Developing the next generation of cardiac markers: Disease-induced modifications of troponin I

doi:10.1016/j.pcad.2004.07.001

Progress in Cardiovascular Diseases

Volume 47, Issue 3, November–December 2004, Pages 207-216

https://doi.org/10.1016/j.pcad.2004.07.001 Get rights and content

Abstract

Troponin I (TnI) and Troponin T (TnT) have evolved into arguably the two most important diagnostic markers for acute myocardial injury. Part of their diagnostic utility lies in the uniquely important roles that both TnI and TnT play in the calcium-dependent regulation of cardiac muscle contraction. Both proteins undergo extensive physiologic regulation, principally through phosphorylation, as well as specific disease-induced pathologic modifications, including phosphorylation, oxidation, and proteolysis. Many, if not all, of these protein modifications in some way modulate contractility, and when detected in serum may therefore provide important information about both the disease state and functional status of the heart. However, the complexity of the TnI (and TnT) forms in the serum is large, which leads to difficulty in detecting all of the Tn subunits in serum, and hence interpreting the biologic significance of each modified product. But, as diagnostic tools and modalities improve, our ability to monitor and detect specific disease-induced modified forms of proteins will inevitably lead to better and more specific diagnoses and therapies.

Section snippets

TnI the molecule and analyte

Contraction of the cardiac myocyte is achieved through the calcium-dependent interaction of the thick filament (primarily composed of myosin) and the thin filament (primarily composed of filamentous actin and tropomyosin (Tm)) (see reviews in Kim et al2 and Thomas et al3). The trimeric troponin complex, composed of TnI, troponin T (TnT, also a diagnostic marker for acute myocardial infarction [AMI]), and troponin C (TnC), regulates this calcium-dependent interaction, and thereby the myocyte and

Harnessing diagnostic information from the physical form of serum TnI

Of primary importance for the design and use of any biomarker is determining what form(s) of TnI or TnT is actually detected in serum. Whereas currently available TnI diagnostics each detect TnI, it is unclear exactly what form of TnI is being found.1, 10, 11 It is important to recognize that the tight interactions between TnI, TnT, and TnC affect the physical form of TnI (e.g., as a monomer, or part of a dimeric or trimeric complex) found in serum following its release from the myocyte. The

TnI modification products in human myocardium

Despite discrepancies between large and small animal models, TnI degradation can occur in patient populations. Myocardial biopsies obtained from patents undergoing coronary bypass surgery demonstrated TnI degradation, both before and after application of the coronary cross clamp.46, 52 Interestingly, the number of TnI degradation products differed between patients, some with a maximum of 8 fragments observed (above 10 kDa) and some with only 1 fragment, corresponding to TnI residues 1–192,

Conclusion: how can TnI modification be made into a useful diagnostic?

There are three major stumbling blocks to the design and implementation of diagnostics based on the detection of modified TnI products. First, and perhaps most important, is the lack of comprehensive tools to observe and quantify all the possible products. As previously discussed, phosphorylation and proteolysis present specific challenges to antibody-based detection, such that it is highly unlikely to find even a single pair of antibodies that can recognize all forms of TnI (for use in

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Citation Excerpt :
These biomarkers are useful in diagnosing acute myocardial infarction due to prolonged release in the blood [2]. Because cardiac troponin is a cardiac-specific biomarker, it helps isolate cardiac injury from skeletal muscle or other organ damage [7]. The use of biosensors for early detection of CVD has increased in recent years.
Troponin T, which is a biomarker of cardiovascular disease, is vital for acute myocardial infarction (AMI). AMI is the most well-known cause of death in the world. In the present work, we designed a novel immunosensor using disposable, cheap, and very conductive graphite paper electrodes (GP). The proposed immunosensor was modified with hydrochloric acid (HCl) for immobilization of anti-TnT antibody. After carboxyl group formation on the surface, the GP electrodes were incubated with EDC/NHS pair as a cross-linker. Electrochemical impedance spectroscopy (EIS), single frequency impedance (SFI), cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques were used for electrochemical characterization of the label-free immunosensor. Furthermore, the fabrication process of the proposed immunosensor was examined by using scanning electron microscopy (SEM). The proposed electrochemical immunosensor had a wide detection range (0.5 fg - 1000 fg/mL), and low limit of detection (LOD) and low limit of quantification (LOQ) at 1.28 fg/mL and 4.29 fg/mL, respectively. The designed immunosensor exhibited very good sensitivity, reproducibility, repeatability, reusability and long storage life. Additionally, the immunosensor was successfully applied to detection of TnT in human serum.
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We propose a design strategy for affinity-based biosensors using nanowires for sensing and measuring biomarker concentration in biological samples. Such sensors have been shown to have superior properties compared to conventional biosensors in terms of LOD (limit of detection), response time, cost, and size. However, there are several parameters affecting the performance of such devices that must be determined. In order to solve the design problem, we have developed a comprehensive model based on stochastic transport equations that makes it possible to optimize the sensing behavior.
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For precision medicine to be successful, the identification and measurement of proteins that are reflective of a personal health status is key both for the development and use of circulating biomarkers and to define mechanistic pathways involved in drug responses. Prior to clinical translation, it is essential to accurately and reproducibly detect protein concentrations, their isoforms or polymorphism expression, and the many potential cotranslational and posttranslational modifications on 100–10,000 individuals. Over the last 20 years, the proteomic field has matured with respect to its approaches, methods, and instrumentation to allow for increased number of measurements per analysis. Through the use of the current, state-of-art, high precision, and accuracy mass spectrometry technology, quantitative proteomics is becoming a quantitative method able to address the needs of precision medicine.
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Acute myocardial infarction or myocardial infarction (MI) is a major health problem, due to diminished flow of blood to the heart, leads to higher rates of mortality and morbidity. Data from World Health Organization (WHO) accounted 30% of global death annually and expected more than 23 million die annually by 2030. This fatal effects trigger the need of appropriate biomarkers for early diagnosis, thus countermeasure can be taken. At the moment, the most specific markers for cardiac injury are cardiac troponin I (cTnI) and cardiac troponin T (cTnT) which have been considered as ‘gold standard’. Due to higher specificity, determination of the level of cardiac troponins became a predominant indicator for MI. Several ways of diagnostics have been formulated, which include enzyme-linked immunosorbent assay, chemiluminescent, fluoro-immunoassays, electrical detections, surface plasmon resonance, and colorimetric protein assay. This review represents and elucidates the strategies, methods and detection levels involved in these diagnostics on cardiac superior biomarkers. The advancement, sensitivity, and limitations of each method are also discussed. In addition, it concludes with a discussion on the point-of care (POC) assay for a fast, accurate and ability of handling small sample measurement of cardiac biomarker.
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PAK3 has also been linked to cTnT phosphorylation, although no sites could be identified and the observed increased Ca2 +-sensitivity was attributed to cTnI phosphorylation [60]. Recently, scientists have debated the physiological relevance of many of these identified phosphorylation sites of cTnT and other myofilament proteins [72,73]. The above reviewed studies all used purified or recombinant troponin, which was in vitro phosphorylated by several selected kinases and subsequently reconstituted.
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.
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^☆: Supported through NHLBI Contract #N01-HV-28180, and by a grant from the Donald W. Reynolds Foundation. JLM was supported by a Postdoctoral Fellowship from the Canadian Institutes of Health Research.

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Developing the next generation of cardiac markers: Disease-induced modifications of troponin I☆

Abstract

Section snippets

TnI the molecule and analyte

Harnessing diagnostic information from the physical form of serum TnI

TnI modification products in human myocardium

Conclusion: how can TnI modification be made into a useful diagnostic?

J Biol Chem

FEBS Lett

J Mol Biol

Biophys J

J Biol Chem

J Biol Chem

J Biol Chem

Am J Pathol

J Mol Cell Cardiol

J Mol Cell Cardiol

J Am Coll Cardiol

J Biol Chem

The role of cardiac troponin in the recent redefinition of acute myocardial infarction

Clin Lab Sci

Regulation of contraction in striated muscle

Physiol Rev

Altered interactions among thin filament proteins modulate cardiac function

J Mol Cell Cardiol

Covalent and noncovalent modification of thin filament actionThe essential role of troponin in cardiac muscle regulation

Circ Res

The special structure and function of troponin I in regulation of cardiac contraction and relaxation

Adv Exp Med Biol

Binding of cardiac troponin I 147–163 induces a structural opening in human cardiac troponin C

Biochemistry

Standardization of cardiac troponin I assaysRound robin of ten candidate reference materials

Clin Chem

Characterization of cardiac troponin subunit release into serum after acute myocardial infarction and comparison of assays for troponin T and I

Clin Chem

Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry

Clin Chem

The phosphorylation of troponin I from cardiac muscle

Biochem J

Cardiac troponin I, isolated from bovine heart, contains two adjacent phosphoserines. A first example of phosphoserine determination by derivatization to S-ethylcysteine

Eur J Biochem

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

Biochemistry

Effects of protein kinase A phosphorylation on signaling between cardiac troponin I and the N-terminal domain of cardiac troponin C

Biochemistry

Conformation of the N-terminal segment of a monocysteine mutant of troponin I from cardiac muscle

Biochemistry

Phosphorylation-induced distance change in a cardiac muscle troponin I mutant

Biochemistry

Structural mapping of single cysteine mutants of cardiac troponin I

Proteins