Troponin T isoforms modulate calcium dependence of the kinetics of the cross-bridge cycle: studies using a transgenic mouse line

Alternative splicing of troponin T (TnT) in striated muscle during development results in expression of different isoforms, with the splicing of a 5(') exon of TnT resulting in the expression of low-molecular-weight basic adult TnT isoforms and high-molecular-weight acidic embryonic TnT isoforms. Although other differences exist, the main differences between cardiac TnT (cTnT) and fast skeletal muscle TnT (fTnT) are in the NH(2) terminus, with fTnT being less acidic than cTnT. A transgenic mouse line expressing chicken fTnT in the heart was used to investigate the functional significance of TnT NH(2)-terminal charge differences on cardiac muscle contractility. The rates of force redevelopment (k(tr)) at four levels of Ca(2+) activation were recorded for skinned left ventricular trabeculae from control and transgenic mice. The k(tr) vs Ca(2+) relationship was different in control mice and transgenic mice, suggesting that the structure of TnT, and possibly the NH(2)-terminal region, is involved in determining the kinetics of cross-bridge cycle. These results suggest that isoform shifts in TnT may be an important molecular mechanism for determining the Ca(2+) dependence of cardiac muscle contractility.     

鸡形目鸟类成熟胸肌中特异性表达的fTnT同工异构型及其生理学意义

脊椎动物中的肌钙蛋白T（tropnin T,TnT）分为心肌型TnT（cardiac TnT,cTnT）、快肌型TnT（fast skeletal TnT,fTnT）和慢肌型TnT（slow skeletal TnT,sTnT）,且每种TnT又因mRNA可变剪接(alternative mRNA splicing)形成了多种同工异构型,其中fTnT的同工异构型形式最为复杂。某些鸟类如鸡形目鸟类的成熟快肌（尤其是胸部快肌）中特异性表达的TnT同工异构型有如下特点：（1）N端区含有过渡金属离子结合位点——Tx元件（一般为4~7个重复的H-E/A-E-A-H序列）;（2）与哺乳动物及雏鸟fTnT相比,其C端区外显子16有很高的表达率。本文还就鸡形目鸟类成熟胸肌中表达的fTnT同工异构型可能具有的生理学意义及应用前景进行了探讨。 The Fast TnT Isoforms Specifically Expressed in Avian Adult Pectoral Muscles of Galliforms and Physiological Significance DUAN Ying-li,YU Shu-yang,LI Ning National Laboratories for Agrobiotechnology,China Agricultural University,Beijing 100094,China Abstract:Three homologous genes have evolved to encode the cardiac,slow and fast skeletal muscle troponin Ts(TnTs) in the vertebrate.Multiple isoforms in each type of TnT are generated through alternative mRNA splicing during the development and the modality of the fast skeletal TnT isoforms is the most complex.The TnT isoforms specifically expressed in avian adult fast skeletal muscle (especially in the adult pectoral muscle) of Galliforms have been characterized as follows:1.There exist a cluster of transition metal ion binding sites ［generally 4~7 repeats of a sequence motif His-(Glu/Ala)- Glu-Ala-His,designated as Tx］ in the NH2-terminal variable region.2.Compared with mammalian TnT and the neonatal or young avian TnT,these avian pectoral muscle TnTs prefer to express exon 16 in the COOH-terminal variable region.Furthermore,possible effects of the pectoral fTnT isoforms on the physiological activity are discussed in this article. Key words：Aves; troponin T; isoform     

Expression of cDNAs encoding mouse cardiac troponin T isoforms: characterization of a large sample of independent clones

We have isolated 52 mouse cardiac troponin-T-encoding cDNA clones (TnT) by specific antibody screening of a λZAPII expression library. Sequencing data from the large sample of independent cDNAs demonstrated relationships among the expression of four alternatively-spliced exons of the cardiac TnT gene, producing seven classes of cDNAs encoding four protein isoforms differing in two variable regions. In the N-terminal variable region and next to the embryonic-specific exon 4, an alternatively spliced exon 3a was identified in 20% of the adult isoforms. The alternatively spliced exon 12, corresponding to a central variable region between the two functional domains of TnT, was found in approx. 79% of the 52 mouse cardiac TnT cDNAs with a single base mutation completely abolishing the splicing at an internal acceptor site. Three novel alternative splicing acceptor sites in the 5'-untranslated portion of exon 2 have been identified with different frequencies.     

Role of the fetal and alpha/beta exons in the function of fast skeletal troponin T isoforms: correlation with altered Ca2+ regulation associated with development

In mammalian fast skeletal muscle, constitutive and alternative splicing from a single troponin T (TnT) gene produce multiple developmentally regulated and tissue specific TnT isoforms. Two exons, alpha (exon 16) and beta (exon 17), located near the 3' end of the gene and coding for two different 14 amino acid residue peptides are spliced in a mutually exclusive manner giving rise to the adult TnTalpha and the fetal TnTbeta isoforms. In addition, an acidic peptide coded by a fetal (f) exon located between exons 8 and 9 near the 5' end of the gene, is specifically present in TnTbeta and absent in the adult isoforms. To define the functional role of the f and alpha/beta exons, we constructed combinations of TnT cDNAs from a single human fetal fast skeletal TnTbeta cDNA clone in order to circumvent the problem of N-terminal sequence heterogeneity present in wild-type TnT isoforms, irrespective of the stage of development. Nucleotide sequences of these constructs, viz. TnTalpha, TnTalpha + f, TnTbeta - f and TnTbeta are identical, except for the presence or absence of the alpha or beta and f exons. Our results, using the recombinant TnT isoforms in different functional in vitro assays, show that the presence of the f peptide in the N-terminal T1 region of TnT, has a strong inhibitory effect on binary interactions between TnT and other thin filament proteins, TnI, TnC and Tm. The presence of the f peptide led to reduced Ca2+-dependent ATPase activity in a reconstituted thin filament, whereas the contribution of the alpha and beta peptides in the biological activity of TnT was primarily modulatory. These results indicate that the f peptide confers an inhibitory effect on the biological function of fast skeletal TnT and this can be correlated with changes in the Ca2+ regulation associated with development in fast skeletal muscle.     

Evolution of a Metal-Binding Cluster in the NH2-Terminal Variable Region of Avian Fast Skeletal Muscle Troponin T: Functional Divergence on the Basis of Tolerance to Structural Drifting

This study investigated the evolution of a transition metal ion-binding cluster ([H–X–X–X–H] _n ; Tx) in the alternatively spliced NH₂-terminal variable region of avian pectoral muscle troponin T (TnT). Encoded by avian fast skeletal muscle TnT-specific P exons, Tx-like structures were expressed in the breast muscle TnT's of almost all birds examined. Their presence results in the developmentally up-regulated high molecular weight pectoral muscle TnT. Sequence analysis and metal affinity chromatography revealed that in Galliformes and Craciformes, the Tx structure evolved into multiple H–X–X–X–H pairs with a high-affinity metal-binding capacity. Turkey, chicken, quail, and curassow breast muscle TnT's contain nine, seven, four, and three consecutive or closely located metal-binding sites, respectively, in the NH₂-terminal region. The metal-binding affinity of the Tx element increased as the number of His pairs increased due to the duplication of P exons and the conversion of other exon sequences. The data show two related components of avian pectoral muscle TnT evolution: a larger, more acidic NH₂-terminal segment and a cluster of transition metal-binding sites, both of which may have functional significance for their selection value. The evolution of the Tx segment in the NH₂-terminal variable region of avian pectoral muscle TnT demonstrates a functional divergence on the basis of tolerance to structural drifting. Received: 2 May 2000 / Accepted: 5 September 2000     

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

Troponin T (TnT) is an essential protein in the Ca²⁺ 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 +dF/dt_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 Ca²⁺ 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.     

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, TnT1f, TnT2f and TnT3f, 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. TnT2f is the fast TnT sequenced by Pearlstone et al. The larger species TnT1f 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. TnT3f 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.     

Genomic sequence and structural organization of mouse slow skeletal muscle troponin T gene

Three muscle type-specific troponin T (TnT) genes are present in vertebrate to encode a number of protein isoforms via alternative mRNA splicing. While the genomic structures of cardiac and fast skeletal muscle TnT genes have been documented, this study cloned and characterized the slow skeletal muscle TnT (sTnT) gene. Complete nucleotide sequence and genomic organization revealed that the mouse sTnT gene spans 11.1kb and contains 14 exons, which is smaller and simpler than the fast skeletal muscle and cardiac TnT genes. Potentially representing a prototype of the TnT gene family, the 5'-region of the sTnT gene contains fewer unsplit large exons, among which two alternatively spliced exons are responsible for the NH2-terminal variation of three sTnT isoforms. The sTnT gene structure shows that the alternatively spliced central segment found in human sTnT cDNAs may be a result from splicing using an alternative acceptor site at the intron 11-exon 12 boundary. Together with the well-conserved protein structure, the highly specific expression of sTnT in slow skeletal muscles indicates a differentiated function of this member of the TnT gene family. The determination of genomic structure and alternative splicing pathways of sTnT gene lays a foundation to further understand the TnT structure-function evolution as well as contractile characteristics of different types of muscle fiber.     

Transitions from fetal to fast troponin T isoforms are coordinated with changes in tropomyosin and α-actinin isoforms in developing rabbit skeletal muscle

In adult fast skeletal muscle, specific combinations of thin filament and Z-line protein isoforms are coexpressed. To determine whether the expression of these sets of proteins, designated the TnT1f, TnT2f, and TnT3f programs, is coordinated during development, we characterized the transitions in troponin T (TnT), tropomyosin (Tm), and α-actinin isoforms that occur in developing fetal and neonatal rabbit skeletal muscle. Two coordinated developmental transitions were identified, and a novel pattern of thin filament expression was found in fetal muscle. In fetal muscle, new TnT species—whose protein and immunochemical properties suggest that they are the products of a new TnT gene—are expressed in combination with β₂ Tm and α-actinin_1f/8. This pattern, which is found in both back and hindlimb muscles, is specific to fetal and early neonatal muscle. Just prior to birth, there is a transition from the fetal program to the isoforms that define the TnT3f program, TnT_3f, and αβ Tm. Like the fetal program, expression of the TnT3f program appears to be a general feature of muscle development, because it occurs in a variety of fast muscles as well as in the slow muscle soleus. The transition to adult patterns of thin filament expression begins at the end of the first postnatal week. Based on studies of erector spinae, the isoforms comprising the TnT2f program, TnT_2f, α₂ Tm, and α-actinin_2f, appear and increase coordinately at this time. The transitions, first to the TnT3f program, and then to adult patterns of expression indicate that synthesis of the isoforms comprising each program is coordinated during muscle specialization and throughout muscle development. In addition, these observations point to a dual role for the TnT3f program, which is the major thin filament program in some adult muscles, but appears to bridge the transition from developmentally to physiologically regulated patterns of thin filament expression during late fetal and early neonatal development.     

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: - and -tropomyosin decreased and -tropomyosin increased, resulting in an / 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. troponin T; fatigue resistance; troponin I; tropomyosin; myosin; hindlimb-suspended rat; Western blot protein quantification     

Autoregulation of Fox protein expression to produce dominant negative splicing factors

The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (FoxΔRRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These FoxΔRRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform.     

Differential expression and developmental regulation of a novel alpha-dystrobrevin isoform in muscle

Alpha-dystrobrevin is a dystrophin-related protein expressed primarily in skeletal muscle, heart, lung and brain. In skeletal muscle, alpha-dystrobrevin is a component of the dystrophin-associated glycoprotein complex and is localized to the sarcolemma, presumably through interactions with dystrophin and utrophin. Alternative splicing of the alpha-dystrobrevin gene generates multiple isoforms which have been grouped into three major classes: alpha-DB1, alpha-DB2, and alpha-DB3. Various isoforms have been shown to interact with a variety of proteins; however, the physiological function of the alpha-dystrobrevins remains unknown. In the present study, we have cloned a novel alpha-dystrobrevin cDNA encoding a protein (referred to as alpha-DB2b) with a unique 11 amino acid C-terminal tail. Using RT PCR with primers specific to the new isoform, we have characterized its expression in skeletal muscle, heart, and brain, and in differentiating C2C12 muscle cells. We show that alpha-DB2b is expressed in skeletal muscle, heart and brain, and that exons 12 and 13 are alternatively spliced in alpha-DB2b to generate at least three splice variants. The major alpha-DB2b splice variant expressed in adult skeletal muscle and heart contains exons 12 and 13, while in adult brain, two alpha-DB2b splice variants are expressed at similar levels. This is consistent with the preferential expression of exons 12 and 13 in other alpha-dystrobrevin isoforms in skeletal muscle and heart. Similarly, in alpha-DB1 the first 21 nucleotides of exon 18 are preferentially expressed in skeletal muscle and heart relative to brain. We also show that the expression of alternatively spliced alpha-DB2b is developmentally regulated in muscle; during differentiation of C2C12 cells, alpha-DB2b expression switches from an isoform lacking exons 12 and 13 to one containing them. We demonstrate similar developmental upregulation of exons 12, 13, and 18 in alpha-DB1 and of exons 12 and 13 in alpha-DB2a. Finally, we show that alpha-DB2b protein is expressed in adult skeletal muscle, suggesting that it has a functional role in adult muscle. Together, these data suggest that alternatively spliced variants of the new alpha-dystrobrevin isoform, alpha-DB2b, are differentially expressed in various tissues and developmentally regulated during muscle cell differentiation in a fashion similar to that previously described for alpha-dystrobrevin isoforms.     

Adaptation by alternative RNA splicing of slow troponin T isoforms in type 1 but not type 2 Charcot-Marie-Tooth disease

Slow troponin T (TnT) plays an indispensable role in skeletal muscle function. Alternative RNA splicing in the NH₂-terminal region produces high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms of slow TnT. Normal adult slow muscle fibers express mainly HMW slow TnT. Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral polyneuropathies caused by various neuronal defects. We found in the present study that LMW slow TnT was significantly upregulated in demyelination form type 1 CMT (CMT1) but not axonal form type 2 CMT (CMT2) muscles. Contractility analysis showed an increased specific force in single fibers isolated from CMT1 but not CMT2 muscles compared with control muscles. However, an in vitro motility assay showed normal velocity of the myosin motor isolated from CMT1 and CMT2 muscle biopsies, consistent with their unchanged myosin isoform contents. Supporting a role of slow TnT isoform regulation in contractility change, LMW and HMW slow TnT isoforms showed differences in the molecular conformation in conserved central and COOH-terminal regions with changed binding affinity for troponin I and tropomyosin. In addition to providing a biochemical marker for the differential diagnosis of CMT, the upregulation of LMW slow TnT isoforms under the distinct pathophysiology of CMT1 demonstrates an adaptation of muscle function to neurological disorders by alternative splicing modification of myofilament proteins. muscle adaptation; demyelination; force and velocity     

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

Fetal rat skeletal muscles express a troponin T (TnT) isoform similar to the TnT isoform expressed in the embryonic heart with respect to electrophoretic mobility and immunoreactivity with cardiac TnT-specific monoclonal antibodies. Immunoblotting analyses reveal that both the embryonic and the adult isoforms of cardiac TnT are transiently expressed during the neonatal stages. In addition, other TnT species, different from both cardiac TnTs and from the TnT isoforms expressed in adult muscles, are present in skeletal muscles during the first two postnatal weeks. By immunocytochemistry, cardiac TnT is detectable at the somitic stage and throughout embryonic and fetal development, and disappears during the first weeks after birth, persisting exclusively in the bag fibers of the muscle spindles. Cardiac TnT is re-expressed in regenerating muscle fibers following a cold injury and in mature muscle fibers after denervation. Developmental regulation of this TnT variant is not coordinated with that of the embryonic myosin heavy chain with respect to timing of disappearance and cellular distribution. No obligatory correlation between the two proteins is likewise found in regenerating and denervated muscles.