TROPOnin FRAGMentation in Myocardial Injury Study

Overview

Troponin T (TnT) is a part of the troponin protein complex that principally exists in cardiac and skeletal muscle cells and is widely used as diagnostic biomarker for myocardial injury and, thus, myocardial infarction (MI). Elevated TnT levels can, however, be observed in the presence of other clinical conditions such as heart failure, sepsis and kidney failure and the contemporary high-sensitivity TnT test may yield false positive results when performing diagnostics for suspected MI in these patients. Recent data have demonstrated that in the presence of MI, TnT gradually undergoes fragmentation into smaller fragments. It has been suggested that in the presence of e.g. chronic kidney disease or physical exercise the released TnT is predominantly in the form of smaller fragments. However, the clinical significance of TnT fragmentation is unknown and, thus, we sought to investigate the prevalence of fragmentation of TnT in different patient cohorts.

Modern cardiac troponin T (cTnT) tests are highly sensitive in diagnosing AMI and myocardial damage. Atrial fibrillation and many non-cardiac conditions and even strenuous exercise are associated with elevated cTnT levels which are problematic to clinicians and may lead to redundant use of diagnostic coronary angiography or "overdiagnosis" of ACS in the emergency room workup. The cardiac troponin (cTn) complex is part of the thin filaments of myocardium. Small part (approximately 5% of total content) of troponins are cytosolic. These smaller cytosolic fragments may more easily traverse across cell membranes that have become leaky for some reason but not irreversibly damaged. Only cytoplasmic forms of troponin are contained in the subcellular blebs which lyse upon early cTn release into the circulation. Bleb formation may explain how troponin can appear in blood in the absence of cell necrosis. Still many aspects of their intramyocardial degradation, their tissue release, and their degradation within and elimination from the human circulation are still incompletely understood. In the early hours of AMI, troponin is found in its full-size complex, but the complexed cTnT degrades in a time-dependent pattern after the first hours. Small molecular weight troponin fragments originating from the cytoplasm may traverse across leaky cell membranes not completely damaged in various conditions without irreversible myocardial cell necrosis. Commercial cTnT test detects all these fragments and may thus lead to false diagnosis of AMI. At present, there is limited information based on small studies using complicated gel filtration chromatography and Western blotting showing cTnT fragmentation in later phases of AMI and in renal failure. These time-consuming analytical methods are, however, not suitable for clinical purposes in the emergency room. ANALYTICAL METHODS In this study we test a novel sensitive time-resolved immunofluorometric assay, which has been developed at the biotechnology unit of the Department Biochemistry, University of Turku, which enables us to measure cTnT from blood samples in its intact or only slightly fragmented form. Thus, we can exclude the highly fragmented short forms of cTnT from the analysis. This selectivity can be achieved with specific capture and tracer antibodies that bind to carefully chosen epitopes located closer to the opposite ends of the cTnT molecule. In the assay one of the antibodies is attached to a surface and acts as an antigen capturing antibody. The capture-antigen pair can be detected with tracer antibody, thus forming capture-antigen-tracer complex. In our case this tracer antibody has been labeled with europium chelates which provide sensitive detection by time-resolved fluorometry. The aim of Tropo-Fragment study is to use the described preliminary assay format in the clinical studies to evaluate: * TnT fragmentation and its time course in STEMI and NSTEMI * TnT fragmentation in renal failure, sepsis, strenuous exercise, stroke, atrial fibrillation, takotsubo, myocarditis and healthy subjects