Vertebrate Troponin Research Articles

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.

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry

Muscle contraction is tightly regulated by Ca(2+) binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca(2+) binding site in the NH(2) domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D(2)O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D(2)O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca(2+) binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca(2+)-saturated TnC NH(2) domain. More notably, Ca(2+) also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca(2+) sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin.

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry

Troponin is the thin filament protein that confers tight, Ca2+-dependent control over muscle contraction. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin, using amide hydrogen exchange-mass spectrometry, in the presence of either saturation or non-saturation of the regulatory Ca2+ binding site in the NH2-domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D2O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, more than six hours, identifying the most stable portion of the trimeric troponin complex. Regulatory site Ca2+ binding altered dynamic properties (i.e., H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca2+-saturated TnC NH2-domain. More notably, Ca2+ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH-domain that contains the regulatory Ca2+ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto troponin's highly extended structure indicates contacts important in conformational change: in the low Ca2+ state the TnI region that effects inhibition bends back and interacts with the end of the TnT-TnI coiled-coil, as previously suggested by intermediate resolution X-ray data of skeletal muscle troponin. Thus, troponin-mediated Ca2+ sensitive regulation of muscle contraction consists of Ca2+-triggered switching between alternative sets of intra-troponin interactions.

Structure–function relationships of molluscan troponin T revealed by limited proteolysis

Molluscan troponin regulates muscle contraction through a novel Ca 2+-dependent activating mechanism associated with Ca 2+-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin I. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin I, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca 2+-dependent activation of the Mg-ATPase activity of rabbit-actomyosin–scallop-tropomyosin. In addition, the actin–tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca 2+ concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin–tropomyosin filaments and is essential for regulation.

Comparative studies on the functional roles of N‐ and C‐terminal regions of molluskan and vertebrate troponin‐I

Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca(2+)-dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akazara scallop TnI (ATnI(232-292)), which contains the inhibitory region (residues 104-115 of rabbit TnI) and the regulatory TnC-binding site (residues 116-131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase. However, it did not interact with TnC, even in the presence of Ca(2+). These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI(130-252), which contains the structural TnC-binding site (residues 1-30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca(2+)-dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI-fragment (RTnI(1-116)) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca(2+)-dependent activation.

Imaging transcription in vivo: distinct regulatory effects of fast and slow activity patterns on promoter elements from vertebrate troponin I isoform genes.

Firing patterns typical of slow motor units activate genes for slow isoforms of contractile proteins, but it remains unclear if there is a distinct pathway for fast isoforms or if their expression simply occurs in the absence of slow activity. Here we first show that denervation in adult soleus and EDL muscles reverses the postnatal increase in expression of troponin I (TnI) isoforms, suggesting that high-level transcription of both genes in mature muscles is under neural control. We then use a combination of in vivo transfection, live muscle imaging and fluorescence quantification to investigate the role of patterned electrical activity in the transcriptional control of troponin I slow (TnIs) and fast (TnIf) regulatory sequences by directly stimulating denervated muscles with pattern that mimic fast and slow motor units. Rat soleus muscles were electroporated with green fluorescent protein (GFP) reporter constructs harbouring 2.7 and 2.1 kb of TnIs and TnIf regulatory sequences, respectively. One week later, electrodes were implanted and muscles stimulated for 12 days. The change in GFP fluorescence of individual muscle fibres before and after the stimulation was used as a measure for transcriptional responses to different patterns of action potentials. Our results indicate that the response of TnI promoter sequences to electrical stimulation is consistent with the regulation of the endogenous genes. The TnIf and TnIs enhancers were activated by matching fast and slow activity patterns, respectively. Removal of nerve-evoked activity by denervation, or stimulation with a mismatching pattern reduced transcriptional activity of both enhancers. These results strongly suggest that distinct signalling pathways couple both fast and slow patterns of activity to enhancers that regulate transcription from the fast and slow troponin I isoforms.

Molecular evolution of the vertebrate troponin I gene family.

In the higher vertebrates troponin I (TnI) is encoded by three related genes, each of which is expressed specifically in one of the three major sarcomeric muscle cell classes, i.e. cardiomyocytes or fast or slow skeletal muscle fibers. The TnIcardiac isoform contains an "extra" block of proline-rich protein sequence near the N-terminus encoded by an exon that has no counterpart in the TnIfast and TnIslow genes. All three TnI isoforms appear to be orthologously related between birds and mammals, indicating that the TnI gene family was already established in its modern form in the early reptile common ancestor to birds and mammals. Analysis of ascidian TnI suggests that early vertebrate ancestors contained a single TnI gene and that the gene duplications that established the family occurred after the ascidian/vertebrate divergence. Evidence from organisms representing evolutionary intermediates between ascidians and reptiles is incomplete and does not yet delineate the exact order and timing of the TnI gene duplication events. However it does appear that early tetrapods already contained specialized TnI genes encoding long and short isoforms and that multiple differentially expressed TnI genes were present in the vertebrate lineage before the teleost/tetrapod divergence. Ascidians and the protostome invertebrate Drosophila produce long and short TnI isoforms (the longer isoforms containing a proline-rich block of extra sequence near the N-terminus) by an alternative RNA splicing mechanism from a single gene. It is likely that the alternative splicing mechanism is an ancestral feature, and that during vertebrate evolution this mechanism was abandoned in favor of transcriptional regulatory mechanisms directing tissue-specific expression of multiple genes separately encoding long and short TnI isoforms.

Distinct Troponin T Genes Are Expressed in Embryonic/Larval Tail Striated Muscle and Adult Body Wall Smooth Muscle of Ascidian

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the sessile adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actomyosin Mg2+-ATPase activity in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only <60% identity with each other. Northern blotting and whole mount in situ hybridization revealed that these isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.

Ca(2+)-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction.

The steric model of muscle regulation holds that tropomyosin strands running along thin filaments move away from myosin-binding sites on actin when muscle is activated. Exposing these sites would permit actomyosin interaction and contraction to proceed. This compelling and widely cited model is based on changes observed in X-ray diffraction patterns of skeletal muscle following activation. Although analysis of X-ray patterns can suggest models of filament structure, unambiguous interpretation is not possible. In contrast, three-dimensional reconstruction of thin-filament electron micrographs could, in principle, offer direct confirmation of the predicted tropomyosin movement, but so far tropomyosin in skeletal muscle has been resolved definitively only in the 'on' state but not in the 'off' state. Thin filaments from the arthropod Limulus have a similar composition to those from vertebrate skeletal muscle, and troponin-tropomyosin is distributed in both species with the same characteristic 38-nm periodicity. Limulus thin filaments activate skeletal muscle myosin ATPase at micromolar Ca2+ concentrations and confer a high calcium dependence on the enzyme. Arthropod and vertebrate troponin subunits form functional hybrids in vitro and the respective tropomyosins are functionally interchangeable, arguing for a common mechanism of thin-filament-linked regulation in the two phyla. Here we report that tropomyosin is readily resolved in native filaments of troponin-regulated Limulus muscle in both the 'on' and 'off' states, and demonstrate tropomyosin movement, providing support for the importance of steric effects in muscle activation.

Cloning, structural analysis, and expression of the human fast twitch skeletal muscle troponin C gene.

The gene encoding human fast skeletal muscle troponin C (TnC) was cloned, mapped, and sequenced. The locations of intron positions in this gene were compared to those in the related genes for mouse slow skeletal TnC and vertebrate and nonvertebrate calmodulins. We detected strikingly similar purine-rich DNA sequences on the coding strand in the basal promoter of the genes for fast and slow troponin C and chicken calmodulin II which may represent conserved regulatory elements in genes of the vertebrate troponin C/calmodulin gene family. We mapped the transcriptional start site of the gene and analyzed the expression of TnC test genes in the myogenic cell lines C2, L8, and H9c2(2-1) and in the human fibroblast line HuT12. Constructs comprising 4.7 or 6.2 kilobase pairs of 5'-flanking sequence (including the genuine transcriptional start site) upstream of the chloramphenicol acetyltransferase gene as reporter expressed the hybrid gene in C2 cells but not in nonmuscle cells. Surprisingly, no expression was found in cell lines L8 and H9c2(2-1) despite the fact that all three muscle cell lines vigorously express the endogenous TnC fast mRNA after differentiation. The discrepancy between the expression of endogenous genes and the test gene in these cell lines indicates different requirements for regulatory elements in different myogenic cells.

Amino Acid Sequence of Crayfish Troponin I

Troponin I is the actomyosin ATPase inhibitory subunit present in the thin filament regulatory complex. The complete amino acid sequence of crayfish tail muscle troponin I has been determined. The protein is composed of 201 amino acid residues and has a molecular weight of 23,547. The N terminus is blocked, likely by an acetyl group. Crayfish troponin I shows a rather low (20-25%) sequence identity with vertebrate troponin Is as compared to the 60-82% identity within the vertebrate phylum. Similar to vertebrate cardiac troponin I, crayfish troponin I contains a 30-residue-long N-terminal extension. In crayfish troponin I, this segment bears significant sequence homology with the heavy or light chains of particular myosins. The actin-binding domain of crayfish troponin I, which displays 57% sequence homology with vertebrate troponin Is, possesses 2 unusual trimethyllysine residues. The consensus sequence of this domain in five troponin Is is as follows: D-L-R-G-K-F-X-R*-P-X-L-R*-R*-V, where R+ stands for Arg/Lys, R* for Arg/trimethyllysine, and X for any amino acid residue. Troponin I possesses two Ca2+-dependent interactive sites for troponin C; one partly overlaps with the actin binding domain and is highly conserved, and the other, corresponding to the 30-residue-long segment following the N-terminal extension in vertebrate cardiac and crayfish troponin I, is poorly conserved in the different troponin Is. Troponin I also interacts with troponin T. The consensus sequence for the interacting site on troponin I is as follows: h-D- -X-D- -R+-Y-D-h-E-h, where h stands for a hydrophobic residue, D- for Asp/Glu, R+ for Arg/Lys, and X for any residue. The five troponin Is further possess one more 15-residue-long segment of high sequence identity near the C terminus. Its evolutionary conservation suggests that this domain is involved in protein-protein interaction.

Troponin from nematode: purification and characterization of troponin from Ascaris body wall muscle

1. 1. A troponin-like protein was isolated from body wall muscle of Ascaris and separated into three components, the mol. wts of which were approx. 58,000, 36,000 and 20,000 respectively. 2. 2. The three components were designated as troponin- T (TNT), troponin- I (TNI) and troponin- C (TNC) in order of mol. wt, since each component had properties similar to the respective components of vertebrate skeletal-muscle troponin. 3. 3. Ascaris troponin were localized on actin filaments with a 44 nm repeat, an approximately 4 nm longer repeat than vertebrate troponin.

Troponin-like proteins from muscles of the scallop, Aequipecten irradians.

Ca2+ regulation of molluscan actomyosin adenosine triphosphatase is known to be associated with the myosin molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, however, also suggests the possible presence of troponin, a thin-filament-linked Ca2+-regulatory complex. In the present study, scallop troponin and tropomyosin were prepared and complexed with rabbit actin; the resulting synthetic thin filaments form a Ca2+-dependent actomyosin adenosine triphosphatase with Ca2+-insensitive rabbit myosin, indicating that the troponin in scallops is potentially functional. Scallop troponin I was isolated and mixed with chicken troponin C and troponin T, forming a functional hybrid troponin complex, indicating that scallop and vertebrate troponins may act by a common mechanism. Densitometry of sodium dodecyl sulphate/polyacrylamide gels reveals that in synthetic thin filaments there are larger amounts of troponin than are present in native thin filaments. Amounts present in the intact muscle were not determined.

The stoichiometry of the components of arthropod thin filaments

Limulus thin filaments confer calcium sensitivity on calcium-independent myosins and contain in addition to actin and tropomyosin, three troponin components. The molar ratio of actin:tropomyosin: troponin sub-unit T (TN-T): troponin sub-unit C (TN-C) is approximately 7:1:1:1, as in vertebrates, but twice the amount of the troponin sub-unit I (TN-I) may be present. Arthropod troponin binds approximately 1 mole Ca/mol troponin, a significantly smaller amount than bound by vertebrate troponin.

Hybrid troponin reconstituted from vertebrate and arthropod subunits.

TROPONIN, a regulatory protein modifying muscular contraction is found in all the chordate striated muscles as well as in many of the invertebrate muscles which have been studied1,3. The complex of troponin and tropomyosin is bound to the actin containing thin filaments, and both components are required for thin filament-linked regulation1–3,10. In the absence of calcium the troponin–tropomyosin complex blocks the myosin combining sites, inhibiting ATPase activity and therefore contraction. When present in micromolar amounts, calcium binds to troponin and releases the inhibition. Vertebrate troponin consists of three subunits: TN-I, TN-C, and TN-T (ref. 4). TN-1 by itself (molecular weight 24,000) inhibits the actin–myosin interaction irrespective of calcium ion concentration. TN-C (molecular weight 18,000) binds calcium ions and this releases the TN-I imposed inhibition. TN-T (molecular weight 37,000) attaches the subunit complex to tropomyosin. The troponin found on arthropod thin filaments is also composed of subunits3,5,6. Components with chain weights of 29,000 and 18,000 have features analogous to those of vertebrate TN-I and TN-C respectively6. A third component is also found in arthropod troponin preparations (chain weight 55,000–59,000), but its function is not yet fully understood5,6. For convenience, this chain will be referred to here as TN-T. In spite of the overall similarity of vertebrate and arthropod troponin, invertebrate troponin differs from vertebrate troponin in calcium binding properties, and binds one-half to one-quarter of the calcium in conditions where the thin filament is maximally switchedon3,6. Moreover, the thin filament-based regulation in invertebrates is usually coupled with a myosin-linked regulatory system which is apparently not present in chordate striated muscle3,10.