Alexander S. Streng

Posttranslational modifications of cardiac troponin T: An overview

Cardiac troponin (cTn) is an important sarcomeric protein complex situated on the thin filament and is involved in the regulation of cardiac muscle contraction. This regulation is primarily controlled by Ca(2+) binding to troponin C and in addition fine-tuned by the posttranslational modification of cTnI and cTnT. The vast majority of cTnT modifications involve the phosphorylation by protein kinase C (PKC) or other kinases and the N-terminal cleavage by caspase and calpain. In vitro studies employing reconstituted detergent-skinned fiber bundles and cell culture generally show a detrimental effect of cTnT phosphorylation on muscle contraction, which is backed by some in vivo studies finding increased cTnT phosphorylation in heart failure, but contradicted by others. In addition, N-terminal cleavage of cTnT is thought to be another factor influencing cardiac contraction. Time-dependent degradation of cTnT has been observed in human serum upon myocardial infarction. These molecular changes might influence the immunoreactivity of cTnT in the clinical immunoassay and have consequences for the clinical interpretations of these measurements. No consensus has yet been reached on the occurrence and extent of these observations and their underlying processes are subject of intense scientific debate. This review will focus on discussing these modifications, their implications on physiology and disease and summarizes the complex interplays of different enzymes on the molecular forms of cTnT and their associated effects.

Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T.

UNLABELLED Cardiac troponin T (cTnT) is a highly cardiospecific protein commonly used in the diagnosis of acute myocardial infarction (AMI), but is subject to proteolytic degradation upon its release in the circulation. In this study, a targeted mass spectrometry assay was developed to detect peptides which are differentially present within the different degradation products. cTnT was spiked in human serum and incubated at 37 °C to induce proteolytic degradation. Isolation and fractionation of cTnT and its fragments from serum were performed using immunoprecipitation and SDS-PAGE. Bands migrating to 37 kDa (intact cTnT), 29 kDa (primary fragment), and 19, 18, and 16kDa (secondary fragments) were excised, digested, and subsequently analysed using targeted selected ion monitoring on a UHPLC-coupled quadrupole-Orbitrap mass spectrometer. Sixteen precursor ions from a total of 11 peptides unique to cTnT were targeted. Precursor ions were detectable up until 1200 ng/L cTnT, which is a typical cTnT concentration after AMI. With tandem-MS and relative quantification, we proved the formation of cTnT fragments upon incubation in human serum and identified differentially present peptides in the fragment bands, indicative of N- and C-terminal proteolytic cleavage. These findings are of importance for the development of future cTnT assays, calibrators, and quality control samples. BIOLOGICAL SIGNIFICANCE In this study we have developed a gel-based targeted mass spectrometry assay which is able to differentiate between different molecular forms of cTnT. The unravelling of the molecular presentation of cTnT in human serum is of importance in the field of clinical chemistry, where this highly specific and sensitive biomarker is being measured on a routinely basis in patient samples. Knowledge of the amino acid sequence of the different cTnT fragments may aid in the development of improved calibrators and quality control samples. In addition, different fragmentation patterns may be indicative of different underlying pathologies. New antibodies for future assays targeting specific areas of cTnT can thus be created based on this information. This assay will be used in future experiments to assess the fragmentation pattern of cTnT in serum of multiple patient groups in our laboratory.

Cardiac troponin in ischemic cardiomyocytes: intracellular decrease before onset of cell death.

AIM Cardiac troponin I (cTnI) and T (cTnT) are the most important biomarkers in the diagnosis of acute myocardial infarction (AMI). Nevertheless, they can be elevated in the absence of AMI. It is unclear if such elevations represent irreversible cardiomyocyte-damage or leakage from viable cardiomyocytes. Our objective is to evaluate whether cTn is released from viable cardiomyocytes in response to ischemia and to identify differences in the release of cTn and its molecular forms. METHODS AND RESULTS HL-1 cardiomyocytes (mouse) were subjected to ischemia (modeled by anoxia with glucose deprivation). The total contents and molecular forms of cTn were determined in culture media and cell lysates. Cell viability was assessed from the release of lactate dehydrogenase (LDH). Before the release of LDH, the intracellular cTn content in ischemic cells decreased significantly compared to control (52% for cTnI; 23% for cTnT) and was not matched by a cTn increase in the medium. cTnI decreased more rapidly than cTnT, resulting in an intracellular cTnT/cTnI ratio of 25.5 after 24 h of ischemia. Western blots revealed changes in the relative amounts of fragmented cTnI and cTnT in ischemic cells. CONCLUSIONS HL-1 cardiomyocytes subjected to simulated ischemia released cTnI and cTnT only in combination with the release of LDH. We find no evidence of cTn release from viable cardiomyocytes, but did observe a significant decrease in cTn content, before the onset of cell death. Intracellular decrease of cTn in viable cardiomyocytes can have important consequences for the interpretation of cTn values in clinical practice.

Origin of Cardiac Troponin T Elevations in Chronic Kidney Disease

Plasma concentrations of cardiac troponins, the preferred biomarkers for the diagnosis of acute myocardial infarction, are often persistently elevated in patients with chronic kidney disease (CKD). The origin of these elevations is unknown: Is it the heart, by increased release, or the kidneys, by decreased renal elimination? In clinical practice, this equivocal view on troponin elevations in patients with reduced glomerular clearance underlies countless clinical discussions among physicians and may delay rapid initiation of adequate treatment when these patients present with chest pain. In the present study, we aimed to discriminate between increased cardiac release and reduced renal elimination as the main process underlying this phenomenon. Specifically, we used the recently demonstrated rhythmic diurnal oscillation pattern of troponin T as a model to assess the contribution of impaired renal elimination to persistently elevated cardiac troponin levels in patients with CKD.1 The diurnal troponin T rhythm is characterized by gradually decreasing concentrations throughout daytime and rising concentrations during nighttime.1 If decreased renal clearance, and not increased production, is the key driver of elevated troponins in patients with CKD, the increased half-life and subsequent accumulation of cardiac troponin T will fade its diurnal rhythm. …

Validation, optimisation, and application data in support of the development of a targeted selected ion monitoring assay for degraded cardiac troponin T

Cardiac troponin T (cTnT) fragmentation in human serum was investigated using a newly developed targeted selected ion monitoring assay, as described in the accompanying article: “Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T” [1]. This article presents data describing aspects of the validation and optimisation of this assay. The data consists of several figures, an excel file containing the results of a sequence identity search, and a description of the raw mass spectrometry (MS) data files, deposited in the ProteomeXchange repository with id PRIDE: PXD003187.

Cardiac troponin T degradation in serum is catalysed by human thrombin

Cardiac troponin T (cTnT) has been shown to be present in fragmented forms in human serum after acute myocardial infarction (AMI). While calpain-1 and caspase-3 have been identified as intracellular proteases able to cleave the N-terminus of cTnT, it is still unclear which proteases are responsible for the extensive and progressive cTnT fragmentation observed in serum of AMI-patients. In this pilot study we have investigated the possibility that human thrombin may be involved in this process. Purified human cTnT was spiked in unprocessed and deproteinated serum in the presence or absence of either purified human thrombin or PPACK thrombin inhibitor. After immunoprecipitation, SDS-PAGE and Western blotting we observed an increase in cTnT fragmentation when purified thrombin was added to deproteinated serum. Consequently, the addition of thrombin inhibitor to unprocessed serum resulted in a decrease of cTnT fragmentation. Our results suggest that multiple enzymes are involved in cTnT degradation, and that thrombin plays an important role.

Large Variation in Measured Cardiac Troponin T Concentrations after Standard Addition in Serum or Plasma of Different Individuals

To the Editor: During acute myocardial infarction (AMI), 1 cardiac troponin T (cTnT) is released from the damaged myocardium. One would expect a strong correlation between concentrations of cTnT measured in the blood after AMI and the extent of myocardial damage. However, various studies have shown a rather moderate correlation between infarct size and cTnT concentrations (1, 2). Frequently considered explanations for this phenomenon are methodological or focused on physiological factors associated with differences in release and elimination of cTnT (2). In contrast, less attention has been paid to the possible presence of inherent factors in the blood, such as (auto)antibodies or proteases, that might modify cTnT or interfere with the assay. We hypothesized that these factors may be ubiquitously present in the general population and might affect the measured concentrations of cTnT in individuals. We measured cTnT, cTnI, creatine kinase MB isoenzyme (CK-MB), and myoglobin concentrations in sera from 24 healthy volunteers (age range 22–54 years) before and after the addition of serum from a patient suffering from AMI (age 59 years, cTnT concentration approximately 19.5 μg/L). All participants gave written informed consent and leftover material was used in accordance with the code of proper secondary use of human tissue …

Thrombin Activation via Serum Preparation is Not the Root Cause for Cardiac Troponin T Degradation

To the Editor: We read with interest the recent article by Katrukha et al. concerning thrombin-mediated degradation of human cardiac troponin T (cTnT) in serum (1). In their study, Katrukha and coworkers confirmed the finding of Streng et al. that human coagulation factor II (thrombin) is a strong mediator of cTnT degradation (2). They compared cTnT degradation in serum and heparin plasma collected simultaneously from the same patients with acute myocardial infarction (AMI), revealing substantial differences in cTnT fragment composition. They also performed in vitro experiments in which troponin T, thrombin, and hirudin were supplemented in various combinations. In addition, the cleavage site in cTnT was localized by use of mass spectrometry and probing of different proteolytic fragments with the use of various antibodies. Their main conclusion was that the primary 29-kDa cTnT fragment was mainly formed because of thrombin activation during serum preparation. We believe that the evidence for thrombin as one of the main proteases for cTnT degradation is convincing. However, we disagree with their final conclusion because there is published evidence suggesting …