Immunohistochemical studies on regulation of alternative splicing of fast skeletal muscle troponin T: non-uniform distribution of the exon x3 epitope in a single muscle fiber

Troponin T (TnT) isoforms of chicken fast skeletal muscle are classified into two types, breast-muscle-type (B-type) and leg-muscle-type (L-type) isoforms. These isoforms are produced from a single gene by differential alternative splicing of pre-mRNA. We investigated immunohistochemically the distribution of B-type TnT isoforms in chicken leg muscle (musculus biceps femoris), using anti-exon x3 that was raised against a synthetic peptide corresponding to exon x3 and recognized B-type, but not the L-type, TnT isoforms. Mosaic patterns of immunostaining showing locally different expression of B-type TnT isoforms in a single fiber were observed among fibers, and the non-uniform distribution of the isoforms was also detected in sectioned fibers and myofibrils from the muscle. The results indicated that regulation of pre-mRNA splicing of fast skeletal muscle TnT was different not only among the muscle fibers but also within a single fiber, suggesting that heterogeneous myonuclei in regulation of alternative splicings occur in a single muscle fiber.     

Expression of multiple troponin T isoforms in chicken breast muscle regeneration induced by sub-serous implantation

Chicken fast-muscle type (F-type) troponin T (TnT) isoforms are classified into two types, leg-muscle type (L-type) and breast-muscle type (B-type), which are generated by exclusion and inclusion of exon x series-derived sequences in mRNAs, respectively. The B-type isoforms are further classified into neonatal breast-muscle (BN), young chicken breast-muscle (BC), and adult chicken breast-muscle (BA) subtypes. It is known that the multiple F-type TnT isoforms are transiently expressed in the breast muscle tissue during normal development. To examine whether the transition of the isoforms was fixed in muscle cell lineage, breast muscle pieces (pectoralis major) of 1-day old chicks were cultured under gizzard serous membrane of the same chicks for 60 days at the longest. TnT isoform expression of the implants was monitored by immunoblotting and immunostaining using anti-F-type TnT against both L-type and B-type isoforms, anti-exon x3 against only B-type isoforms, and anti-S-type TnT against slow-muscle-type (S-type) isoforms. Muscle fibers in the implant degenerated first, and then new myotubes expressing L-type isoforms were formed by the fusion of myoblasts from surviving satellite cells. When the maturation of the myotubes into myofibers proceeded, BN-, BC-, and BA-subtype isoforms were expressed in the order of developmental stage specific-manner, indicating that the order of appearance of these isoforms was fixed in muscle cell lineage. In immunostaining of the implants recovered on the 60th day after implantation, at least three kinds of the regenerated myofibers were observed, expressing mainly B-type, both B-type and L-type, and only L-type isoforms. The immunohistochemical results suggested that the regulation of alternative splicing of F-type TnT pre-mRNAs was different among individual myofibers, and that the regulation was programmed in myogenic cells, probably satellite cells, which were the primary source of the fibers.     

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.     

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     

Immunochemical analysis of troponin T isoforms in adult, embryonic, regenerating, and denervated chicken fast skeletal muscles

Using monoclonal antibodies (McAbs) which can distinguish between breast- and leg-type troponin T (TnT), we studied the spatial distribution of TnT isoforms in adult chicken fast skeletal muscles. The breast (pectoralis major) and leg (iliotibialis posterior) muscles were composed predominantly of homogeneous fibers containing breast- and leg-type TnT, respectively. The posterior latissimus dorsi muscle was composed of heterogeneous fibers of at least two types, namely breast and leg types. In developing and regenerating fast muscles, only leg-type TnT was expressed at early stages, and later breast-type TnT appeared either transiently or permanently. This led ultimately to several distinct adult fast muscle breast/leg TnT isoform profiles. Since both types of TnT were synthesized in embryonic and regenerating muscles with nerves intact as well as in regenerating muscles with nerves resected, the switching on of their expression during fast muscle development appears to be independent of nerves. However, its full development ("fine tuning" of the protein isoform distribution within the fast fiber types) and the maintenance of the adult state are presumed to be dependent on the nerves, since, although regenerating fibers in denervated muscles could exhibit the early and then the later embryonic stainabilities, they again returned to the early embryonic state; further, the denervation of adult muscles caused the replacement of TnT isoform from the adult to the early embryonic state.     

Tx-TnT在不同发育时期的艾维茵鸡和洪山鸡肌肉组织中的表达分析

以艾维茵鸡和湖北省地方鸡种洪山鸡为实验材料 ,借助特异性识别Tx残基肽的单克隆抗体 6B8,采用Western杂交方法 ,检测Tx TnT异构体在洪山鸡和艾维茵鸡 7个发育时期 (孵化第 14d、初生 1日龄、7、14、2 1、2 8和35日龄 )的胸肌和腿肌中的表达差异 ,并与胸肌重进行相关分析。结果表明 ,Tx TnT在腿肌和孵化第 14d的胸肌中均不表达 ,在初生 1日龄后胸肌中的表达随发育逐步增长 ,统计分析发现 ,Tx TnT在艾维茵鸡和洪山鸡胸肌中的表达量具有显著差异 (P <0 0 5 ) ,与胸肌重具有显著相关 (P <0 0 5 )。     

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     

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.     

Many isoforms of fast muscle troponin T from chicken legs

Troponin T from fast muscle of chicken legs was found to be composed of about 40 kinds of isoforms by two-dimensional polyacrylamide gel electrophoresis in conjunction with immunoblotting tests with an antiserum to chicken breast muscle troponin T. Almost all of the isoforms were found in the myofibril preparation and troponin preparation from the leg muscle, and they showed complex-forming ability with troponin I and troponin C. These isoforms existed in most of the fast muscle except pectoralis and posterior latissimus dorsi muscles, and they changed in composition during development. The breast muscle troponin T also showed different types of isoforms in the period soon after hatching. Since proteolysis was completely inhibited during two-dimensional gel electrophoresis and since the many isoforms were observed consistently in various muscles of chicken leg, they are most probably products of mRNAs generated by differential gene splicing.     

Conformational modulation of slow skeletal muscle troponin T by an NH(2)-terminal metal-binding extension

TroponinT (TnT) is an essential element in the thin filamentCa²⁺-regulatory system controlling striated musclecontraction. Alternative RNA splicing generates developmental andmuscle type-specific TnT isoforms differing in the hypervariableNH₂-terminal region. Using avian fast skeletal muscle TnTcontaining a metal-binding segment, we have demonstrated a role of theNH₂-terminal domain in modulating the conformation of TnT(Wang J and Jin JP. Biochemistry 37: 14519-14528,1998). To further investigate the structure-function relationship ofTnT, the present study constructed and characterized a recombinantprotein in which the metal-binding peptide present in avian fastskeletal muscle TnT was fused to the NH₂ terminus of mouseslow skeletal muscle TnT. Metal ion or monoclonal antibody binding tothe NH₂-terminal extension induced conformational changes in other domains of the model TnT molecule. This was shown by thealtered affinity to a monoclonal antibody against the COOH-terminal region and a polyclonal antiserum recognizing multiple epitopes. Protein binding assays showed that metal binding to theNH₂-terminal extension had effects on the interaction ofTnT with troponin I, troponin C, and most significantly, tropomyosin.The data indicate that the NH₂-terminal Tx [4-7repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His] extension confers a specific conformational modulation in the slowskeletal muscle TnT.

     

Expression pattern of skeletal muscle troponin T isoforms is fixed in cell lineage.

The expression of fast-muscle-type troponin T isoforms in chicken skeletal muscles was studied by two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting. According to the pattern of troponin T isoform expression, chicken fast muscle was classified into two groups: One group expressed breast-fast-muscle-type troponin T in addition to leg-fast-muscle-type troponin T, the other expressed only leg-fast-muscle-type troponin T. To the former group belong breast and wing fast muscles and some of the back fast muscles, and to the latter group belong the fast muscles in leg, abdomen, and neck. Transplantation of breast muscle into leg was performed in order to change the physical environment and to investigate the mechanism of isoform expression. Histological observation of the transplant revealed severe degeneration of muscle cells, followed by differentiation of myoblasts in which breast-muscle-type troponin T was eventually expressed. The results showed that the pattern of troponin T isoform expression is primarily fixed in the cell lineage, although nerves modulate it.     

Structure and evolution of the alternatively spliced fast troponin T isoform gene.

The vertebrate fast skeletal muscle troponin T gene, TnTf, produces a complexity of isoforms through differential mRNA splicing. The mechanisms that regulate splicing and the physiological significance of TnTf isoforms are poorly understood. To investigate these questions, we have determined the complete sequence structure of the quail TnTf gene, and we have characterized the developmental expression of alternatively spliced TnTf mRNAs in quail embryonic muscles. We report the following: 1) the quail TnTf gene is significantly larger than the rat TnTf gene and has 8 non-homologous exons, including a pectoral muscle-specific set of alternatively spliced exons; 2) specific sequences are implicated in regulated exon splicing; 3) a 900-base pair sequence element, composed primarily of intron sequence flanking the pectoral muscle-specific exons, is tandemly repeated 4 times and once partially, providing direct evidence that the pectoral-specific TnT exon domain arose by intragenic duplications; 4) a chicken repeat 1 retrotransposon element resides upstream of this repeated intronic/pectoral exon sequence domain and is implicated in transposition of this element into an ancestral genome; and 5) a large set of novel isoforms, produced by regulated exon splicing, is expressed in quail muscles, providing insights into the developmental regulation, physiological function, and evolution of the vertebrate TnTf isoforms.     

Transitions from fetal to fast troponin T isoforms are coordinated with changes in tropomyosin and alpha-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 alpha-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 beta 2 Tm and alpha-actinin1f/s. 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, TnT3f, and alpha beta 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, TnT2f, alpha 2 Tm, and alpha-actinin2f, 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 the late fetal and early neonatal development.     

Toad Heart Utilizes Exclusively Slow Skeletal Muscle Troponin T: AN EVOLUTIONARY ADAPTATION WITH POTENTIAL FUNCTIONAL BENEFITS

The three isoforms of vertebrate troponin T (TnT) are normally expressed in a muscle type-specific manner. Here we report an exception that the cardiac muscle of toad (Bufo) expresses exclusively slow skeletal muscle TnT (ssTnT) together with cardiac forms of troponin I and myosin as determined using immunoblotting, cDNA cloning, and/or LC-MS/MS. Using RT-PCR and 3'- and 5'-rapid amplification of cDNA ends on toad cardiac mRNA, we cloned full-length cDNAs encoding two alternatively spliced variants of ssTnT. Expression of the cloned cDNAs in Escherichia coli confirmed that the toad cardiac muscle expresses solely ssTnT, predominantly the low molecular weight variant with the exon 5-encoded NH(2)-terminal segment spliced out. Functional studies were performed in ex vivo working toad hearts and compared with the frog (Rana) hearts. The results showed that toad hearts had higher contractile and relaxation velocities and were able to work against a significantly higher afterload than that of frog hearts. Therefore, the unique evolutionary adaptation of utilizing exclusively ssTnT in toad cardiac muscle corresponded to a fitness value from improving systolic function of the heart. The data demonstrated a physiological importance of the functional diversity of TnT isoforms. The structure-function relationship of TnT may be explored for the development of new treatment of heart failure.     

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.     

Nonmammalian vertebrate skeletal muscles express two triad junctional foot protein isoforms.

Mammalian skeletal muscles express a single triad junctional foot protein, whereas avian muscles have two isoforms of this protein. We investigated whether either case is representative of muscles from other vertebrate classes. We identified two foot proteins in bullfrog and toadfish muscles on the basis of (a) copurification with [3H]epiryanodine binding; (b) similarity to avian muscle foot proteins in native and subunit molecular weights; (c) recognition by anti-foot protein antibodies. The bullfrog and toadfish proteins exist as homooligomers. The subunits of the bullfrog muscle foot protein isoforms are shown to be unique by peptide mapping. In addition, immunocytochemical localization established that the bullfrog muscle isoforms coexist in the same muscle cells. The isoforms in either bullfrog and chicken muscles have comparable [3H]epiryanodine binding capacities, whereas in toadfish muscle the isoforms differ in their levels of ligand binding. Additionally, chicken thigh and breast muscles differ in the relative amounts of the two isoforms they contain, the amounts being similar in breast muscle and markedly different in thigh muscle. In conclusion, in contrast to mammalian skeletal muscle, two foot protein isoforms are present in amphibian, avian, and piscine skeletal muscles. This may represent a general difference in the architecture and/or a functional specialization of the triad junction in mammalian and nonmammalian vertebrate muscles.