Analysis of Troponin Fragments: The Start of a New Era—Perhaps?

Cardiac troponin (cTn) has been the marker advocated for the evaluation of patients with cardiovascular disease since 2000, when its use was mandated to evaluate patients with ischemic heart disease (1). Over the years, its use has increased and new novel, more high sensitivity assays have been developed (2). These assays are configured to optimize the detection of cTn—that is, maximize the amount of troponin captured so that the signal is as robust as possible for use in evaluating patients. This may lead some to think that the assays are entirely specific for one particular protein, i.e., cTn T (33.5 Kd) or I (23.5 Kd). However, as with most proteins, there are a multiplicity of fragments elaborated acutely or subsequently developed during processing either in the target organ in which they reside or once they are released into the circulation (3). This has been known for years, and there have been suggestions that if one could understand the fragments, one might be able to facilitate distinctions between a variety of diseases where cTn is released (3). These innovative approaches, however, have been difficult to implement because of the need for increases in the sensitivity of detection of the fragments, which are present in far lower amounts than the overall quantity of cTn (3). Studies in this area have now led to the development of novel assays that utilize antibodies to specific areas of cTn to allow larger and smaller fragments to be distinguished (4). It is hypothesized that bigger fragments are more likely to be originally released after myocardial injury and that smaller fragments only occur subsequently as the proteins are processed in the blood (4). If this is true, and there are data to suggest that it is the case (5–7), one might have an earlier marker of troponin release by looking for a specific peptide form. The report in this issue of the journal by a group from Turku in Finland describes the development of a more sensitive assay for what they call long troponin T (4). The antibodies used in the assays are directed against epitopes in such a way that the carboxyterminal end of the protein is captured along with part of amino terminus as well (8). This is in contradistinction to the commonly used clinical assay for cTnT where the epitopes for detection are in the central area of the molecule and are close together (4). The authors suggest that the ability to detect these longer forms might be an earlier way to diagnose disease (4, 8). The group tested an earlier version of their assay with more limited sensitivity in patients with myocardial infarction (MI) and compared them to patients with chronic renal dysfunction who have a variety of small cTnT fragments thought due to degradation of both the amino and carboxy terminal ends of the protein (5–8). They were able to use the ratio of the so-called long form to the smaller products as a way of distinguishing patients with MI from those with chronic renal failure (8). These findings fit with the concept expressed above that over time the percentage of longer forms in the circulation diminishes and the smaller forms increase. The newly developed assay now has taken an important additional step by utilizing upconversion luminescence to increase the sensitivity of detection (4). This technique uses nanoparticles that convert low-energy signals into higher-energy signals in the visible range (9), enabling detection of protein forms that are found in much lower concentrations in the circulation. However, it remains unclear how much additional information this new assay provided when applied to the samples previously studied (4), perhaps because the prior cohort was selected to include patients with higher values for cTnT. A further increase in sensitivity may be required to provide sufficient sensitivity in the context of cTnT levels that are closer to the normal range, as is the case in many patients. From the point of view of first principles, the ability to improve the sensitivity of detection to allow measurement of these various forms should facilitate diagnostic insights in regard to not only the timing but perhaps also the mechanisms of cTn release. However, there are a variety of other issues that need to be taken into account. These include: Prior data (which was mostly with cTnI) suggests that some processing occurs in the myocardium (2). If that is true for cTnT as well, then it could mean that, in some patients, short forms could be detected due to degradation in the myocardium before release into plasma, mimicking the pattern anticipated by late degradation in plasma from long troponin. This issue requires some additional information about exactly what occurs in the myocardium vis-à-vis what occurs in the blood, and the relative importance of both. This issue could have clinical implications. Acute ST elevation myocardial infarction (STEMI) is usually associated with total occlusion of a coronary artery (10) so that there is a paucity of blood flow to the ischemic territory, meaning that protein is not washed out to the same extent and may stay within the myocardium for a much longer period of time, If so, protein degradation might be more likely to occur there. On the other hand, the majority on non-ST segment elevation myocardial infarction (NSTEMI) patients have open arteries and measurement of different cTn forms may have more benefit in this setting. Further work will be required to clarify this issue. It is attractive to think that, because of the biologic activity shown when one overexpresses troponin fragments in vivo (3), these fragments could be used to define differences in disease mechanisms (2). However, we do not know how much overlap there is in terms of which fragments are elaborated in which disease processes. It has recently been shown that the increases in cTnT that occur with extreme exercise tend to be smaller forms (11). This raises a question as to whether the elaboration of cTn fragments may be related to the mechanism of myocardial injury rather than just degradation of longer forms of troponin in plasma. The mechanism of release in extreme exercise appears to be a result of increases in myocardial filling pressures associated with exercise. These increases in the left heart cause increases in pulmonary pressure and acute pressure overload of the right ventricle (RV) with RV dilation (12). It is possible/likely, therefore, that the release of troponin is via a mechanism of increased preload as described by Canty et al. (13). This is a calpain-mediated mechanism whereby cTn is first proteolyzed and then released, with subsequent cell death due to apoptosis. It may be that the cTn fragments elaborated via an apoptotic pathway are different from those elaborated in other ways and provide predominantly smaller fragments. This could complicate the clinical use of novel assays as it is known that apoptosis is a mechanism of cell death in many cardiovascular situations, including MI (14). These issues need to be elucidated before we start using these probes diagnostically. It is also clear that thrombin can proteolyze cTnT at the amino terminus (15). Ischemic heart disease is known be associated with increases in thrombin activity, and this may provide an additional confounding factor in vivo. Finally, each cardiovascular disease has its own unique time course. Some cardiac diseases occur acutely, whereas others may present more chronically. Therefore, there may be time for degradation to occur with ongoing processes. Even with acute ischemic heart disease, there can be a stuttering process with opening and closing of an infarct-related artery leading to intermittent perfusion and reperfusion. This will make the interpretation of cTnT fragmentation more complex. Nonetheless, it is important to acknowledge that cTn fragmentation has been investigated to a limited extent because of the lack of sensitivity in the assays that were available, and the assay described in this issue of the journal represents an important advance in this regard. It is very likely that similar research on the fragments elaborated with cTnI could provide additional synergistic information to that provided by cTnT. Thus, advances in technology may now allow us to begin to again consider how cTn fragment analysis could improve our ability to time and characterize clinical events. Equally importantly, such advances may allow us to distinguish the mechanisms of cTn release so that distinctions can be made between direct myocardial injury, supply–demand abnormality, and acute coronary occlusion (14). Time will tell whether this will herald the start of a new era or a false dawn, and we should look forward to hearing more about these important issues in the future. The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Upon manuscript submission, all authors completed the author disclosure form. None declared. A.S. Jaffe presently or in the past has consulted for smost of the major diagnostic companies who make high-sensitivity cardiac troponin assays, including Abbott, Siemens, Rochester, Radiometer, ET Healthcare, Sphingotec, SpinChip, LuminaRX, and Moderna. A.S. Jaffe receives royalties from UpToDate.