Anti-troponin antibodies following myocardial infarction.

Recent improvements in medical and surgical coronary revascularization techniques have significantly improved outcomes for patients with acute myocardial infarction (MI). However, large infarctions are often followed by a poorly understood process of pathological ventricular remodelling, which fails to return the heart to its premorbid state. Although it remains incompletely understood, there is increasing interest in the role of the immune system in this process. One hypothesis is that released cardiac proteins become the focus of an immune response that results in the formation of functionally significant autoantibodies. This review summarizes the current literature, both human and animal, relating to the formation and clinical relevance of anti-troponin antibodies (ATAs) in patients with MI.

Molecular interference in antibody-antigen interaction studied with magnetic force immunoassay.

Molecular interferences are an important challenge in biotechnologies based on antibody-antigen interactions, such as sandwich immunoassays. We report how a sandwich immunoassay with magnetic particles as label can be used to probe interference by surfactants. Surfactants are often used to improve the performance of immunoassays, however the surfactants can affect the involved proteins and the mechanism of action of surfactant molecules on the antibody-antigen system is mostly unknown. As an example, we investigated molecular interference by a nonionic surfactant (Pluronic F-127) in a cardiac troponin (cTn) sandwich immunoassay with two monoclonal antibodies. The influence of the surfactant below the critical micelle concentration (0.00-0.04%) on dissociation properties was quantified in a magnetic tweezers setup, where a force is applied to the molecules via magnetic particle labels. The force-dependent dissociation curves revealed the existence of two distinct cTn-dependent bond types, namely a weak bond attributable to non-specific binding of cTn, and a strong bond attributable to the specific binding of cTn. The dissociation rate constant of the strong bonds increased with the surfactant concentration by about a factor of two. Circular dichroism spectroscopy data showed that the nonionic surfactant influences the conformation of cTn while not noticeably affecting the two monoclonal antibodies. This suggests that the surfactant-induced increase of the dissociation rate of the specific sandwich-type cTn binding may be related to a conformational change of the antigen molecule. The described methodology is an effective tool to study the influence of surfactants and other interferences on assays based on protein interactions.

Troponin: messenger or actor?

Cardiac troponin (cTn) is a biomarker of myocardial damage. New generations of high-sensitivity assays find circulating cTn in virtually all subjects. Multiple studies in various populations and patient groups have found higher levels of cTn to be predictive of future heart failure. The author proposes that initial myocardial damage from various mechanisms may lead to anti-cTn antibodies that participate in ongoing myocardial damage that eventually results in heart failure.

False positive troponin result caused by a true macrotroponin.

BACKGROUND: A 35-year-old female presented with complaints of tiredness, shortness of breath and recent onset of chest pain. The cardiac troponin I (cTnI) concentration was elevated at 6.4 µg/L (Abbott Architect reference value: <0.03 µg/L) with a normal ECG. Physical examination, radiological imaging and routine laboratory investigations did not provide an explanation for the elevated cTnI concentration. METHODS: Use of different troponin assays, dilutions with assay diluent, addition of mouse serum, polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC) were used to investigate possible interference. RESULTS: Troponin concentrations were below the level of detection when measured using all other troponin immunoassays. Serial dilutions of sample and addition of mouse serum did not alter the results. However, PEG precipitation and GFC showed the presence of a high molecular weight immunoreactive protein. Using GFC and protein-A IgG precipitation, the interference could be identified as a macrocomplex containing IgG and (fragments of) cTnI. CONCLUSIONS: We report a false positive cTnI result caused by a true macrotroponin, containing IgG and (fragments of) cTnI. This macrotroponin was only immunoreactive in the Abbott Architect cTnI immunoassay. Clinicians should be aware of analytical interference when troponin results are constantly elevated in the absence of (non)coronary causes of an increased troponin.

Anti-troponin autoantibodies and the cardiovascular system.

Autoimmunity plays a role in the pathogenesis of numerous cardiac diseases such as dilated cardiomyopathy (DCM), myocarditis, rheumatic fever and atherosclerosis. An autoimmune response may appear following myocardial injury and exposure to self-antigens. Anti-heart autoantibodies have been identified in patients with heart disease and in low titres of healthy individuals. Troponin is the preferred marker in detecting acute coronary syndrome. In recent years, anti-troponin autoantibodies were identified in patients with DCM and ischaemic cardiomyopathy. The presence of anti-troponin autoantibodies in the serum may cause a false-negative evaluation of troponin levels and delay treatment of acute coronary syndrome. The role of anti-troponin autoantibodies in humans and their possible involvement in the pathogenesis of heart disease remains controversial. Previous studies have demonstrated that anti-troponin autoantibodies can cause cardiac dysfunction in animal models. This paper reviews current knowledge on anti-troponin autoantibodies. Future research should focus on the pathogenic role of anti-troponin autoantibodies and reactive T cells and the possible benefits of targeted therapy in acute coronary syndrome, post-myocardial infarction, myocarditis, DCM and heart failure.

Circulating immunoreactive cardiac troponin forms determined by gel filtration chromatography after acute myocardial infarction.

BACKGROUND: Cardiac troponin I (cTnI) and cTnT measurements are used in the diagnosis of acute myocardial infarction (AMI). Together with troponin C (TnC), the cTnI and cTnT forms make up the ternary cTnT-cTnI-TnC (TIC) complex found within myocardium. Whether cTn occurs in the circulation after AMI as ternary TIC, binary cTnI-TnC (IC) complexes, or free troponin forms has not been thoroughly investigated. METHODS: Blood samples from 10 AMI patients were collected at hospital admission and then at 12, 24, and 48 h after onset of chest pain. Serum was subjected to gel filtration chromatography and cTnT (Roche cTnT) and cTnI (Siemens Centaur UltraTnI and Beckman Access AccuTnI) concentrations were measured in the gel filtration chromatography fractions. RESULTS: cTnT was present predominantly as free cTnT and cTnI as binary IC complex. These 2 forms were present at every time point. Lesser quantities of TIC complex (6%-32% of total cTnT and <50% of total cTnI) were detected in 4 patients at varying times. Minor quantities of a high molecular mass form of cTnI were detected occasionally. No free cTnI was found. Both cTnI assays identified a similar pattern of cTnI forms. CONCLUSIONS: After AMI, cTnI is present in serum as TIC and IC complexes. cTnT may be present as a combination of TIC and free cTnT or exclusively as free cTnT.

Cardiac troponins and autoimmunity: their role in the pathogenesis of myocarditis and of heart failure.

Despite the widespread use of cardiac troponins as biomarkers for the diagnosis and quantitation of cardiac injury, the effect of troponin release and a possible autoimmune response to the troponins is unknown. Other investigators reported that programmed cell death-1 (PD-1)-receptor deficient mice developed severe cardiomyopathy with autoantibodies to troponin I. We found that immunization of genetically susceptible mice with troponin I but not troponin T induced a robust autoimmune response leading to marked inflammation and fibrosis in the myocardium. At later times, antibodies to cardiac myosin were detected in troponin-immunized mice. The severity of inflammation correlated with expression of chemokines RANTES, MIP-2, IP-10 and MCP-1 in the myocardium. Prior immunization with troponin I increased the severity of experimental infarctions, indicating that an autoimmune response to troponin I aggravates acute cardiac damage. Cardiac inflammation, fibrosis and functional impairment were transferred from immunized to naive recipients by CD4+ T cells, and the cytokine profile suggested both Th2 and Th17 profiles in A/J mice. Finally we identified an 18-mer of troponin I containing an immuno-dominant epitope.

Autoimmune myocarditis: past, present and future.

Heart failure has become an increasingly prevalent disorder with considerable morbidity and mortality. While many causal mechanisms such as inherited cardiomyopathies, ischemic cardiomyopathy or muscular overload are easily identified in clinical practice, the molecular mechanisms that determine the progression of heart failure or ventricular remodelling are largely unknown. Autoimmune responses and inflammation are involved in the pathogenesis of many cardiovascular diseases. There is compelling evidence that inflammatory mechanisms may contribute to progressive heart failure. Thus, myocardial infiltration of lymphocytes and mononuclear cells, increased expression of pro-inflammatory chemokines and cytokines and circulating autoantibodies are frequently observed in myocarditis and dilated cardiomyopathy. In this review we give an overview on myocarditis and describe why diagnosis and treatment of myocarditis in the clinic can be difficult. We present current animal models and describe possible experimental approaches to improve diagnosis. Finally, we give an outlook on possible drug targets by describing the latest findings in the animal models focussing on chemokines and cytokines, T cell responses and interactions, tolerance induction and the development of autoantibodies.

Nasal vaccination with troponin reduces troponin specific T-cell responses and improves heart function in myocardial ischemia-reperfusion injury.

Myocardial ischemia with subsequent reperfusion (MI/R) can lead to significant myocardial damage. Ischemia initiates inflammation at the blood-microvascular endothelial cell interface and contributes significantly to both acute injury and repair of the damaged tissue. We have found that MI/R injury in mice is associated with a cellular immune response to troponin. Myocardial cells exclusively synthesize troponin and release the troponin into the bloodstream following injury. Mucosally administered proteins induce T cells that secrete anti-inflammatory cytokines such as IL-10 and transforming growth factor beta at the anatomical site where the protein localizes. We found that nasal administration of the three subunits of troponin (C, I and T isoforms), given prior to or 1 h following MI/R, decreased infarct size by 40% measured 24 h later. At 1.5 months following MI/R, there was a 50% reduction in infarct size and improvement in cardiac function as measured by echocardiography. Protection was associated with a reduction of cellular immunity to troponin. Immunohistochemistry demonstrated increased IL-10 and reduced IFN-gamma in the area surrounding the ischemic infarct following nasal troponin. Adoptive transfer of CD4+ T cells to mice from nasally troponin-treated mice 1 h after the MI/R decreased infarct size by 72%, whereas CD4+ T cells from IL-10-/- mice or nasally BSA-treated mice had no effect. Our results demonstrate that IL-10-secreting CD4+ T cells induced by nasal troponin reduce injury following MI/R. Modulation of cardiac inflammation by nasal troponin provides a novel treatment to decrease myocardial damage and enhance recovery after myocardial ischemia.

Development of troponin autoantibodies in experimental coxsackievirus B3 myocarditis.

BACKGROUND: Autoantibodies against various endogenous proteins are found in myocarditis. Troponin autoantibodies are detected in patients with chronic dilated cardiomyopathy, but their presence in myocarditis remains unknown. We set out to study the presence of troponin autoantibodies in experimental viral myocarditis. MATERIALS AND METHODS: BALB/c mice infected with coxsackievirus B3 Nancy strain were followed-up at days 1-7 and 2, 4, 8 and 12 weeks after infection. Levels of circulating cardiac troponin I and circulating troponin autoantibodies were measured. Transthoracic echocardiography was performed. Myocarditis was histopathologically graded and cardiomyocyte apoptosis was quantified (TUNEL). RESULTS: Histopathologically relatively mild acute myocarditis followed by persistent cardiomyocyte damage was observed. Rate of cardiomyocyte apoptosis was the highest on day 5 (0.16 +/- 0.01% vs. 0.03 +/- 0.01% in controls, P < 0.001). Circulating troponin I levels were increased to day 5 (45.2 +/- 6.5 ng mL(-1), P < 0.005 vs. controls). Troponin autoantibodies were detected from 2 weeks after infection (20% of animals had autoantibodies at 2 weeks, 60% at 4 and 8 weeks and 20% at 12 weeks, P < 0.05 vs. controls). Fractional shortening remained decreased after acute myocarditis (0.36 +/- 0.02 at 4 weeks, 0.30 +/- 0.02 at 8 and 12 weeks vs. 0.41 +/- 0.01 before infection, P < 0.01) parallel to development of troponin autoantibodies. CONCLUSION: Troponin autoantibodies are formed in experimental virus induced myocarditis following troponin I release and cardiomyocyte apoptosis. The definite role of these autoantibodies remains to be further characterized.

Troponin revisited 2008: assay performance.

Troponin quality specifications describing the pre-analytical, analytical and post-analytical performance of cardiac troponin (cTn) assays are important for both manufacturers of cTn assays and laboratories that routinely test for cTn. Pre-analytical requirements refer not only to acceptable sample type for analysis and the stability of cTn but also to the proper handling of specimens prior to analysis to avoid pre-analytical false positive results. Analytical issues that may contribute to differences between cTn assays include analytical sensitivity and imprecision at low cTn concentration, antibody specificity and immunoreactivity of plasma cTn forms, assay specificity and the presence of falsely positive and negative interferences, and for cTnI the lack of standardised measurement, all which may impact on patient cTn results. Current second generation cTnI and fourth generation cTnT assays generally have an imprecision of around 20% coefficient of variation (CV) at the 99th percentile of the reference population, which is greater than the recommended imprecision of 10% CV. As the next generation of more analytically sensitive cTn assays are developed it can be anticipated that cTn upper reference limits will decrease by approximately 10-fold. Monitoring assay imprecision at ultra low cTn concentrations will require that the laboratory uses a quality control close to this level and a negative control to monitor baseline drift. Establishment of cTn reference ranges will require reference populations to be cardio-healthy to enable differentiation from community populations who are at increased cardiovascular risk. Close collaboration between the laboratory and local clinicians is required to ensure adequate clinical validation of more sensitive cTn assays.

The prevalence of autoantibodies to: myosin, troponin, tropomyosin and myoglobin in patients with circulating triiodothyronine and thyroxine autoantibodies (THAA).

OBJECTIVE: Anti-thyroglobulin, anti-thyroid-peroxidase and anti-TSH receptor antibodies have been observed with high frequency in autoimmune thyroid diseases. Thyroid hormone auto-antibodies (THAA): anti-thyroxine (T4) and anti-triiodothyronine (T3), conversely, have been reported rarely. In both hyperthyroidism and hypothyroidism, patients suffer from muscle weakness and function disorders. The aim of our study was the evaluation of the occurrence rate of autoantibodies targeting muscle proteins in a group of 24 patients with circulating anti-T3 and/or anti-T4 autoantibodies. The control group consisted of 41 healthy blood donors. METHODS: In polyethylene tubes coated with muscle antigens: actin, myosin, myoglobin, troponin and tropomyosin solid-phase radioimmunoassay was performed to detect autoantibodies. A reaction with 125I-labelled staphylococcus protein A was used for the detection of antibodies bound to the antigens on the tubes. RESULTS: We found a high occurrence of antibodies to muscle proteins in patients with THAA. Anti-myoglobin autoantibodies were most frequent (54.2% of subjects), the binding index values was very high and exceeded normal values two to four fold. Anti-myosin autoantibodies were detected in 50% of subjects; anti-troponin autoantibodies in 33.3%, and anti-tropomyosin autoantibody in 3 patients (12.5%). Differences between the patients and the controls were statistically significant. The antibody binding index to actin was low and statistically insignificant. CONCLUSIONS: Our study indicates that muscle protein antibodies, especially to myoglobin, myosin and troponin, are very frequently present in patients with autoimmune thyroid disease and circulating anti-T3 and anti-T4 autoantibodies, as well as in most cases of chronic thyroiditis with clinical symptoms of hypothyroidisms.

Quantitative measurement of cardiac markers in undiluted serum.

Two mycocardial infarction biomarkers, myoglobin (MG) and cardiac troponin I (cTnI), were quantified at biological levels and in undiluted serum without sample pretreatment using surface plasmon resonance (SPR) sensors. To achieve detection of biomarkers in undiluted serum (72 mg/mL total protein concentration), minimization of the nonspecific signal from the serum protein was achieved by immobilizing the antibody for the biomarkers on an N-hydroxysuccinimide activated 16-mercaptohexadecanoic acid self-assembled monolayer. This monolayer reduces the nonspecific signal from serum proteins in such a manner that short exposure of the sensor to serum prior to analysis prevents any further nonspecific adsorption during analysis. Thus, sensing of MG and cTnI was achieved on the basis of the difference between signals from the active sensor and a reference sensor that captured background interference. This resulted in direct measurement of these biomarkers in undiluted serum. Detection limits for both markers were below 1 ng/mL, which is below the threshold needed to detect myocardial infarction. Detecting biomarkers in the low ng/mL range without signal amplification in such a complex matrix as serum corresponds to a selectivity of 108. The root-mean-square-error (RMSE) of calibration was below 2 ng/mL.

[Acute coronary syndrome and inflammation. Biomarkers for diagnostics and risk stratification]. / Akutes Koronarsyndrom und Entzündung. Biomarker zur Diagnostik und Risikostratifizierung.

Inflammation plays a pivotal role in atherosclerosis and coronary heart disease. Inflammatory processes of the coronary arterial wall are involved in plaque formation, progression and, finally, plaque instability consecutively leading to the clinical manifestations of stable coronary artery disease or acute coronary syndromes (unstable angina, non-ST elevation and ST elevation myocardial infarction). Acute coronary syndromes result from plaque rupture or erosion leading to local thrombus formation with consecutive necrosis of myocytes due to ischemia, which is associated with widespread and diffuse pancoronary and panmyocardial inflammation. Accordingly, markers of myocardial necrosis (e. g., cardiac troponins) do have crucial diagnostic and prognostic value. In case of troponin-negative acute coronary syndromes, however, markers of inflammation emerged as potentially useful tools for risk stratification. C-reactive protein has been shown to serve as a powerful predictor of future cardiovascular events following acute coronary syndromes, even if troponins are not (yet) positive. Moreover, a variety of pro- (soluble CD40 ligand, placental growth factor, interleukin-6, pregnancy-associated plasma protein A, myeloperoxidase, monocyte chemoattractant protein-1) and anti-inflammatory markers (interleukin-10, activin A) have been suggested to provide relevant prognostic information in patients with acute coronary syndrome. However, the clinical utility of these novel markers has not been established so far.

First immunocytochemical study of echinoderm smooth muscle: the Antarctic cushionstar Odontaster validus Koehler (Echinodermata, Asteroidea).

Our immunocytochemical observations reveal that the muscle present in the tips of the arms of the Antarctic cushionstar Odontaster validus contains caldesmon and calponin but not troponin. Thus, the muscle clearly belongs to the smooth muscle category. Distributions of contractile proteins such as actin, myosin (the latter a typical vertebrate muscle filament protein), paramyosin, and miniparamyosin (the latter two being characteristic of thick invertebrate muscle filaments) were also determined immunocytochemically. The results suggest that the thin filaments of the starfish smooth muscle are similar to those of the vertebrate muscle, but that the thick filaments differ from those of vertebrates and possess traits that are also seen in the muscle organization of invertebrates. The absence from the O. validus muscle of titin and nebulin, proteins so far known almost exclusively from the striated vertebrate muscle, comes as no surprise, but immunoreactivity to mini-titin (a protein of the same family as titin and its replacement in invertebrates) was strong and unambiguously recognizable between filaments. Odontaster validus' histochemical characteristics may be a reflection of the phylogenetic position of the echinoderms as deuterostome invertebrates or they may express an adaptation of the muscle to the harsh environmental conditions under which it has to function in the Antarctic water.

Measurement of cardiac troponins.

The cardiac troponins form part of the regulatory mechanism for muscle contraction. Specific cardiac isoforms of cardiac troponin T and cardiac troponin I exist and commercially available immunoassay systems have been developed for their measurement. A large number of clinical and analytical studies have been performed and the measurement of cardiac troponins is now considered the 'gold standard' biochemical test for diagnosis of myocardial damage. There have been advances in understanding the development and structure of troponins and their degradation following myocardial cell necrosis. This has contributed to the understanding of the problems with current assays. Greater clinical use has also highlighted areas of analytical and clinical confusion. The assays are reviewed based on manufacturers' information, current published material as well as the authors' in-house experience.

Cardiac troponin T isoforms expressed in renal diseased skeletal muscle will not cause false-positive results by the second generation cardiac troponin T assay by Boehringer Mannheim.

The purpose of this study was to determine whether the two monoclonal anti-cardiac troponin T (cTnT) antibodies (MAbs) used in the second generation cTnT assay by Boehringer Mannheim (BM, capture Ab, M11.7; detection Ab, M7) would detect cTnT isoforms expressed in human skeletal muscle in response to chronic renal disease (CRD). cTnT expression was examined in skeletal muscle biopsies obtained from 45 CRD patients, as well as nondiseased human heart (n = 3) and skeletal muscle (n = 3). cTnT proteins were resolved by modified 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with the following anti-cTnT MAbs: M11.7; M7; JS-2, Lakeland Biomedical; and 13-11, Duke University. All four antibodies detected the cTnT isoforms (Ta, Te) expressed in human myocardium. In 20 of 45 skeletal muscle biopsies, MAb M11.7 recognized its epitope in one to three proteins, molecular mass 34-36 kDa, designated Te, Td, and Tc; the strongest signal was that of Te. The same proteins were recognized by MAbs JS-2 and 13-11. The BM M7 antibody did not detect the cTnT isoforms in the molecular mass range of 34-36 kDa. However, MAb M7 did detect a cTnT isoform, molecular mass 39 kDa, in 2 of 45 biopsies. This isoform had an electrophoretic mobility similar to the predominant heart cTnT isoform, Ta. We conclude that cTnT isoforms are expressed in the skeletal muscle of CRD patients. However, given the epitopes recognized by the BM MAbs M7 and M11.7 and the variable presence of these cTnT isoforms in skeletal muscle, the second generation BM cTnT assay will not detect these isoforms if they are released from skeletal muscle into the circulation.

Troponin-T is a calcium-binding protein in insect muscle: in vivo phosphorylation, muscle-specific isoforms and developmental profile in Drosophila melanogaster.

Two sets of muscle polypeptides showing calcium-binding capacity and intense labelling in vivo with 32P were purified and characterized from Drosophila melanogaster adult extracts. The polypeptides exhibit crossed immunoreactivity and share similar biochemical properties such as those involved in purification. They have been identified as isoforms of troponin-T (TnT) by sequence analysis of a cDNA clone isolated from an embryonic library. The two sets of TnT polypeptides correspond to the fibrillar and non-fibrillar muscle isoforms, respectively. The non-fibrillar muscle isoforms separate into two bands which are differentially expressed during development. Analysis of TnT isoforms in bee thoraces indicates that the expression of the fibrillar muscle isoform correlates with the acquisition of functional flight capability. In vivo labelling experiments reveal that the two TnT sets are readily phosphorylated. The Drosophila TnTs show calcium-binding properties by three different types of assays. Our results suggest that this property could be specific to insect TnTs and may be related to the long, extremely acidic polyglutamic carboxy-terminus present in these polypeptides, which does not occur in non-arthropod TnTs.