mutants in troponin-I (TnI) and troponin-T (TnT) genes cause a "hypercontraction" muscle phenotype, suggesting that this condition arises from defects in Ca(2+) regulation and actomyosin-generated tension. We have tested the hypothesis that missense mutations of the myosin heavy chain gene, Mhc, which suppress the hypercontraction of the TnI mutant held-up(2) (hdp(2)), do so by reducing actomyosin force production. Here we show that a "headless" Mhc transgenic fly construct that reduces the myosin head concentration in the muscle thick filaments acts as a dose-dependent suppressor of hypercontracting alleles of TnI, TnT, Mhc, and flightin genes. The data suggest that most, if not all, mutants causing hypercontraction require actomyosin-produced forces to do so. Whether all Mhc suppressors act simply by reducing the force production of the thick filament is discussed with respect to current models of myosin function and thin filament activation by the binding of calcium to the troponin complex.

Differential expression of mutually exclusive exons of the fast skeletal muscle troponin T gene in the chicken wing and leg muscles.

To determine the physiological significance of developmentally regulated and muscle tissue-specific expression of troponin T (TnT) isoforms and also to elucidate mechanisms of alternative splicing, we investigated splicing patterns of fast skeletal muscle TnT by sequencing cDNAs obtained from the chicken wing and leg muscles. The chicken fast skeletal muscle TnT gene has 27 exons, and produces potentially 2(15) transcripts if alternative exons are randomly spliced. For the fourteen alternative exons in the 5'-region of the gene, we found 7 and 12 splice variants in the wing biceps brachii and leg gastrocnemius, respectively. Out of 19, 12 variants were newly found in this study. Twenty two different splice variants were reported by the previous studies, but this number has now increased to 34 in the chicken TnT gene, suggesting that alternative splicing is not random, but strictly regulated. We also investigated splicing patterns of mutually exclusive exons 16 and 17 in the 3'-region of the gene. Approximately equal amounts of cDNAs containing exons 16 and 17 were detected in proximal regions of adult biceps brachii and gastrocnemius, respectively, whereas cDNA with exon 17 dominated in distal regions. Only cDNA containing exon 17 was found in both proximal and distal regions of 5-day-old gastrocnemius. The inclusion of exon 16 into cDNAs was detected in the proximal region of 15-day-old gastrocnemius and increased during development.

Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy.

A splice donor site mutation in intron 15 of the cardiac troponin T (TnT) gene has been shown to cause familial hypertrophic cardiomyopathy (HCM). In this study, two truncated human cardiac TnTs expected to be produced by this mutation were expressed in Escherichia coli and partially (50-55%) exchanged into rabbit permeabilized cardiac muscle fibers. The fibers into which a short truncated TnT, which lacked the COOH-terminal 21 amino acids because of the replacement of 28 amino acids with 7 novel residues, had been exchanged generated a Ca(2+)-activated maximum force that was slightly, but statistically significantly, lower than that generated by fibers into which wild-type TnT had been exchanged when troponin I (TnI) was phosphorylated by cAMP-dependent protein kinase. A long truncated TnT simply lacking the COOH-terminal 14 amino acids had no significant effect on the maximum force-generating capability in the fibers with either phosphorylated or dephosphorylated TnI. Both these two truncated TnTs conferred a lower cooperativity and a higher Ca(2+) sensitivity on the Ca(2+)-activated force generation than did wild-type TnT, independent of the phosphorylation of TnI by cAMP-dependent protein kinase. The results demonstrate that the splice donor site mutation in the cardiac TnT gene impairs the regulatory function of the TnT molecule, leading to an increase in the Ca(2+) sensitivity, and a decrease in the cooperativity, of cardiac muscle contraction, which might be involved in the pathogenesis of HCM.

The structure of the avian fast skeletal muscle troponin T gene: seven novel tandem-arranged exons in the exon x region.

To elucidate the mechanism that produces enormous molecular diversity in troponin T (TnT) of fast skeletal muscle, we determined the 5'-half genomic sequence of the chicken fast muscle TnT gene. The sequence of ca. 16 kb included seven exons (exons 1, 2, 3, 4, w, 5, and 6), which have been reported previously and presumed by sequencing TnT cDNAs. Additionally we found six 15 nt and one 18 nt sequences in the region between exons 5 and 6 (i.e. the exon x region). They were encompassed by consensus splice donor and acceptor sites and preceded by putative branch sites, and designated herein as exons xa to xg. Our result shows that the sequence derived from exons x1, x2, and x3, the exons presumed previously by cDNA sequencing, is actually encoded by the seven exons xa to xg, establishing the precise gene structure in the exon x region. Based on our data, together with that on the 3'-half genomic sequence of the quail fast muscle TnT gene, we conclude that the avian fast skeletal muscle TnT gene includes 27 exons, 16 of which are alternatively spliced.