Acetogenin-induced fibrotic heart disease from avocado (Persea americana, Lauraceae) poisoning in horses.

Poisoning by avocado (Persea americana) has been confirmed in sheep, goats, dogs, rabbits and ostriches. The clinical signs and lesions are attributed to the acetogenin, persin. Little is known regarding the epidemiology, clinical signs, lesions and therapy caused by acetogenin-induced heart damage. During the two-year study, we investigated a horse farm with six horses that often fed themselves with P. americana leaves or mature fruit pulp and skin on the ground. Two horses died, and one underwent necropsy, histopathology, and immunohistochemistry using the anti-cardiac troponin C (cTnC). Grossly and histopathologically, there was severe cardiac fibroplasia. Immunohistochemically, there was a multifocal decrease or negative expression in the cTnC cardiomyocytes' cytoplasm. Persea americana leaves were confirmed in the alimentary tract using botanical anatomy and molecular techniques. The chemical investigation by (LC-ESI-MS) revealed the presence of the acetogenins, persin and avocadene 1-acetate from P. americana. Persin was present in leaves and fruits (seed and pulp), while avocadene 1-acetate was found in leaves and fruits (seed, peel, and pulp) with a higher concentration in the pulp. Four other horses have been examined by electrocardiogram, echocardiogram and serum Troponin 1 (cTnI). To establish a causal effect of consumption of P. Americana and heart fibroplasia in horses, long-time experiments must be carried out.

X-ray structure of a human cardiac muscle troponin C/troponin I chimera in two crystal forms.

The X-ray crystal structure of a human cardiac muscle troponin C/troponin I chimera has been determined in two different crystal forms and shows a conformation of the complex that differs from that previously observed by NMR. The chimera consists of the N-terminal domain of troponin C (cTnC; residues 1-80) fused to the switch region of troponin I (cTnI; residues 138-162). In both crystal forms, the cTnI residues form a six-turn α-helix that lays across the hydrophobic groove of an adjacent cTnC molecule in the crystal structure. In contrast to previous models, the cTnI helix runs in a parallel direction relative to the cTnC groove and completely blocks the calcium desensitizer binding site of the cTnC-cTnI interface.

Biomarker Assays for Personalised Stroke Risk Assessment in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation is a well-known independent risk factor for stroke yet there is no international consensus on guidelines regarding the introduction of anticoagulation in patients deemed at intermediate risk (e.g. CHA2DS2-VASc of 1). The evolution of cardiac biomarkers such as highly sensitive troponins and B-type natriuretic peptide as well as data on D-dimers, may offer incremental enhancements for personalized thromboembolism risk assessment. These markers provide prognostic data for risk of cardiovascular morbidities associated with atrial fibrillation and offer additional specificity for assessing stroke and thromboembolic risk. These assays may therefore enhance risk prognosis in atrial fibrillation alongside conventional stroke risk stratification tool patients. We seek to explore the application of personalised risk assessment using the biomarkers to aid the clinician treating the patient with atrial fibrillation deemed to be at intermediate risk of stroke. CONCLUSION: The stroke risk assessment of a patient with an intermediate risk of stroke (CHA2DS2- VASc score 1) may be improved by using cardiac biomarkers such as highly sensitive troponin, BNP and D-dimers. We explore the application of these biomarkers to provide personalised risk assessment to help a patient with AF decide on whether to commence anticoagulation.

Troponins, intrinsic disorder, and cardiomyopathy.

Cardiac troponin is a dynamic complex of troponin C, troponin I, and troponin T (TnC, TnI, and TnT, respectively) found in the myocyte thin filament where it plays an essential role in cardiac muscle contraction. Mutations in troponin subunits are found in inherited cardiomyopathies, such as hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). The highly dynamic nature of human cardiac troponin and presence of numerous flexible linkers in its subunits suggest that understanding of structural and functional properties of this important complex can benefit from the consideration of the protein intrinsic disorder phenomenon. We show here that mutations causing decrease in the disorder score in TnI and TnT are significantly more abundant in HCM and DCM than mutations leading to the increase in the disorder score. Identification and annotation of intrinsically disordered regions in each of the troponin subunits conducted in this study can help in better understanding of the roles of intrinsic disorder in regulation of interactomes and posttranslational modifications of these proteins. These observations suggest that disease-causing mutations leading to a decrease in the local flexibility of troponins can trigger a whole plethora of functional changes in the heart.

Functional Divergence in Teleost Cardiac Troponin Paralogs Guides Variation in the Interaction of TnI Switch Region with TnC.

Gene duplication results in extra copies of genes that must coevolve with their interacting partners in multimeric protein complexes. The cardiac troponin (Tn) complex, containing TnC, TnI, and TnT, forms a distinct functional unit critical for the regulation of cardiac muscle contraction. In teleost fish, the function of the Tn complex is modified by the consequences of differential expression of paralogs in response to environmental thermal challenges. In this article, we focus on the interaction between TnI and TnC, coded for by genes that have independent evolutionary origins, but the co-operation of their protein products has necessitated coevolution. In this study, we characterize functional divergence of TnC and TnI paralogs, specifically the interrelated roles of regulatory subfunctionalization and structural subfunctionalization. We determined that differential paralog transcript expression in response to temperature acclimation results in three combinations of TnC and TnI in the zebrafish heart: TnC1a/TnI1.1, TnC1b/TnI1.1, and TnC1a/TnI1.5. Phylogenetic analysis of these highly conserved proteins identified functionally divergent residues in TnI and TnC. The structural and functional effect of these Tn combinations was modeled with molecular dynamics simulation to link divergent sites to changes in interaction strength. Functional divergence in TnI and TnC were not limited to the residues involved with TnC/TnI switch interaction, which emphasizes the complex nature of Tn function. Patterns in domain-specific divergent selection and interaction energies suggest that substitutions in the TnI switch region are crucial to modifying TnI/TnC function to maintain cardiac contraction with temperature changes. This integrative approach introduces Tn as a model of functional divergence that guides the coevolution of interacting proteins.

Chronic calcium imaging of neurons in the mouse visual cortex using a troponin C-based indicator.

This protocol describes the use of the genetically encoded troponin C-based calcium indicator TN-XXL to chronically monitor the functional properties of single neocortical neurons in the mouse visual cortex. A cranial window is implanted over the brain of a mouse expressing TN-XXL in pyramidal neurons of the cerebral cortex. Several days later, the visual cortex is mapped and photographed to facilitate repeated imaging of the same region using two-photon microscopy. Initial two-photon imaging may be done ∼2 wk after the window is implanted. We show the application of this technique for long-term in vivo imaging of stimulus response properties. Beyond providing functional information, long-term imaging of TN-XXL-labeled neurons also enables the simultaneous monitoring of structural properties down to the level of single dendritic spines.

"Troponin elevation in coronary ischemia and necrosis".

Detection of a rise and/or fall of cardiac troponin (cTn) is the cornerstone in the diagnosis of myocardial infarction (MI). For the acute risk, it is hypothesized that cTn mirrors activated coagulation and platelet reactivity and indicates the presence of a ruptured plaque, which may help to identify patients at high risk who benefit particularly from aggressive pharmacological treatment and early invasive strategy. High-sensitivity assays using the 99th percentile as the threshold for positivity can achieve sensitivity at presentation of 90 % or more, and performance further improves with subsequent measurements within 3 to 6 h. By 3 h, negative predictive values of almost 100 % have been reported. However, use of assays with higher sensitivity lead ultimately to a loss of clinical specificity. Thus, other conditions than MI, such as stroke, pulmonary embolism, sepsis, acute perimyocarditis, Takotsubo, acute heart failure and tachycardia also can go with elevated troponin levels. The detection of brief rise and subsequent fall of troponin concentration in marathon runners, and even in healthy subjects, after a standardized exercise test has cast doubts on the hypothesis that troponin is released only upon irreversible damage. This kind of troponin leakage may originate from a cytosolic compartment of the cells and not from the necrosis of thin filaments.

[A case of an allergic reaction due to Anisakis simplex possibly after the ingestion of squid--successful detection of four A. simplex allergens, Ani s 1, Ani s 2, Ani s 12 and troponin C-like protein].

A 62-year-old man ingested dressed salmon and its roe (ikura) and grilled mackerel and one hour later further ingested raw tuna and squid as an evening meal at a bar. Soon after the ingestion of raw seafood, he showed wheals, loss of consciousness and low blood pressure. Specific serum IgE to the nematode Anisakis simplex was positive but those to some seafoods were negative. Moreover, a skin prick test using the crude extract was positive for A. simplex but negative for the seafoods, which he ingested on the day of the above episode. When the A. simplex extract was analyzed by IgE-binding immunoblot analysis using the patient serum, two highly intense protein bands were recognized at 18 and 17 kDa, one intense band at 35 kDa and two weak bands at 28 and 26 kDa. ELISA with 11 natural or recombinant A. simplex allergens (Ani s 1-6, 8, 9, 11 and 12 and troponin C-like protein) showed that the patient serum strongly reacted to Ani s 1 and Ani s 12 and weakly to Ani s 2 and troponin C-like protein. Based on these results, he was diagnosed as IgE-mediated A. simplex allergy due to four allergens (Ani s 1, Ani s 2, Ani s 12 and troponin C-like protein), possibly infested in the raw squid which he had ingested just before manifestation of allergic reactions.

"Outer-sphere to inner-sphere" redox cycling for ultrasensitive immunosensors.

This paper reports chemical-chemical (CC) and electrochemical-chemical-chemical (ECC) redox cycling, for use in ultrasensitive biosensor applications. A triple chemical amplification approach using an enzymatic reaction, CC redox cycling, and ECC redox cycling is applied toward electrochemical immunosensors of cardiac troponin I. An enzymatic reaction, in which alkaline phosphatase converts 4-aminophenyl phosphate to 4-aminophenol (AP), triggers CC redox cycling in the presence of an oxidant and a reductant, and electrochemical signals are measured with ECC redox cycling after an incubation period of time in an air-saturated solution. To obtain high, selective, and reproducible redox cycling without using redox enzymes, two redox reactions [the reaction between AP and the oxidant and the reaction between the oxidized form of AP (4-quinone imine, QI) and the reductant] should be fast, but an unwanted reaction between the oxidant and reductant should be very slow. Because species that undergo outer-sphere reactions (OSR-philic species) react slowly with species that undergo inner-sphere reactions (ISR-philic species), highly OSR-philic Ru(NH(3))(6)(3+) and highly ISR-philic tris(2-carboxyethyl)phosphine (TCEP) are chosen as the oxidant and reductant, respectively. The OSR- and ISR-philic QI/AP couple allows fast redox reactions with both the OSR-philic Ru(NH(3))(6)(3+) and the ISR-philic TCEP. Highly OSR-philic indium-tin oxide (ITO) electrodes minimize unwanted electrochemical reactions with highly ISR-philic species. Although the formal potential of the Ru(NH(3))(6)(3+)/Ru(NH(3))(6)(2+) couple is lower than that of the QI/AP couple, the endergonic reaction between Ru(NH(3))(6)(3+) and AP is driven by the highly exergonic reaction between TCEP and QI (via a coupled reaction mechanism). Overall, the "outer-sphere to inner-sphere" redox cycling in the order of highly OSR-philic ITO, highly OSR-philic Ru(NH(3))(6)(3+)/Ru(NH(3))(6)(2+) couple, OSR- and ISR-philic QI/AP couple, and highly ISR-philic TCEP allows high, selective, and reproducible signal amplification. The electrochemical data obtained by chronocoulometry permit a lower detection limits than those obtained by cyclic voltammetry. The detection limit of an immunosensor for troponin I in serum, calculated from the anodic charges in chronocoulometry, is ca. 10 fg/mL.

Transgenic model of cardiac rhabdomyosarcoma formation.

OBJECTIVES: Cardiac rhabdomyosarcomas are rare, and the pathogenesis of this detrimental disease is widely unknown. Most data are obtained from case reports or small series, and models for systematic pathogenetic studies are lacking. We aimed to establish a transgenic mouse model of cardiac rhabdomyosarcoma formation. METHODS: Standard techniques were used to construct a minigene comprised of the 5' region of the 1.4-kb SM22alpha gene (expressed in embryonic cardiac muscle) and the 2.7-kb SV40 T antigen early region. This T antigen fragment includes the coding sequences for the binding sites of p53 and the proteins of the pRb family. Genotyping of transgenic mice was performed by means of polymerase chain reaction, and phenotypic expression was evaluated by means of immunohistochemistry. RESULTS: Transgenic mice were studied at the age of approximately 8 to 12 weeks. Cardiac tumors were found of variable size in the left or right sides of the heart and were associated with T antigen expression. Histologic analysis revealed a 3.1-fold enhanced cell density, enlarged cell nuclei, and a 3.4-fold enhanced DNA content. Phenotypic characterization of cardiac tumors resulted in positive staining for desmin, smooth muscle alpha-actin, troponin C, and Myo D1, which met the criteria for rhabdomyosarcomas. CONCLUSIONS: To the best of our knowledge, the present study is the first description of a mouse model of cardiac rhabdomyosarcoma formation based on genetic modulation. Our model will be a valuable tool for illuminating the pathogenesis of cardiac rhabdomyosarcomas and will allow the testing of new therapeutic approaches to fight this dreadful disease.

Cardiac and noncardiac, particularly neuromuscular, disease with troponin-T positivity.

OBJECTIVES: Although elevated cardiac troponin T is caused by myocardial damage in the vast majority of the cases (primary cardiac causes), noncardiac disease with secondary damage to the myocardium (secondary cardiac causes) is being increasingly recognised. The present study aimed to retrospectively evaluate the frequency of primary cardiac and secondary cardiac causes of troponin-T positivity, in particular how often troponin-T positivity is associated with neuromuscular disorders. RESULTS: Of 16,944 troponin-T determinations in a secondary centre between April 2004 and April 2005, troponin T was positive in 1408 of them (8.3%). Of these, 622 were included for evaluation. Troponin-T positivity was associated with elevated creatine kinase in 54.5% and with creatinine >2 mg/dl (177 micromol/l) in 16.6% of the tests. The most frequent primary cardiac causes of troponin-T positivity were myocardial ischaemia (59%), atrial fibrillation (23%), and heart failure (22%). The most frequent secondary cardiac causes of troponin-T positivity were renal insufficiency (22%), chronic obstructive lung disease (10%), and acute stroke (4%). There was one cause for troponin-T positivity in 249 cases and more than one in 373 cases. A neurologist saw patients with troponin-T positivity in 9.5% of the cases. Troponin-T positivity was associated with a neuromuscular disorder in 6.3% of the cases. Causes of troponin-I positivity were also frequently causes of troponin-T positivity. CONCLUSIONS: Ischaemic heart disease is the most frequent cause of troponin-T positivity, followed by heart failure and renal insufficiency. Many causes previously described to be only responsible for troponin-I positivity also cause troponin-T elevation. Troponin-T positivity is frequently associated with neuromuscular disorders, most likely due to cardiac involvement of these conditions.

Assessment of the interaction between a synthetic epitope of troponin C and its specific antibody using a label-free faradaic impedimetric immunosensor and alpha-Keggin silicotungstic heteropolyacid as a redox probe.

The development of an immunosensor for the direct probing of the interaction between a cysteine-modified synthetic peptide, which corresponds to the epitope cTnC-89-98 of troponin C, and its specific antibody is described. Following immobilization of the peptide onto gold electrodes through the formation of a self-assembled monolayer, the alteration of the interfacial properties of the electrodes upon peptide-antibody interaction was traced by faradaic electrochemical impedance spectroscopy (EIS) using a silicotungstic heteropolyacid, H(4)SiO(4).12WO(3), as a redox probe. The electrochemical behaviour of the redox probe was evaluated with cyclic voltammetry and EIS. The effect of milk protein or 4-mercaptophenol, which was used as post-blocking agents, on the performance of the immunosensor, was investigated. Treatment with 4-mercaptophenol resulted in immunoeffective electrodes that successfully tested in anti-serum samples. An optimum dilution ratio of the samples, where the effect of the matrix on the measuring signal is negligible, was also determined.

Troponina: Interpretación Clínica / No disponible

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Bioenzymatic detection of troponin C using micro-opto-electro-mechanical systems.

Diagnosis and monitoring of critical diseases such as acute myocardial infarction (AMI) require a quantitative analysis of biological molecules. A high-throughput identification of these biological molecules can be generated by using micro-electro-mechanical systems (MEMS) structures like simple cantilever beams, which respond to the intermolecular forces resulting from binding these molecules. Biochemical markers like troponin C are considered the primary markers for myocardial injury and have generated considerable interest. A 26-residue lytic membrane protein of bee venom melittin (ME) is chosen to interact with rabbit skeletal muscle troponin C (TnC) on the surface of the cantilever beams. An optical beam deflection method is employed to identify the enzymatic reaction on the surface of the cantilever due to these proteins. Identification of these proteins is also done using fluorescence spectroscopy (FS) to compliment the optical monitored deflection method. A second set of proteins like horse radish peroxide (HRP) and hydrogen peroxide (H2O2) are applied to atomic force microscopy (AFM) cantilever beams to study their behavior under the enzymatic reactions of proteins. Identification of these proteins is done using Fourier transform infrared spectroscopy (FTIR). An analytical model of the cantilever beam is developed, and its mode shapes are studied by employing orthogonal polynomials in the classic Rayleigh-Ritz method. The surface stress caused by the enzymatic reaction of the proteins that leads to pure bending on the top surface of the cantilever is evaluated. The information provided by the experimental and analytical modeling reported in this work will be useful in the development of a portable biosensor for the detection of AMI.

Biochemical analysis and immunohistochemical determination of cardiac troponin for the postmortem diagnosis of myocardial damage.

Cardiac disease is the most common cause of sudden death in Western nations. In forensic practice there is a need for more sensitive diagnostic methods for the postmortem diagnosis of myocardial damage. The aim of this study was to analyse the diagnostic efficacy of biochemical markers in cadaver fluids in conjunction with histological studies and the immunohistochemical determination of cardiac troponin C (cTnC) and cardiac troponin T (cTnT) levels in myocardial tissue fixed in formol and included in paraffin. We studied 50 cadavers (43 males and 7 females) with a mean age of 47.5 years (SD 19.2; range 12 to 87 years). Cases were chosen according to the postmortem interval, cause of death, and circumstances of death. Pericardial fluid and serum were tested in duplicate for cardiac troponin I (cTn I), myoglobin and CKMB by immunoassay system using commercial kits. In myocardial tissue, histological studies were performed with hematoxylin and eosin (HE), Masson's trichrome staining and immunohistochemical techniques involving streptavidin-biotin-peroxidase were performed. The results pointed to statistically significant differences for all the biochemical markers in pericardial fluid. The highest levels were obtained in the group of cadavers who had died from myocardial infarction. The immunohistochemical expression of cTnC was detected in 86% of cases; it was strongly positive and usually diffuse. The expression of cTnT, was much less frequent (46% of cases) and less intense. It was concluded that the immunohistochemical determination of cTnC and cTnT levels in myocardial tissue may be used as an index of myocardium damage.

Stunned peri-infarct canine myocardium is characterized by degradation of troponin T, not troponin I.

OBJECTIVE: Degradation of cardiac troponin I (cTnI) has been proposed to represent the underlying molecular mechanism responsible for post-ischemic contractile dysfunction of viable but 'stunned' myocardium. However, this concept is largely derived from models of brief, sublethal ischemia essentially devoid of necrosis, and there is speculation that defects in cTnI may be model-dependent. Accordingly, our primary aim was to evaluate the integrity of cardiac troponins-i.e., cTnI, as well as cTnT and cTnC-in viable but stunned peri-infarct tissue. In addition, we addressed the as-yet unexplored issue of whether the profound reduction of infarct size evoked by brief preconditioning ischemia (PC) was accompanied by a favorable attenuation in ischemia/reperfusion-induced degradation of cTnI, cTnT or cTnC in the remaining viable subepicardium. METHODS: Anesthetized open-chest dogs received 10 min of PC ischemia or a comparable control period, followed by 1 h of sustained coronary occlusion and 3 h of reperfusion. Subepicardial biopsies from the center of the soon-to-be ischemic territory were obtained at baseline and at 30 min and 3 h post-reflow, and myofilament protein integrity (intact cTnI, cTnT and cTnC, as well as degradation bands and covalent complexes) were assessed by Western immunoblotting. In addition, in all dogs, wall thickening was measured by echocardiography, collateral blood flow was assessed during sustained occlusion by injection of radiolabeled microspheres, and infarct size was delineated by tetrazolium staining. RESULTS: Although PC was, as expected, cardioprotective (infarct size of 2 +/- 1% of the risk region vs. 17 +/- 6% in controls; p < 0.05), both control and PC groups exhibited profound and comparable contractile dysfunction following reflow (mean wall thickening reduced to 20-22% of baseline values). There was, however, no significant degradation of cTnI in the viable but stunned, peri-infarct tissue. We did observe degradation of cTnT in the stunned subepicardium, an effect that was attenuated in dogs that received antecedent PC ischemia. However, there was no correlation between post-ischemic wall thickening and the immunoreactivity of the intact cTnT band, or wall thickening and the intensity of the cTnT degradation products. CONCLUSIONS: Our results suggest cTnI degradation is not a universal determinant of post-ischemic myocardial stunning. Moreover, the dissociation between cTnT degradation and wall thickening argue against a direct 'cause-and-effect' relationship between proteolysis of cTnT and acute, post-ischemic contractile dysfunction of stunned peri-infarct myocardium.

Altered cardiac function and minimal cardiac damage during prolonged exercise.

PURPOSE: The purpose of the present study was to examine markers of cardiac function and cardiac damage during a simulated half-ironman triathlon in highly trained athletes. METHODS: Nine highly trained male triathletes volunteered for the study (mean +/- SD; age: 33 +/- 3 yr; height: 1.8 +/- 0.1 m; body mass: 77.7 +/- 3.2 kg). The subjects completed a half-ironman triathlon; 1.9-km swim in an indoor 20-m pool, followed by a laboratory-based 90-km cycle and 21.1-km run. Venous blood samples were drawn and echocardiographic assessment completed before the start of exercise, immediately after each stage, and 24 h postexercise. Serum was analyzed for total creatine kinase activity (CK), creatine kinase isoenzyme MB(mass) (CK-MB(mass)), and cardiac troponin T (cTnT). Left ventricular systolic (stroke volume, and systolic blood pressure/end systolic volume ratio (SBP/ESV)) and diastolic (ratio of early [E] to late [A], ventricular filling E:A) measurements were derived from echocardiographic assessment. RESULTS: The mean completion time of the half-ironman triathlon was 301 +/- 28 min. Left ventricular contractility (SBP/ESV) was significantly reduced after the half-ironman triathlon (P < 0.05). A significant reduction in E:A was observed after the run phase of the half-ironman triathlon (P < 0.05). Significant increases in CK and CK-MB(mass) were observed during and after the half-ironman triathlon (P < 0.05), and cTnT was elevated in four subjects over the course of the half-ironman triathlon. CONCLUSIONS: The physiologic stress imposed by the half-ironman triathlon resulted in a reduced left ventricular contractility and altered diastolic filling, coupled with minimal cardiac damage in a number of highly trained male triathletes. The mechanisms behind such altered cardiac function and cardiac damage after prolonged exercise are yet to be elucidated.

A single-fibre study of the relationship between MHC and TnC isoform composition in rat skeletal muscle.

In the present study, we investigated the possibility that MHC (myosin heavy chain) and TnC (troponin C) isoforms exist in specific combinations in rat-skeletal-muscle fibres. Single fibres (numbering 245) from soleus (predominantly slow-twitch) and sternomastoid (predominantly fast-twitch) muscles of adult rats were analysed for MHC and TnC isoform composition, using alanine-SDS/PAGE for separating MHC isoforms, and a novel method (based on the previously reported influence of Ca2+ on the mobility of Ca2+-binding proteins in SDS gels) for unequivocal identification of TnC isoforms in single-fibre segments. In this study, all fibres that contained only one MHC isoform (slow or fast) contained only the matching TnC isoform and all fibres that contained multiple fast MHC isoforms contained only the fast TnC isoform. Fibres expressing both slow and fast MHC isoforms displayed either both TnC isoforms or only one TnC isoform of a type depending on the relative proportion of fast/slow MHC present. Our results suggest a close relationship between MHC and TnC isoform composition in non-transforming skeletal muscles of adult rat.