Troponin T isoforms and posttranscriptional modifications: Evolution, regulation and function

Troponin-mediated Ca²⁺-regulation governs the actin-activated myosin motor function which powers striated (skeletal and cardiac) muscle contraction. This review focuses on the structure–function relationship of troponin T, one of the three protein subunits of the troponin complex. Molecular evolution, gene regulation, alternative RNA splicing, and posttranslational modifications of troponin T isoforms in skeletal and cardiac muscles are summarized with emphases on recent research progresses. The physiological and pathophysiological significances of the structural diversity and regulation of troponin T are discussed for impacts on striated muscle function and adaptation in health and diseases.     

The Ca/Mg sites of troponin C modulate crossbridge-mediated thin filament activation in cardiac myofibrils

The Ca²⁺/Mg²⁺ sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca²⁺ was bound to the N-terminal regulatory Ca²⁺-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca²⁺. NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1 mM or 30 μM free Mg²⁺. ATPase activity was maximal when sites III and IV were occupied by Mg²⁺ and it steadily declined as Ca²⁺ displaced Mg²⁺. The data suggest that in the absence of Ca²⁺ at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg²⁺ rather than Ca²⁺. This finding could be relevant to the contraction–relaxation kinetics of cardiac muscle. As Ca²⁺ dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca²⁺ bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.     

Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle

Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction-associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinase, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin-linked contractile machinery in smooth muscle.     

Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.     

Kinetic analysis of the interactions between troponin C (TnC) and troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.     

Differences in myofilament calcium sensitivity in rat psoas fibers reconstituted with troponin T isoforms containing the alpha- and beta-exons

The carboxy terminus of fast skeletal muscle troponin T (fsTnT) is highly conserved. However, mutually exclusive splicing of exons 16 and 17 in the fsTnT gene results in the expression of either the alpha- or beta-fsTnT isoform. The alpha-isoform is expressed only in adult fast skeletal muscle, whereas the beta-isoform is expressed in varying quantities throughout muscle development. Reconstitution of detergent-skinned adult rat psoas muscle fibers with rat fast skeletal troponin complexes containing either fsTnT isoform demonstrated that reconstitution with alpha-fsTnT resulted in greater myofilament Ca(2+) sensitivity than reconstitution with beta-fsTnT, without changes to Ca(2+)-activated maximal tension, ATPase activity or tension cost. The observed isoform-specific differences in myofilament Ca(2+) sensitivity may be due to changes in the transition of the thin-filament regulatory unit from the off to the on state, possibly due to altered interactions of the C-terminus of fsTnT with troponins I and/or C.     

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

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

Structural basis for the in situ Ca sensitization of cardiac troponin C by positive feedback from force-generating myosin cross-bridges

The in situ structural coupling between the cardiac troponin (cTn) Ca²⁺-sensitive regulatory switch (CRS) and strong myosin cross-bridges was investigated using Förster resonance energy transfer (FRET). The double cysteine mutant cTnC(T13C/N51C) was fluorescently labeled with the FRET pair 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid (IAEDENS) and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and then incorporated into detergent skinned left ventricular papillary fiber bundles. Ca²⁺ titrations of cTnC(T13C/N51C)_AEDENS/DDPM-reconstituted fibers showed that the Ca²⁺-dependence of the opening of the N-domain of cTnC (N-cTnC) statistically matched the force−Ca²⁺ relationship. N-cTnC opening still occurred steeply during Ca²⁺ titrations in the presence of 1 mM vanadate, but the maximal extent of ensemble-averaged N-cTnC opening and the Ca²⁺-sensitivity of the CRS were significantly reduced. At nanomolar, resting Ca²⁺ levels, treatment with ADP·Mg in the absence of ATP caused a partial opening of N-cTnC. During subsequent Ca²⁺ titrations in the presence of ADP·Mg and absence of ATP, further N-cTnC opening was stimulated as the CRS responded to Ca²⁺ with increased Ca²⁺-sensitivity and reduced steepness. These findings supported our hypothesis here that strong cross-bridge interactions with the cardiac thin filament exert a Ca²⁺-sensitizing effect on the CRS by stabilizing the interaction between the exposed hydrophobic patch of N-cTnC and the switch region of cTnI.     

35 kDa fragment of h-caldesmon conserves two consensus sequences of the tropomyosin-binding domain in troponin T

Using a tropomyosin-coupled affinity column, we have demonstrated a direct association between the chymotryptic 35 kDa fragment of h-caldesmon, which is located at the C-terminal of the parent molecule, and gizzard tropomyosin. We have subsequently determined the nucleotide sequence of cDNA clones encoding the 35 kDa fragment from the cDNA library prepared from chick embryo gizzards, and have deduced the amino acid sequence. Calculating from the predicted sequence, the 35 kDa fragment is composed of 306 amino acid residues. In agreement with the tropomyosin-binding ability, the 35 kDa fragment conserves two consensus sequences of the tropomyosin-binding domain in troponin T. These results suggest that the 35 kDa fragment of h-caldesmon, at least in part, has a common property to the striated muscle troponin T.     

Myeloperoxidase and troponin T are linked with myocardial infarction among young Indians

It is of interest to document the correlation of myeloperoxidase (MPO) and Troponin T (TnT) in young Indian patients with myocardial infarction using epidemiological data. A study with 220 (110 case and 110 control) participants with Acute Coronary Syndrome (both males and females) was completed. Patients were identified using a pre-designed PROFORMA at the OPD and IPD of the Medicine Department at the Index Medical College & Hospital, Malwanchal University and Moti Lal Nehru Medical College and Swaroop Rani Nehru Hospital in India. MPO and TnT were measured using the Sandwich ELISA (Sandwich Enzyme linked immunosorbent Assay) kit. TnT (pg/ml) (Mean ± SD) were high in the case group than control 134.7 ± 8.57 vs 96.6 ± 2.82; F = 145.9; t = 44.3; p < 0.01). Moreover, MPO was 3-fold high in case group than control (157±30.1 and 42.4±14.82, respectively). The differences was found statistically significant (p<0.001). This suggests a link between TnT and MPO among young Indian patients with myocardial infarction.     

Recurrent and founder mutations in the Netherlands: mutation p.K217del in troponin T2, causing dilated cardiomyopathy

Background. About 30% of dilated cardiomyopathy (DCM) cases are familial. Mutations are mostly found in the genes encoding lamin A/C, beta-myosin heavy chain and the sarcomeric protein cardiac troponin-T (TNNT2). Mutations in TNNT2 are reported in approximately 3% of DCM patients. The overall phenotype caused by TNNT2 mutations is thought to be a fully penetrant, severe disease. This also seems to be true for a recurrent deletion in the TNNT2 gene; p.K217del (also known as p.K210del). Methods. We compared the phenotype of all Dutch patients identified as carrying the TNNT2 p.K217del mutation with those described in the literature. All index patients underwent cardiological evaluation. Family screening was done in all described families. Results. Six DCM patients carrying the TNNT2 p.K217del mutation were identified from four Dutch families. Mean age of disease manifestation was 33 years. Heart transplantation was required in three of them at ages 12, 18 and 19 years. These outcomes are comparable with those described in the literature. Conclusion. Carriers of the TNNT2 p.K217del mutation in our Dutch families, as well as in families described in the literature before, generally show a severe, early-onset form of DCM. (Neth Heart J 2010;18:478-85.)     

Role of troponin T in disease

Several striated muscle myopathies have been directly linked to mutations in contractile and associated proteins. Troponin T (TnT) is one of the three subunits that form troponin (Tn) which together with tropomyosin is responsible for the regulation of striated muscle contraction. All three subunits of cardiac Tn as well as tropomyosin have been associated with hypertrophic cardiomyopathy (HCM). However, TnT accounts for most of the mutations that cause HCM in these regulatory proteins. To date 30 mutations have been identified in the cardiac TnT (CTnT) gene that results in familial HCM (FHC). The CTnT gene has also been associated with familial dilated cardiomyopathy (DCM). CTnT deficiency is lethal due to impaired cardiac development. A recessive nonsense mutation in the gene encoding slow skeletal TnT has been associated with an unusual, severe form of nemaline myopathy among the Old Order Amish. How each mutation leads to the diverse clinical symptoms associated with FHC, DCM or nemaline myopathy is unclear. However, the use of animal model systems, in particular transgenic mice, has significantly increased our knowledge of normal and myopathic muscle physiology. In this review, we focus on the role of TnT in muscle physiology and disease. (Mol Cell Biochem 263: 115–129, 2004)     

Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

Correlation of molecular and functional effects of mutations in cardiac troponin T linked to familial hypertrophic cardiomyopathy: an integrative in silico/in vitro approach

Nearly 70% of all of the known cTnT mutations that cause familial hypertrophic cardiomyopathy fall within the TNT1 region that is critical to cTn-Tm binding. The high resolution structure of this domain has not been determined, and this lack of information has hindered structure-function analysis. In the current study, a coupled computational experimental approach was employed to correlate changes in cTnT dynamics to basic function using the regulated in vitro motility assay (R-IVM). An in silico approach to calculate forces in terms of a bending coordinate was used to precisely identify decreases in bending forces at residues 105 and 106 within the proposed cTnT "hinge" region. Significant functional changes were observed in multiple functional properties, including a decrease in the cooperativity of calcium activation, the calcium sensitivity of sliding speed, and maximum sliding speed. Correlation of the computational and experimental findings revealed an association between TNT1 flexibility and the cooperativity of thin filament calcium activation where an increase in flexibility led to a decrease in cooperativity. Further analysis of the primary sequence of the TNT1 region revealed a unique pattern of conserved charged TNT1 residues altered by the R92W and R92L mutations and may represent the underlying "structure" modulating this central functional domain. These data provide a framework for further integrated in silico/in vitro approaches that may be extended into a high-throughput predictive screen to overcome the current structural limitations in linking molecular phenotype to genotype in thin filament cardiomyopathies.     

Troponin replacement in permeabilized cardiac muscle Reversible extraction of troponin I by incubation with vanadate

Calcium-dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate-free, low-Ca²⁺ solution (pCa> 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca2²⁺ (pCa 4.5 prior to incubation with vanadate. Transfer back to a high Ca²⁺ solution elicited no additional force (83.2% ± 18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (>90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.     

The mobility of troponin C and troponin I in muscle

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca²⁺ concentration. The binding of Ca²⁺ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τ_r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg²⁺ by Ca²⁺ at these sites (τ_r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca²⁺ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd.     

Primary cultures of endothelial cells of the rat liver

Summary In the soleus muscle of the normal rat the number of cells containing fast troponin I decreased and those containing slow troponin I increased after birth until less than 10% stained for the fast form in the adult muscle. On denervation of soleus muscle this pattern of change was reversed with the result that the majority of cells stained for fast troponin I. The change was more rapid when denervation was carried out at 12 weeks rather than at 52 weeks of age. Denervation of extensor digitorum longus and tibialis anterior muscles produced little change in the distribution of fast and slow troponin I over a period of 12 weeks. After long periods (>24 weeks) of denervation of these fast muscles, fast troponin I was observed in cells in which originally only slow troponin I could be detected. Similar results to those obtained with troponin I in both fast and slow muscles were obtained using antibodies to the fast and slow forms of troponin C and troponin T.     

Characterization of two tropomyosin isoforms from the fast skeletal muscle of bluefin tuna Thunnus thynnusorientalis

Fast skeletal muscle tropomyosin (TM) of tunas is composed of nearly equimolar amount of two isoforms designated α-TM and β-TM expediently based on their migration behavior in SDS-PAGE, whereas corresponding TMs from the other fish species are homogenous (α-type). The presence of β-TM is thus specific to tunas so far. The amino acid sequence of β-TM from bluefin tuna Thunnus thynnus orientalis, which has not been revealed to date unlike α-TM, was successfully obtained in this study by cDNA cloning. The coding region of β-TM cDNA comprised of an open reading frame of 855 bp encoding 284 amino acid residues, like most of the TMs. Unexpectedly, the sequence of β-TM showed high similarity to those of other vertebrate α-type TMs including tuna α-TM. Phylogenetic analysis also showed that β-TM has the closest relationship with α-TM of tuna. This fact was quite unlike the relation of mammalian α- and β-TMs. Based on the distribution of amino acid substitutions, it was suggested that tuna TM isoforms are the products of different genes. By thermodynamic analysis of native and reconstituted TMs, it was demonstrated that β-TM is less thermostable than α-TM. Proteolytic digestion also supported the lower stability of the former.