The components of troponin from chicken fast skeletal muscle. A comparison of troponin T and troponin I from breast and leg muscle.

The three components of troponin were prepared from chicken breast and leg muscle. The troponin I and T components were separated by chromatography on DEAE-cellulose after citraconylation and without the use of urea-containing buffers. The troponin I and C components were similar to their counterparts from rabbit fast skeletal muscle, and a comparison of the troponin I components from breast and leg muscle by amino acid analysis, gel electrophoresis and peptide 'mapping' provides strong evidence for the identity of these proteins. The molecular weights of the troponin T components from breast and leg muscle were 33 500 and 30 500 respectively, determined by gel filtration. A comparison of these two proteins by methods similar to those used for the troponin I components suggested that they differed only in the N-terminal region of the sequence, the breast-muscle troponin T having an extra length of polypeptide chain of approx. 24 residues that is rich in histidine and alanine. The N-terminal hexapeptide sequence, however, is the same in both proteins and is (Ser,Asx,Glx)Thr-Glu-Glu. The genetic implications of these findings are considered.     

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.     

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.     

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.     

Amino acid sequences of porcine fast and slow troponin T isoforms

In this study, 10 troponin T isoforms from adult porcine skeletal muscle messenger RNA were clarified. These were eight fast- and two slow-type isoforms. Fast-type isoforms had three and two variable exons in the N-terminal and the C-terminal region respectively. Slow-type isoforms had one variable exon in the N-terminal region.     

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.     

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

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

Expression of multiple troponin T variants in neonatal chicken breast muscle

The types of troponin-T (TNT) expressed in neonatal chicken breast muscle were examined by two-dimensional gel electrophoresis (2-D PAGE), immunoblotting, and peptide mapping. When troponin from neonatal chicken breast muscle or whole lysate of the muscle was displayed on 2-D PAGE, multiple spots were observed in the TNT region on the gel. They differed slightly from those in adult breast- and leg-type TNT, but were positively stained with the antibody specific for TN-T. These results indicate that multiple spots observed in the TNT region are all TNT isoforms. The TNT isoforms in the neonatal breast muscle were classified into two groups, based on size. Each group contained about five variants. The first group with a larger size was in the molecular weight range of adult breast TNT, while the smaller-sized second group was in the molecular weight range of adult leg TNT. Overall peptide map patterns of variants in the first group and also that of adult breast TNT resembled each other, whereas those of variants in the second group were similar to that of adult leg TNT. The TNT of adult breast-type appeared at about 2- to 3-weeks posthatch, and thereafter became a major TNT isoform.     

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.     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

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.     

Differential pH effect on calcium-induced conformational changes of cardiac troponin C complexed with cardiac and fast skeletal isoforms of troponin I and troponin T

The aim of this study is to investigate the molecular events associated with the deleterious effects of acidosis on the contractile properties of cardiac muscle as in the ischemia of heart failure. We have conducted a study of the effects of increasing acidity on the Ca(2+) induced conformational changes of pyrene labelled cardiac troponin C (PIA-cTnC) in isolation and in complex with porcine cardiac or chicken pectoral skeletal muscle TnI and/or TnT. The pyrene label has been shown to serve as a useful fluorescence reporter group for conformational and interaction events of the N-terminal regulatory domain of TnC with only minimal fluorescence changes associated with C-terminal domain. Results obtained show that the significant decreases at pH 6.0 of site II Ca(2+) affinity of PIA-cTnC when complexed as a binary complex with either cTnI or cTnT are significantly reduced when cTnI is replaced with sTnI or cTnT with sTnT. However, this effect is appreciably diminished when the cTnI and cTnT in the ternary complex are replaced by sTnI and sTnT. The smaller effects in the ternary complex of replacing both cTnI and cTnT by their skeletal counterparts on depressing the Ca(2+) affinity from pH 7.0 to 6.0 arise from TnI replacement. Thus, changes in TnC conformation resulting from isoform-specific interactions with TnI and TnT could be an integral part of the effect of pH on myofilament Ca(2+)sensitivity.     

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.     

Distribution of fast myosin heavy chain isoforms in thick filaments of developing chicken pectoral muscle

Colloidal gold-conjugated monoclonal antibodies were prepared to stage-specific fast myosin heavy chain (MHC) isoforms of developing chicken pectoralis major (PM). Native thick filaments from different stages of development were reacted with these antibodies and examined in the electron microscope to determine their myosin isoform composition. Filaments prepared from 12-d embryo, 10-d chick, and 1-yr chicken muscle specifically reacted with the embryonic (EB165), neonatal (2E9), and adult (AB8) antimyosin gold-conjugated monoclonal antibodies, respectively. The myosin isoform composition was more complex in thick filaments from stages of pectoral muscle where more than one isoform was simultaneously expressed. In 19-d embryo muscle where both embryonic and neonatal isoforms were present, three classes of filaments were found. One class of filaments reacted only with the embryonic antibody, a second class reacted only with the neonatal-specific antibody, and a third class of filaments were decorated by both antibodies. Similar results were obtained with filaments prepared from 44-d chicken PM where the neonatal and adult fast MHCs were expressed. These observations demonstrate that two myosin isoforms can exist in an individual thick filament in vivo. Immunoelectron microscopy was also used to determine the specific distribution of different fast MHC isoforms within individual filaments from different stages of development. The anti-embryonic and anti-adult antibodies uniformly decorated both homogeneous and heterogeneous thick filaments. The neonatal specific antibody uniformly decorated homogeneous filaments; however, it preferentially decorated the center of heterogeneous filaments. These observations suggest that neonatal MHC may play a specific role in fibrillogenesis.     

Fluorescence spectroscopic analysis of the proximity changes between the central helix of troponin C and the C-terminus of troponin T from chicken skeletal muscle

Recent structural studies of the troponin (Tn) core complex have shown that the regulatory head containing the N-lobe of TnC is connected to the IT arm by a flexible linker of TnC. The IT arm is a long coiled-coil formed by alpha-helices of TnI and TnT, plus the C-lobe of TnC. The TnT is thought to play a pivotal role in the linking of Ca(2+) -triggered conformational changes in thin filament regulatory proteins to the activation of cross-bridge cycling. However, a functional domain at the C-terminus of TnT is missing from the Tn core complex. In this study, we intended to determine the proximity relationship between the central helix of TnC and the TnT C-terminus in the binary and the ternary complex with and without Ca2+ by using pyrene excimer fluorescence spectroscopy and fluorescence resonance energy transfer. Chicken fast skeletal TnC contains a Cys102 at the E helix, while TnT has a Cys264 at its C-terminus. These two cysteines were specifically labeled with sulfhydryl-reactive fluorescence probes. The measured distance in the binary complex was about 19 Angstroms and slightly increased when they formed the ternary complex with TnI (20 Angstroms). Upon Ca2+ binding the distance was not affected in the binary complex but increased by approximately 4 Angstroms in the ternary complex. These results suggest that TnI plays an essential role in the Ca(2+) -mediated change in the spatial relationship between the C-lobe of TnC and the C-terminus of TnT.