Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.     

An interdomain distance in cardiac troponin C determined by fluorescence spectroscopy

The distance between Ca2+-binding site III in the C-terminal domain and Cys35 in the N-terminal domain in cardiac muscle troponin C (cTnC) was determined with a single-tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12-residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48-49 A regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half-width >9 A) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 A for cTnC, but without an effect on the half-width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.     

Nuclear cardiac troponin and tropomyosin are expressed early in cardiac differentiation of rat mesenchymal stem cells

Nuclear actin - which is immunologically distinct from cytoplasmic actin - has been documented in a number of differentiated cell types, and cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) have been detected in association with nuclei of adult human cardiac myocytes. It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. We first tested the hypothesis that nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. We also tested the hypothesis that of these five proteins, only actin is present in nuclei of multipotent, bone marrow-derived mesenchymal stem cells (BM-MSCs) from adult rats in culture, but that cTnC, cTnI, cTnT and cTm appear early and uniquely following cardiomyogenic differentiation. Here we show that nuclear actin is present within nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs. We furthermore show that cTnC, cTnI, cTnT and cTm are not only present in myofilaments of ventricular cardiomyocytes in culture but are also within their nuclei; significantly, these four proteins appear between days 3 and 5 in both myofilaments and nuclei of BM-MSCs treated to differentiate into cardiomyogenic cells. These observations indicate that cardiac troponin and tropomyosin could have important cellular function(s) beyond Ca(2+)-regulation of contraction. While the roles of nuclear-associated actin, troponin subunits and tropomyosin in cardiomyocytes are not known, we anticipate that the BM-MSC culture system described here will be useful for elucidating their function(s), which likely involve cardiac-specific, Ca(2+)-dependent signaling in the nucleus.     

Ca(2+) induces an extended conformation of the inhibitory region of troponin I in cardiac muscle troponin.

The inhibitory region of troponin I (TnI) plays a central regulatory role in the contraction and relaxation cycle of skeletal and cardiac muscle through its Ca(2+)-dependent interaction with actin. Detailed structural information on the interface between TnC and this region of TnI has been long in dispute. We have used fluorescence resonance energy transfer (FRET) to investigate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in cTnI brackets the inhibitory region (residues 130-149), and the distance between the two sites was found to be 19.4 A in free cTnI. This distance was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca(2+) in cTnC. An increase of 9 A in the Trp129-Cys152 separation was observed upon saturation of the Ca(2+) regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended conformation is different from a recent model of the Ca(2+)-saturated skeletal TnI-TnC complex in which the inhibitory region is modeled as a beta-turn. The observed Ca(2+)-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca(2+) activation in cardiac muscle.     

A model of calcium activation of the cardiac thin filament

The cardiac thin filament regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin and tropomyosin. Over the past several decades, many details of the structure and function of the cardiac thin filament and its components have been elucidated. We propose a dynamic, complete model of the thin filament that encompasses known structures of cardiac troponin, tropomyosin, and actin and show that it is able to capture key experimental findings. By performing molecular dynamics simulations under two conditions, one with calcium bound and the other without calcium bound to site II of cardiac troponin C (cTnC), we found that subtle changes in structure and protein contacts within cardiac troponin resulted in sweeping changes throughout the complex that alter tropomyosin (Tm) dynamics and cardiac troponin--actin interactions. Significant calcium-dependent changes in dynamics occur throughout the cardiac troponin complex, resulting from the combination of the following: structural changes in the N-lobe of cTnC at and adjacent to sites I and II and the link between them; secondary structural changes of the cardiac troponin I (cTnI) switch peptide, of the mobile domain, and in the vicinity of residue 25 of the N-terminus; secondary structural changes in the cardiac troponin T (cTnT) linker and Tm-binding regions; and small changes in cTnC-cTnI and cTnT-Tm contacts. As a result of these changes, we observe large changes in the dynamics of the following regions: the N-lobe of cTnC, the mobile domain of cTnI, the I-T arm, the cTnT linker, and overlapping Tm. Our model demonstrates a comprehensive mechanism for calcium activation of the cardiac thin filament consistent with previous, independent experimental findings. This model provides a valuable tool for research into the normal physiology of cardiac myofilaments and a template for studying cardiac thin filament mutations that cause human cardiomyopathies.     

Studying Calcium Ion-Dependent Effect on the Inter-subunit Interaction Between the cTnC N-terminal Domain and cTnI C-terminal Switch Peptide of Human Cardiac Troponin via Chou’s 5-Steps Rule

Human cardiac troponin (cTn) is a calcium ion (Ca2+)-sensitive hetero-trimer complex that consists of three subunits: cTnC, cTnI and cTnT. The protein is a     

Troponin elevation in acute ischemic stroke (TRELAS) - protocol of a prospective observational trial

Background  

Levels of the cardiac muscle regulatory protein troponin T (cTnT) are frequently elevated in patients with acute ischemic stroke and elevated cTnT predicts poor outcome and mortality. The pathomechanism of troponin release may relate to co-morbid coronary artery disease and myocardial ischemia or, alternatively, to neurogenic cardiac damage due to autonomic activation after acute ischemic stroke. Therefore, there is uncertainty about how acute ischemic stroke patients with increased cTnT levels should be managed regarding diagnostic and therapeutic workup.     

The interaction of the bisphosphorylated N-terminal arm of cardiac troponin I-A 31P-NMR study

Cardiac troponin I, the inhibitory subunit of the heterotrimeric cardiac troponin (cTn) complex is phosphorylated by protein kinase A at two serine residues located in its heart-specific N-terminal extension. This flexible arm interacts at different sites within cTn dependent on its phosphorylation degree. Bisphosphorylation is known to induce conformational changes within cTnI which finally lead to a reduction of the calcium affinity of cTnC. However, as we show here, the bisphosphorylated cTnI arm does not interact with cTnC, but with cTnT and/or cTnI.     

Small-angle neutron scattering with contrast variation reveals spatial relationships between the three subunits in the ternary cardiac troponin complex and the effects of troponin I phosphorylation

Small-angle neutron scattering with contrast variation has been used to determine the shapes and dispositions of the three subunits of cardiac troponin and to study the influence of phosphorylation on the structure. Three contrast variation series were collected on three different isotopically labeled variants of the cTnC/cTnI/cTnT(198-298) complex, one of which contained deuterated and bisphosphorylated cTnI. Analysis of the scattering data shows cTnT(198-298) interacting with a single lobe of a somewhat compacted cTnC that sits at one end of an elongated rodlike cTnI, covering about one-third of its length. The cTnT(198-298) sits near the center of the long cTnI axis. The components undergo significant conformational changes and reorientations in response to protein kinase A phosphorylation of cTnI. The rodlike cTnI bends sharply at the end interacting with the cTnC/cTnT(198-298) component, which reorients so as to maintain its contacts with cTnI while undergoing only a relatively small change in shape.     

Cardiac troponin T forms a tetramer in vitro

Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction has led to the development of antibody-based assays for measuring cTnT serum concentration. We examined the behavior of cTnT in solution. Recombinant human cTnT3, the dominant isoform in the adult human heart, was used. The protein was pure and functional, as demonstrated by SDS-PAGE and surface plasmon resonance. cTnT3 was found to bind specifically and in a concentration-dependent manner to cTnC. Routine size exclusion chromatography suggested a higher-than-expected MW for cTnT. Using analytical ultracentrifugation, we found cTnT3 in solution to be mainly in the form of a tightly bound tetramer at concentrations as low as 4 micromol/L. Our sedimentation velocity and transmission electron microscopy results indicate that the tetramer's shape is elongated rather than globular. CTnT's self-association in solution is an important consideration in the design and interpretation of experiments with the aim of understanding the biochemical and biophysical properties of cTnT, its isoforms, and its mutants.     

Molecular effects of familial hypertrophic cardiomyopathy-related mutations in the TNT1 domain of cTnT

Familial hypertrophic cardiomyopathy (FHC) is one of the most common genetic causes of heart disease. Approximately 15% of FHC-related mutations are found in cTnT [cardiac troponin (cTn) T]. Most of the cTnT FHC-related mutations are in or flanking the N-tail TNT1 domain that directly interacts with overlapping tropomyosin (Tm). We investigate two sets of cTnT mutations at opposite ends of TNT1, mutations in residue 92 in the Tm-Tm overlap region of TNT1 and mutations in residues 160 and 163 in the C-terminal portion of TNT1 adjacent to the cTnT H1-H2 linker. Though all the mutations are located within TNT1, they have widely different phenotypes clinically and biophysically. Using a complete atomistic model of the cTn-Tm complex, we identify mechanisms by which the effects of TNT1 mutations propagate to the cTn core and site II of cTnC, where calcium binding and dissociation occurs. We find that mutations in TNT1 alter the flexibility of TNT1, which is inversely proportional to the cooperativity of calcium activation of the thin filament. Further, we identify a pathway of propagation of structural and dynamic changes from TNT1 to site II of cTnC, including TNT1, cTnT linker, I-T arm, regulatory domain of cTnI, the D-E linker of cTnC, and site II cTnC. Mutationally induced changes at site II of cTnC alter calcium coordination that corresponds to biophysical measurements of calcium sensitivity. Finally, we compare this pathway of mutational propagation with that of the calcium activation of the thin filament and find that they are identical but opposite in direction.     

An improved method for exchanging troponin subunits in detergent skinned rat cardiac fiber bundles.

We describe a method for the removal of endogenous troponin (Tn) complex from bundles of detergent-treated cardiac fibers. After 70 min treatment with cTnT-cTnI most of the endogenous Tn complex was removed from fiber bundles. Complete reconstitution of the Tn complex was achieved by reconstituting with cardiac troponin C (cTnC) in fully relaxing conditions. Ca(2+)-dependent maximum force of the fibers treated with cTnT-cTnI or cTnT-cTnI(33-211), which was used to aid in the visualization of the troponin exchange, decreased to 85-90% of the force developed by fibers before the treatment. SDS-PAGE analysis of the cTnT-cTnI(33-211) and the cTnT(77-289)-cTnI(33-211) treated fiber bundles demonstrated that 70-80% of the endogenous Tn subunits were removed. After reconstitution with cTnC, approximately 80-85% of the Ca(2+)-regulated force was restored in cTnT-cTnI/cTnI(33-211) treated fibers. Our results demonstrate that by minimizing the prolonged exposure of skinned cardiac fiber bundles to rigor conditions, successful exchange of all three subunits of the Tn complex can be accomplished with minimal loss of function.     

Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data

OBJECTIVE: We used a new graphical ordinal logit method (GOLDminer) to assess a single cardiac troponin T (cTnT) analysis at the time of admission (first generation monoclonal; Roche BMC Corp., Indianapolis, Indiana), the character of chest pain, and electrocardiographic (ECG)findings in predicting the likelihood of acute myocardial infarction (AMI) in patients presenting with suspected myocardial ischemia. The final diagnosis of AMI was based on serial ECG findings and evolution of CKMB isoenzyme levels in conjunction with clinical findings. SUBJECTS: The study population consisted of 293 consecutive patients who presented at a mean of six hours after onset of chest pain or associated symptoms warranting a "rule-out" for AMI assessment to a university-affiliated community hospital. RESULTS: The odds-ratio for an elevated cTnT (> 0. 1 ng/ml) in AMI was 22.2:1. There was an association between typical chest pain and cTnT (chi square = 78.23, p < .0001) and between abnormal ECG findings and cTnT (chi square = 108, p < .0001). The cTnT yielded diagnostic benefit in addition to chest pain characteristics and ECG findings in AMI. We present the odds-ratios for the combined features in GOLDminer plots. CONCLUSION: We demonstrate how the odds-ratios for AMI are obtained after scaling continuous to ordinal the values for a single cTnT determination alone and with other features in patients presenting with chest pain.     

The contractile apparatus as a target for drugs against heart failure: Interaction of levosimendan,a calcium sensitiser,with cardiac troponin c

Cardiac failure is one of the leading causes of mortality in developed countries. As life expectancies of the populations of these countries grow, the number of patients suffering from cardiac insufficiency also increases. Effective treatments are being sought and recently a new class of drugs, the calcium sensitisers, was developed. These drugs cause a positive inotropic effect on cardio-myocytes by interacting directly with the contractile apparatus. Their mechanism of action is not accompanied by an increase in intracellular calcium concentration at therapeutic doses, as seen for the older generation of positive inotropic drugs, and thus does not induce calcium-related deleterious effects such as arrhythmias or apoptosis. Levosimendan is a novel calcium sensitiser which has been discovered by using cardiac troponin C (cTnC) as target protein. This drug has been proved to be a well-tolerated and effective treatment for patients with severe decompensated heart failure. This review describes the basic principles of muscle contraction, the main components of the contractile apparatus and their roles in the heart contraction. The regulatory proteins troponin C (cTnC), troponin I (cTnI), troponin T (cTnT), and tropomyosin (Tm) and their interactions are discussed in details. The concept of calcium sensitisation is thereafter explained and a few examples of calcium sensitisers and their putative mechanisms are discussed. Finally, the binding of levosimendan to cTnC and its mechanism of action are described and the results discussed under the light of the action of this drug in vitro and in vivo.     

Drug binding to cardiac troponin C.

Compounds that sensitize cardiac muscle to Ca(2+) by intervening at the level of regulatory thin filament proteins would have potential therapeutic benefit in the treatment of myocardial infarctions. Two putative Ca(2+) sensitizers, EMD 57033 and levosimendan, are reported to bind to cardiac troponin C (cTnC). In this study, we use heteronuclear NMR techniques to study drug binding to [methyl-(13)C]methionine-labeled cTnC when free or when complexed with cardiac troponin I (cTnI). In the absence of Ca(2+), neither drug interacted with cTnC. In the presence of Ca(2+), one molecule of EMD 57033 bound specifically to the C-terminal domain of free cTnC. NMR and equilibrium dialysis failed to demonstrate binding of levosimendan to free cTnC, and the presence of levosimendan had no apparent effect on the Ca(2+) binding affinity of cTnC. Changes in the N-terminal methionine methyl chemical shifts in cTnC upon association with cTnI suggest that cTnI associates with the A-B helical interface and the N terminus of the central helix in cTnC. NMR experiments failed to show evidence of binding of levosimendan to the cTnC.cTnI complex. However, levosimendan covalently bound to a small percentage of free cTnC after prolonged incubation with the protein. These findings suggest that levosimendan exerts its positive inotropic effect by mechanisms that do not involve binding to cTnC.     

Exercise improves impaired ventricular function and alterations of cardiac myofibrillar proteins in diabetic dyslipidemic pigs.

Chronic diabetes is often associated with cardiomyopathy, which may result, in part, from defects in cardiac muscle proteins. We investigated whether a 20-wk porcine model of diabetic dyslipidemia (DD) would impair in vivo myocardial function and yield alterations in cardiac myofibrillar proteins and whether endurance exercise training would improve these changes. Myocardial function was depressed in anesthetized DD pigs (n = 12) compared with sedentary controls (C; n = 13) as evidenced by an approximately 30% decrease in left ventricular fractional shortening and an approximately 35% decrease in +dP/dt measured by noninvasive echocardiography and direct cardiac catheterization, respectively. This depression in myocardial function was improved with chronic exercise as treadmill-trained DD pigs (DDX) (n = 13) had significantly greater fractional shortening and +dP/dt than DD animals. Interestingly, the isoform expression pattern of the myofibrillar regulatory protein, cardiac troponin T (cTnT), was significantly shifted from cTnT1 toward cTnT2 and cTnT3 in DD pigs. Furthermore, this change in cTnT isoform expression pattern was prevented in DDX pigs. Finally, there was a decrease in baseline levels of cAMP-dependent protein kinase-induced phosphorylation of the myofibrillar proteins troponin I and myosin-binding protein-C in DD animals. Overall, these results indicate that 20 wk of DD lead to myocardial dysfunction coincident with significant alterations in myofibrillar proteins, both of which are prevented with endurance exercise training, implying that changes in myofibrillar proteins may contribute, at least in part, to cardiac dysfunction associated with diabetic cardiomyopathy.     

Prognostic significance of elevated cardiac troponin-T levels in acute respiratory distress syndrome patients

Background

Elevated levels of biochemical markers of myocardial necrosis have been associated with worsened outcomes in Acute Respiratory Distress Syndrome (ARDS), but there are few prospective data on this relationship. We investigated elevated cardiac troponin T (cTnT) levels and their relationship with outcome in patients with ARDS.

Methods

A prospective cohort study of patients with ARDS was conducted at a tertiary-care academic medical center. Patients had blood taken within 48 hours of ARDS onset and assayed for cTnT. Patients were followed for the outcomes of 60-day mortality, number of organ failures, and days free of mechanical ventilation. Echocardiographic and electrocardiographic (ECG) data were analyzed for signs of myocardial ischemia, infarction, or other myocardial dysfunction.

Results

177 patients were enrolled, 70 of whom died (40%). 119 patients had detectable cTnT levels (67%). Median cTnT level was 0.03 ng/mL, IQR 0–0.10 ng/mL, and levels were higher among non-survivors (P = .008). Increasing cTnT level was significantly associated with increasing mortality (P = .008). The association between increasing cTnT level and mortality remained significant after adjustment in a multivariate model (HR_adj = 1.45, 95% CI 1.17–1.81, P = .001). Elevated cTnT level was also associated with increased number of organ failures (P = .002), decreased number of days free of mechanical ventilation (P = .03), echocardiographic wall motion abnormalities (P = 0.001), and severity of tricuspid regurgitation (P = .04). There was no association between ECG findings of myocardial ischemia or infarction and elevated cTnT.

Conclusions

Elevated cTnT levels are common in patients with ARDS, and are associated with worsened clinical outcomes and certain echocardiographic abnormalities. No association was seen between cTnT levels and ECG evidence of coronary ischemia.     

Structural kinetics of cardiac troponin C mutants linked to familial hypertrophic and dilated cardiomyopathy in troponin complexes

The key events in regulating cardiac muscle contraction involve Ca(2+) binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca(2+) movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca(2+) binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca(2+)-induced opening and closing of the N-domain by F?rster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca(2+). L29Q had no effect on the closing rate of the N-domain triggered by release of Ca(2+), but reduced the Ca(2+)-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca(2+) dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.     

Effects of PKA phosphorylation of cardiac troponin I and strong crossbridge on conformational transitions of the N-domain of cardiac troponin C in regulated thin filaments

Regulation of cardiac muscle function is initiated by binding of Ca2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine-tuned by phosphorylation of cTnI. The objective of the present study is to use a new F?rster resonance energy transfer-based structural marker to distinguish structural and kinetic effects of Ca2+ binding, crossbridge interaction, and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a double-cysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin, and actin. Changes in the distance between Cys13 and Cys51 induced by Ca2+ binding/dissociation were determined by FRET-sensed Ca2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.