Cardiac troponin I inhibitory peptide: location of interaction sites on troponin C

Cardiac troponin I(129-149) binds to the calcium saturated cardiac troponin C/troponin I(1-80) complex at two distinct sites. Binding of the first equivalent of troponin I(129-149) was found to primarily affect amide proton chemical shifts in the regulatory domain, while the second equivalent perturbed amide proton chemical shifts within the D/E linker region. Nitrogen-15 transverse relaxation rates showed that binding the first equivalent of inhibitory peptide to the regulatory domain decreased conformational exchange in defunct calcium binding site I and that addition of the second equivalent of inhibitory peptide decreased flexibility in the D/E linker region. No interactions between the inhibitory peptide and the C-domain of cardiac troponin C were detected by these methods demonstrating that the inhibitory peptide cannot displace cTnI(1-80) from the C-domain.     

Hypoxemia-induced modification of troponin I and T in canine diaphragm.

Impaired muscle function (fatigue) may result, in part, from modification of contractile proteins due to inadequate O(2) delivery. We hypothesized that severe hypoxemia would modify skeletal troponin I (TnI) and T (TnT), two regulatory contractile proteins, in respiratory muscles. Severe isocapnic hypoxemia (arterial partial pressure of O(2) of approximately 25 Torr) in six pentobarbital sodium-anesthetized spontaneously breathing dogs increased respiratory frequency and electromyographic activity of the diaphragm and internal and external obliques, with death occurring after 131-285 min. Western blot analysis revealed proteolysis of TnI and TnT, 17.5- and 28-kDa fragments, respectively, and higher molecular mass covalent complexes, one of which (42 kDa) contained TnI (or some fragment of it) and probably TnT in the costal and crural diaphragms but not the intercostal or abdominal muscles. These modifications of myofibrillar proteins may provide a molecular basis for contractile dysfunction, including respiratory failure, under conditions of limited O(2) delivery.     

A breed-matched case-control study of potential risk-factors for canine pyometra

The objective was to evaluate plausible risk-factors for pyometra, a common disease affecting almost 25% of all (unspayed) female dogs before 10 years of age. Because of the strong breed-predilection, an age- and breed-matched case-control study was undertaken on 87 pairs (pyometra-cases and healthy controls) from five breeds (Rottweiler (n = 13), Collie (n = 8), Golden retriever (n = 24), Labrador retriever (n = 16) and German shepherd dog (n = 26)). The mean age was 7.9 y (range 0.8-13.8 y). Variables analyzed included pseudopregnancy, age at first oestrus, length of and regularity of the interoestrus interval, hormone treatments, nulliparity, number of parities, age at first whelping, previous urinary tract infections and mammary tumours. Data were modelled multivariably using matched-pair conditional logistic regression. Analysing interactions with breed, previous pregnancy was statistically associated with pyometra. When amalgamated, in three breeds previous pregnancy was protective (Rottweiler, Collie, Labrador retriever) and in one breed statistically intermediate (German shepherd dog) when compared to the baseline (Golden retriever). Previous pregnancy was a statistically significant factor that had a protective effect against pyometra in some breeds but not in the Golden retriever breed. These findings indicate that protective- and risk-factors may vary between different breeds. The obvious problem with low power and limited possibility for extrapolation, using few dogs in few breeds, is acknowledged. However, it is suggested that failure to control for the confounding effect of breed, especially in epidemiological studies on dog diseases, may lead to potentially erroneous conclusions.     

Blood lactate levels in 31 female dogs with pyometra

Background  

Canine pyometra is a life-threatening disease common in countries where spaying of dogs is not routinely performed. The disease is associated with endotoxemia, sepsis, systemic inflammatory response syndrome (SIRS) and a 3–4% mortality rate. Blood lactate analysis is clinically valuable in predicting prognosis and survival, evaluating tissue perfusion and treatment response in human and veterinary critical care settings. The aims of the present study were to investigate 1) the blood lactate levels of female dogs with pyometra by a hand-held analyser and 2) if these levels are related with the clinical status or other biochemical or hematological disorders.     

Clinical presentation of canine pyometra and mucometra: a review

Cystic endometrial hyperplasia (CEH) in the bitch can result in either pyometra, hematometra, or hydrometra, and many facets of these uterine diseases can make them difficult to differentiate. The conditions differ in their systemic effects, since pyometra, particularly closed-cervix pyometra, can be a life-threatening condition that must be recognized, managed, and treated expeditiously. Mucometra is an accumulation of sterile intraluminal mucoid fluid, hematometra is an accumulation of sterile, bloody fluid, and hydrometra is an accumulation of sterile, watery fluid; none of which have any significant systemic outward clinical signs. This paper will describe the definitions, signalment, historical findings, incidence, clinical signs, physical exam findings, and diagnostic findings in canine pyometra and mucometra, and hematometra and hydrometra.     

Cardiac contractile impairment associated with increased phosphorylation of troponin I in endotoxemic rats.

The subcellular mechanisms underlying intrinsic myocardial depression during sepsis remain poorly defined, in particular the relative roles of altered intracellular Ca2+ transients versus changes in myofilament properties. We studied contractile function of cardiac myocytes isolated 12 h after induction of endotoxemia (5 mg/kg intravenous E. coli lipopolysaccharide [LPS]) in conscious rats. Cardiomyocytes from LPS-injected rats had depressed twitch shortening compared with control cells (4.10.2% versus 7.80.3%; P2+ transients (peak indo-1 ratio 1.130.02 versus 1.120.02; P = NS). Contractile depression was unaffected by inhibitors of nitric oxide synthase. Steady-state myofilament response to Ca2+, assessed by tetanization of intact cells over a range of [Ca2+], was reduced significantly in the LPS group (P2+ was unaffected by isoproterenol (3 nmol/L) in endotoxemic cells, whereas there was a rightward shift in control cells. A reduction in myofilament response to Ca2+ is the major determinant of intrinsic cardiac depression in systemic endotoxemia. This condition appears to be related to an increase in myocardial troponin I phosphorylation.     

Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals

This study directly demonstrates that cardiac troponin I (cTnI) is a sensitive, specific, and persistent biomarker in laboratory animals. Histopathological and pathophysiological cardiac changes in dogs, rats and mice correlated with increased serum cTnI with various cardiac inotropic agents, and cardiotoxic drugs and with cardiac arrhythmias, tachycardia, cardiac effusion with dyspnoea, and ageing. A comparison of six immunoassays for cTnI and cardiac troponin T (cTnT) to detect and monitor cardiac injury in a rodent model indicated that enzyme-linked immunosorbent (Life Diagnostics Inc and TriChem Resources Inc, West Chester, Philadelphia, USA) and Immulite (Diagnostic Products Corporation, Llanberis, UK) assays had low sensitivity and less than 1% of the dynamic range of Centaur (Bayer Healthcare Diagnostics, Newbury, UK) cTnI and Elecsys (Roche Diagnostics, Basel, Switzerland) and M8 (Bioveris Europe, Whitney, UK) cTnT assays. In dogs, however, the Immulite assay was effective and correlated with the Centaur. Serum concentrations were highly correlated but 10-fold lower for cTnT compared with cTnI with cardiac injury. Centaur assay also detected cTnI in myocardium from marmosets, swine, cattle, and guinea pigs, indicating it to be candidate cardiac biomarker for these species as well. Purified rat cTnI was 50% more reactive than purified human cTnI in the Centaur assay. In the rat, an age- and gender-dependent variation in serum cTnI was found. Male rats aged six and eight months had a 10-fold greater serum cTnI than age-matched females and three-month-old rats. These increases correlated with minimal histopathological change. Isoproterenol-induced serum cTnI increased up to 760-fold the minimal detectable concentration of 0.07 microg/L, within 4-6 h and decreased with a half-life of 6 h, with an expected return to baseline of 60 h. Severity of histopathological change correlated with serum cTnI during the ongoing injury.     

Interaction of troponin C and troponin C fragments with troponin I and the troponin I inhibitory peptide.

We have quantitated the interactions of two rabbit skeletal troponin C fragments with troponin I and the troponin I inhibitory peptide. The calcium binding properties of the fragments and the ability of the fragments to exert control in the regulated actomyosin ATPase assay have also been studied. The N- and C-terminal divalent metal binding domains of rabbit skeletal troponin C, residues 1-97 and residues 98-159, respectively, were prepared by specific cleavage at cysteine-98 and separation by gel exclusion chromatography. Both of the troponin C fragments bind calcium. The calcium affinity of the weak sites within the N-terminal fragment is about an order of magnitude greater than is reported for these sites in troponin C, suggesting interaction between the calcium-saturated strong sites and the weak sites. Stoichiometric binding (1:1) of the troponin I inhibitory peptide to each fragment and to troponin C increased the calcium affinities of the fragments and troponin C. Complex formation was detected by fluorescence quenching or enhancement using dansyl-labeled troponin C (and fragments) or tryptophan-labeled troponin I inhibitory peptide. The troponin C fragments bind to troponin I with 1:1 stoichiometry and approximately equal affinities (1.6 x 10(6) M-1) which are decreased 4-fold in the presence of magnesium versus calcium. These calcium effects are much smaller than is observed for troponin C. The summed free energies for the binding of the troponin C fragments to troponin I are much larger than the free energy of binding troponin C. This suggests a large positive interaction free energy for troponin C binding to troponin I relative to the fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I

Multidimensional heteronuclear magnetic resonance studies of the cardiac troponin C/troponin I(1-80)/troponin I(129-166) complex demonstrated that cardiac troponin I(129-166), corresponding to the adjacent inhibitory and regulatory regions, interacts with and induces an opening of the cardiac troponin C regulatory domain. Chemical shift perturbation mapping and (15)N transverse relaxation rates for intact cardiac troponin C bound to either cardiac troponin I(1-80)/troponin I(129-166) or troponin I(1-167) suggested that troponin I residues 81-128 do not interact strongly with troponin C but likely serve to modulate the interaction of troponin I(129-166) with the cardiac troponin C regulatory domain. Chemical shift perturbations due to troponin I(129-166) binding the cardiac troponin C/troponin I(1-80) complex correlate with partial opening of the cardiac troponin C regulatory domain previously demonstrated by distance measurements using fluorescence methodologies. Fluorescence emission from cardiac troponin C(F20W/N51C)(AEDANS) complexed to cardiac troponin I(1-80) was used to monitor binding of cardiac troponin I(129-166) to the regulatory domain of cardiac troponin C. The apparent K(d) for cardiac troponin I(129-166) binding to cardiac troponin C/troponin I(1-80) was 43.3 +/- 3.2 microM. After bisphosphorylation of cardiac troponin I(1-80) the apparent K(d) increased to 59.1 +/- 1.3 microM. Thus, phosphorylation of the cardiac-specific N-terminus of troponin I reduces the apparent binding affinity of the regulatory domain of cardiac troponin C for cardiac troponin I(129-166) and provides further evidence for beta-adrenergic modulation of troponin Ca(2+) sensitivity through a direct interaction between the cardiac-specific amino-terminus of troponin I and the cardiac troponin C regulatory domain.     

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.     

Alterations in serum selenium levels and their relation to troponin I in acute myocardial infarction

Selenium (Se) is an essential trace element with antioxidant function. The aim of the present study was to estimate the alterations of Se serum level during the acute phase of myocardial infarction and its relation to biomarkers of myocardial necrosis. Serum Se levels were measured at admission and after 24 h in 60 consecutive patients with acute coronary syndrome (both with and without ST elevation). Troponin I (TnI) was assessed at admission and then twice daily for 3 days; patients with normal levels were excluded. Fifty-five patients with acute MI (positive TnI) were included into the analysis. During the first day of hospitalization, patients received standard therapy, including acetylsalicylic acid, clopidogrel, and heparin or enoxaparin; all underwent urgent coronary angiography and percutaneous intervention, when appropriate. Mean Se levels at baseline and 24 h later were comparable (67.1 ± 2.1 vs. 67.2 ± 1.8 μg/L, ns). Linear regression has shown significant correlation between baseline Se levels and peak TnI (y = 3.4x ? 116, r ² = 0.13, P = 0.008). Positive correlation was found also between the peak TnI and the difference from baseline to 24 h (y = 2.2x + 115, r ² = 0.08, P = 0.04). Moreover, close negative correlation was observed between baseline Se levels and the difference from baseline to 24 h (y = ?0.9x + 62.7, r ² = 0.55, P<0.001). Our results have shown marked individual changes in Se levels during the acute phase of MI as well as correlation between Se levels and peak TnI. These results suggest that alterations in serum Se may be related to the extent of myocardial infarction.     

Periodic binding of troponin C.I and troponin I to tropomyosin-actin filaments

We investigated the distribution of troponin C.I and troponin I along tropomyosin-actin filaments by immunoelectron microscopy and found that anti-troponin I antibody formed transverse striations at 38 nm intervals along the bundle of filaments of both troponin C.I-tropomyosin-actin and troponin I-tropomyosin-actin. Since the length of 38 nm corresponds to the repeating period of filamentous tropomyosin along actin double strands, the present study indicates that troponin I is located at a specific region of each tropomyosin, suggesting that a specific interaction between troponin I and tropomyosin is involved in determining the periodic distribution of troponin I along tropomyosin-actin filaments.     

Cardiac troponin I for predicting right ventricular dysfunction and intermediate risk in patients with normotensive pulmonary embolism

Background

Right ventricular dysfunction (RVD) and cardiac troponin I (cTnI) are important tools for risk stratification in pulmonary embolism (PE). We investigate the association of RVD and cTnI in normotensive PE patients and calculate a cTnI cut-off level for predicting RVD and submassive PE.

Methods

Clinical, laboratory, radiological and echocardiagraphic data were analysed. Patients were categorised into groups with or without RVD and compared focussing on cTnI. Effectiveness of cTnI for predicting RVD and submassive PE was tested.

Results

One hundred twenty-nine normotensive PE patients, 71 with and 58 without RVD, were included. Patients with RVD were older (75.0 years (61.3/81.0) vs. 66.0 years (57.7/75.1), P = 0.019). cTnI (0.06 ng/ml (0.02/0.23) vs. 0.01 ng/ml (0.00/0.03), P < 0.0001) and D-dimer values (2.00 mg/l (1.08/4.05) vs. 1.23 mg/l (0.76/2.26), P = 0.016) were higher in PE with RVD. cTnI was associated with RVD (OR 3.95; 95 % CI 1.95–8.02, p = 0.00014). AUC for cTnI diagnosing RVD was 0.79, and for submassive PE0.87. Cut-off values for cTnI predicting RVD and submassive PE were 0.01 ng/ml, with a negative predictive value of 73 %. cTnI was positively correlated with age, D-dimer and creatinine.

Conclusions

In normotensive PE patients, cTnI is helpful for risk stratification and excluding RVD. cTnI elevation is correlated with increasing age and reduced kidney function.     

Lectin binding patterns in normal canine endometrium and in bitches with pyometra and cystic endometrial hyperplasia

Cystic endometrial hyperplasia (CEH) and pyometra in the bitch are dioestral syndromes, supposed to be caused by hormonal disturbances and changes in endometrial steroid hormone receptor levels. Histologically, the endometria show cystic dilated glands and, if bacteria succeed in invading the uterus, pyometra may develop in the following metoestrus. In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of uterine glands and surface epithelium in healthy dogs and in dogs with CEH and pyometra. Lectin binding is a useful tool to identify glycoconjugates, especially of the glycocalyx, which has essential functions in the endometrium during reproduction. Uterine tissue was obtained from 18 healthy bitches in metoestrus or anoestrus and 18 bitches with a clinical diagnosis of CEH or pyometra. Normal endometria showed cycle-dependent changes in SBA, PNA, HPA and UEA binding during metoestrus and anoestrus. LCA did not show cycle-dependent changes and WGA bound to Golgi regions in the apical parts of surface epithelial cells only in metoestrous. Endometria with inflammatory alterations lost cycle-specific lectin binding patterns and, with increasing severity of pathological changes, showed a marked decrease in binding intensity to the glandular and surface epithelial glycocalyx and secretions. In dogs with CEH, unaltered glands with generally strong lectin binding to the glycocoalyx and Golgi regions were found adjacent to altered glands. The decrease of lectin binding in pyometra cases is supposed to be a result of glandular exhaustion after cystic hyperplasia. In addition, bacterial adhesion to sugar residues on the uterine surface epithelium might impede lectin binding.     

Heterogeneity in human cardiac troponin I standards

The LC-MS analysis of recombinant cardiac troponin I (cTnI) and cTnI extracted from human hearts showed a high degree of structural heterogeneity among all samples. The examined recombinant cTnI samples indicated posttranslational modifications, presumably due to their purification (i.e., 2-mercaptoethanol adducts and carbamylation) and related to their expression (i.e., an N-terminal expression tag). The extracted cTnI samples, while having a higher degree of structural heterogeneity, showed less structural variance between samples than the recombinant proteins. The LC-MS analysis of the extracted cTnI samples provided evidence of posttranslational modification by phosphorylation, acetylation, proteolytic cleavage, and intrachain disulfide bond formation.