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Biomarkers
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Possible mechanisms behind cardiac troponin elevations
Ola Hammarsten, Johannes Mair, Martin Möckel, Bertil Lindahl & Allan S.
Jaffe
To cite this article: Ola Hammarsten, Johannes Mair, Martin Möckel, Bertil Lindahl & Allan S. Jaffe (2018) Possible mechanisms behind cardiac troponin elevations, Biomarkers, 23:8, 725-734, DOI:
10.1080/1354750X.2018.1490969
To link to this article: https://doi.org/10.1080/1354750X.2018.1490969
© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Accepted author version posted online: 06 Jul 2018.
Published online: 23 Aug 2018.
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REVIEW
Possible mechanisms behind cardiac troponin elevations
Ola Hammarstena, Johannes Mairb, Martin M€ockelc, Bertil Lindahld and Allan S. Jaffee
aDepartment of Clinical Chemistry and Transfusion Medicine, University of Gothenburg, Gothenburg, Sweden;bDepartment of Internal Medicine III– Cardiology and Angiology, Heart Center, Medical University of Innsbruck, Innsbruck, Austria;cDivision of Emergency Medicine and Department of Cardiology, Charite – Universit€atsmedizin Berlin, Berlin, Germany;dDepartment of Medical Sciences, Uppsala University and Uppsala Clinical Research Center, Uppsala, Sweden;eDepartment of Cardiovascular Medicine, Mayo Clinic and Medical School, Rochester, MN, USA
ABSTRACT
Cardiac-specific troponins are elevated in blood following cardiac injury and are the preferred diagnos- tic biomarkers when acute myocardial infarction is suspected clinically. Cardiac troponin (cTn) eleva- tions are also observed in clinical conditions without obvious connection to cardiac injury. Irrespective of the underlying condition, cTn elevation is linked to a poor prognosis, even if the elevation is stable over time. Here, we explore mechanisms that may lead to cTn elevations, including necrosis, apop- tosis, necroptosis, cell wounds and decreased clearance. The aim is to broaden the perspective of how we interpret unexpected cTn elevations in patients. The cTn elevations may not be able to serve as direct proof of myocardial necrosis especially in the absence of a clear-cut reason for its release.
Abbreviations:
AMI: acute myocardial infarction; cTn: cardiac troponin; cTnI: cardiac troponin I; cTnT: cardiac troponin T; MLKL: mixed lineage kinase domain-like; TUNEL: terminal deoxynucleotidyl transferase nick end labeling
ARTICLE HISTORY Received 22 March 2018 Revised 10 June 2018 Accepted 11 June 2018
KEYWORDS
Cardiac troponin I; cardiac troponin T; myocardial injury; release; reversible;
necrosis; apoptosis;
clearance
Introduction
Cardiac troponin I (cTnI) and cardiac troponin T (cTnT)) are cardiac-specific proteins that are part of the troponin com- plex of thin filaments which form together with thick fila- ments the contractile apparatus, the sarcomere, of cardiomyocytes (Figure 1). Cardiac troponin (cTn) is released and is transiently increased in the blood following an acute myocardial infarction (AMI) and other types of acute myocar- dial injury. The cTns are the preferred biomarkers for the laboratory diagnosis of AMI (Roffi et al. 2016), due to their excellent cardiac specificities, the fact that healthy individuals have only low levels (Cardinaels et al. 2012), and the fact that there are excellent assays available for clinical use.
The cTn elevations are also found in patients with chronic cardiac comorbidities. Recently, increases have been observed in normal individuals in response to what might be considered physiological stress such as exercise, rapid atrial pacing and after the infusion of dobutamine. A large number of studies have shown in non-AMI populations that elevation of the cTn concentration, including some not above the 99th% upper reference limit, is a strong risk marker for death, the development of heart failure and ischaemic heart disease and other non-ischaemic cardiac diseases (Galvani et al. 1997, Aviles et al. 2002, Peacock et al. 2008, Omland
et al.2009, Kawahara et al.2011, Aldous et al.2015, Omland et al. 2015, Roos et al. 2017). At present, we only have evi- dence-based treatment for patients who have had AMI. For other conditions associated with cTn elevations, treatments are frequently advocated based on the assumed pathophysi- ology but data supporting their effectiveness are not avail- able (Roos et al.2017). Herein, we summarize what is known about the possible cellular mechanisms behind cTn release and clearance, which may broaden the perspective on how to interpret cTn values in clinical practice. In most cases, information is available for both cTnT and cTnI (cTn), but in some instances, information is only known for cTnT or cTnI and in those situations, that fact will be made clear.
cTn release by necrosis
The most obvious reason for cTn elevation in a patient, espe- cially if the cTn elevation is transient, is necrosis. In fact, compared with other cell types, cardiomyocytes are more prone to undergo necrosis triggered by situations such as the Ca2þparadox and/or the oxygen paradox (Piper2000).
Myofibrils, which constitute 50–60% of the cardiomyocyte volume, are repeating units of Ca2þ-activated, ATP-consum- ing sarcomeres (Figure 1). Therefore, if Ca2þ leaks into the cardiomyocyte cytoplasm, the sarcomeres contract and
CONTACTOla Hammarsten ola.hammarsten@clinchem.gu.se Newcastle University, Australia, Institution of Biochemistry, Department of Clinical Chemistry and Transfusion Medicine, Bruna straket 16, Sahlgrenska Academy at the University of Gothenburg, SE 413 45 Gothenburg, Sweden
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
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https://doi.org/10.1080/1354750X.2018.1490969
quickly consume all the ATP (Figure 2(A)), resulting in rapid necrosis and release of cTn along with other cellular contents (Piper et al.2003).
A visual representation of the rapid nature of cardiac necro- sis is the‘stone heart syndrome’ after the Ca2þ paradox. If iso- lated hearts are first perfused with a buffer without, and then a buffer with Ca2þ, the entire heart contracts tonically, turning the perfusion buffer red due to the release of myoglobin and the heart becomes a white hard contracting mass of necrotic cardiomyocytes called a‘stone heart’ (Piper2000).
Groundbreaking studies by Jennings showed that ischae- mic cardiomyocytes quickly develop intracellular oedema when ATP levels drop (Jennings et al. 1978). As the sarco- mere is anchored in the plasma membrane via the dys- trophin protein complex, swelling does not result in uniform enlargement of the cardiomyocyte; rather, membrane‘blebs’
(Schwartz et al. 1984) are formed between the dystrophin protein complex anchoring points (Jennings et al.1978, Sage and Jennings 1988) (Figure 3). In this situation, the cardio- myocyte plasma membrane becomes fragile but does not break immediately (Jennings et al. 1983), unless exposed to an acidic or hyposmotic environment (Jennings et al. 1983, Steenbergen et al.1985). However, when contractility occurs after reoxygenation (Vander Heide and Ganote 1987), or when anoxic cardiomyocytes are forced to move by sur- rounding non-ischaemic cardiomyocytes (Steenbergen et al.
1985), or if the ischaemic heart is acutely dilated by a bal- loon in the ventricular chamber (Vander Heide and Ganote 1987), these cells will succumb and result in contraction band necrosis.
Some of this damage can be mitigated by a reduction in contractile performance, so called stunning. Myocyte contrac- tion stops within a few heartbeats following total ischaemia in what is thought to be a mechanism that protects oedema- tous ischaemic cardiomyocytes from contraction-mediated
T I C
(A)
(B)
(C)
Figure 1. Cardiac troponin. (A) Electron microscope image of a cardiomyocyte showing the repeating sarcomeres in myofibrils. (B) The sarcomere contains thick filaments (grey lines), containing ATP consuming myosin interdigitated by thin filaments (black horizontal lines) that bind troponin (green ellipses), and is anchored in Z-bands (black vertical lines). (C) The cardiac troponin complex is composed of cTnT (T) that directly binds to thin filaments (grey bar), cTnI (I) and TnC (C), the Ca2þ-binding subunit.
Figure 2. Possible release mechanisms for cTn discussed in this review (see text for details). (A) Necrosis (B) Apoptosis (C) Necroptosis (D) Cell wound.
Figure 3. Cardiomyocyte necrosis. (A) Phase contrast microscopic images of isolated cardiomyocyte undergoing contraction band necrosis (reproduced from [Garcia-Dorado and Ruiz-Meana2000] with permission). (B) Electron microscope image of an ischaemic cardiomyocyte where intracellular oedema forces the plasma membrane to form‘blebs’ between dystrophin anchoring points in the Z-band of sarcomeres (reproduced from [Sage and Jennings 1988] with permission).
cell damage, termed the ‘smart heart hypothesis’ (St Louis et al.2000, Depre and Vatner2005).
If the resulting membrane damage allows enough Ca2þ to enter the cardiomyocyte cytoplasm, the sarcomeres contract tonically and consume what little ATP is left, limiting the ability of the cardiomyocytes to pump out excess Ca2þ. In the ATP-depleted and Ca2þ-loaded state, myofibrillar short- ening stays fixed in all the sarcomeres, as the cross-bridges between actin and myosin remain in an attached state (Piper et al. 2003), resulting in contraction band necrosis where collapsed sarcomeres and large membrane defects are seen in histological preparations (Figure 3; Jennings 2013).
Unfortunately, much of the literature concerning cardiomyo- cyte necrosis is substantially dated and probably need to be revisited with newer techniques. In addition, during the past 15 years, this simple ‘necrosis by breaking’ picture has been complicated by the realization that programmed necro- sis – necroptosis, described below – also operates in the ischaemically damaged heart (Kung et al. 2011). In essence, the possibility of programmed necrosis occurring at a later stage further complicates the interpretation of the cTn release kinetics following AMI and other myocardial injury.
Nonetheless, in this acute situation, it is thought that most cTn release is due to irreversible cellular injury.
cTn release mechanisms after membrane damage The cTn binds with finite affinity to thin filaments of the sarcomere (Shiraishi et al. 1992) (Figure 1(C)). Although release is immediate after membrane damage, the washout of cTn is slowed when compared to other cardiac injury bio- markers due to what has been called ‘the trapping effect’
(Starnberg et al.2014). This is especially true when there are large volumes of the myocardium that become necrotic, such as after a transmural AMI. These types of insults result in sustained elevations of cTn for days and sometimes weeks (Katus et al.1991, Laugaudin et al.2016). The delayed wash- out of cTn is in contrast to that of cytoplasmic cardiac injury biomarkers (Figure 4), such as lactate dehydrogenase (LDH), creatine kinase (CK) (Katus et al. 1989), CKMB (Katus et al.
1987) and myoglobin (Sylven and Bendz 1978), which have no affinity for cardiac tissue. Thus, if cardiac tissue is crushed in warm plasma, myoglobin and cTn are released immedi- ately but the washout of cTn takes more time, due to its binding to the insoluble sarcomere. Starting within a few
hours, the necrotic cardiac tissue induces local inflammation and accumulation of neutrophils and macrophages that digest damaged tissue, forming granulation tissue and then a stable scar (Pfeffer and Braunwald 1990). During this later process, cTn release is likely, largely due to degradation of myofibrils. Many of these other proteins, such as CK, are locally degraded and not detected by conventional clinical assays. Consequently, even once blood flow is re-established, they no longer can be appreciated (Clark et al.1978).
As in all binding reactions, the washout of cTn is volume dependent and, hence, is faster and more complete if the plasma volume or blood flow is capacious (Starnberg et al.
2014). Increases in cTn levels hours or days after the initial non-reperfused AMI can, in addition to re-infarction, be due to partial restoration of flow (Hermens et al.1990, Voss et al. 1995). Also, if cardiomyocyte necrosis is scattered, as observed after isoproterenol-induced type II infarctions in rats, the washout of cTn from necrotic myocytes could, in principle, be over within 24 hours (York et al. 2007, Zhang et al.2008, Clements et al.2010). The cTn elevations that are cleared within 24 hours thus still can be due to cardiomyo- cyte necrosis.
cTn release by apoptosis and other regulated cell death pathways
All cells, including cardiomyocytes, have built-in ATP-con- suming death programmes that are activated when appropri- ate signals are present. This process is essential for the removal of interdigitate cells in the formation of fingers during embryogenesis (Svandova et al. 2017). However, apoptosis and other death programmes are probably most important in the defence against intracellular parasites like viruses.
Mammalian cells have evolved an array of mechanisms to detect the presence of intracellular pathogens, with many of these pathways culminating in the activation of a cell death response. Some of these responses were found through studies of the cytomegalovirus (CMV) that harbours several genes essential for viral replication dedicated to block apop- tosis and other cell death programmes. Inhibition of cell death not only affords CMV an opportunity to complete the replication cycle, but also restricts the release of cellular con- tent and, hence, limits the inflammatory response required for the generation of the adaptive immune response (Brune and Andoniou2017).
In the unlocking of the multitude of signals that can acti- vate programmed cell death, including TNF receptor activa- tion (Guo et al. 2017), it has become evident that our cells are like primed rat traps, eager to go off at any sign of inva- sion, but – perhaps not intentionally – also in response to oxidative overload, ischaemia and other stressors (Brune and Andoniou2017).
In principle, using its original description by Kerr (Kerr et al.1972) (Figure 2(B)) and the latest definition of apoptosis by the Nomenclature Committee on Cell Death (NCCD) (Galluzzi et al.2012), apoptosis should not result in cTn ele- vations. In fact, it has been suggested that apoptosis cannot cTnT
LDHCK
Time (h)
Relave plasma level
100 200 300
100
50
Figure 4. Kinetics of cardiac troponin T (cTnT), creatine kinase (CK) and lactate dehydrogenase (LDH) from the first published patient with acute myocardial infarction where a cTnT assay was used by Katus [Katuset al.1989].
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be the culprit if the patient has cTn elevations (Takemura et al. 2013). This is because no intracellular content is expected to be released when the apoptotic cell is divided into membrane-enclosed apoptotic bodies with phosphati- dylserine and other‘eat-me’ signals exposed on the surface, resulting in the engulfment and lysosomal degradation of the apoptotic bodies by surrounding cells and sometimes by immune cells (Nagata et al.2010).
It is, however, possible that the myofibrils in cardiomyo- cytes interfere with the formation of tight membrane- enclosed apoptotic bodies, or that apoptotic bodies break and expel their content before they are cleared (Figure 2(B)).
It is also possible that other forms of programmed cell death operate, like necroptosis, which ultimately results in lysis of the cell after the formation of multimers of the protein mixed lineage kinase domain-like (MLKL) that bind to and make holes in cellular membranes (Kung et al.2011) (Figure 2(C)). Although never reported, cardiomyocytes undergoing necroptosis most likely also release cTn as the nuclear pro- tein HMGB1 is released in the process. Our experience is that cTn release from damaged primary cardiomyocytes always overlaps with the release of other intracellular proteins, like LDH and myoglobin (Starnberg et al.2014).
Unfortunately, many studies of apoptosis in the human heart following AMI have methodological problems (Takemura et al. 2013). In addition, most studies are per- formed on hearts from dead patients, possibly adding a selection bias and effects secondary to the cause of death (Abbate et al. 2003). Sometimes, studies have neglected the fact that only 30% of the cells in the normal heart are cardio- myocytes and even less in granulation tissue, because of accumulation of apoptosis-prone immune cells (Saraste et al.
1997, Wakabayashi et al.2015, Jose Corbalan et al. 2016). In addition, no attempt was made to relate the extent of appar- ent apoptosis to the cTn levels in these human studies.
Consequently, we do not know if, or to what extent, apop- tosis contributes to acute or stable cTn elevations in humans.
However, increased levels of caspase-3, thought to be a mediator of apoptosis, are found systemically in patients with AMI (Agosto et al.2011).
What is clear, however, is that the cells in human hearts seem to undergo some kind of apoptosis-like process during an extended period following AMI, also in areas not affected by ischaemia (Abbate et al. 2008), possibly involved in the thinning of the remote myocardium that is often observed after a large MI (Pfeffer and Braunwald 1990). Similar apop- tosis-like processes have been found in human biopsy stud- ies of other cardiac pathologies like heart failure (Narula et al.1999). In these studies, cardiac cells display several hall- marks of apoptosis, including terminal deoxynucleotidyl transferase nick end labeling (TUNEL)-positive nuclei and cas- pase activation co-localizing in cells expressing myocyte-spe- cific proteins that correlate with cardiac disease and outcome (Abbate et al. 2003, Biondi-Zoccai et al. 2004).
Some authors have faith in methods to detect apoptosis not involving electron microscopy (Saraste and Pulkki 2000).
However, according to other authors, TUNEL and other DNA- end labelling methods cannot unequivocally prove that
apoptosis has occurred. This controversy has led to some cases where the interpretation of the published results has been questioned because of discrepancies between the methods (Takemura et al.2013).
There is a large body of studies in mice where cardiomyo- cyte apoptosis or necroptosis can be induced by genetic manipulation, like limited induction of the apoptosis-induc- ing protease caspase-8, which has been shown to result in the development of heart failure (Wencker et al. 2003) and larger infarcts after coronary occlusion. If the apoptotic or necroptic programme is inhibited by genetic or pharmaco- logical means, the almost uniform finding is that the size of experimental infarcts is reduced (Yaoita et al. 1998, Holly et al. 1999, Hochhauser et al. 2003, Abbate et al. 2008) and mice are protected from the development of heart failure (Wencker et al.2003, Kitamura et al.2014, Wang et al.2014).
There are a few studies reporting apoptosis following ischae- mic challenges in larger animals (Goussev et al. 1998). One study has shown a correlation between sustained cTnI eleva- tions and the amount of TUNEL-positive cells in pigs after brief periods of ischaemia (Weil et al.2017).
At this stage, it is safe to conclude that some type of pro- grammed cell death operates in the heart and that necropto- sis could contribute to cTn elevations in patients; however, the current data are not sufficient to make strong statements about how programmed cell death influences cTn elevations more chronically in patients. However, it is fair to speculate that increases in myocardial stress due to volume expansion (Feng et al. 2001) and potentially subendocardial ischaemia due to supply–demand imbalance or transient increases in pulmonary pressures could result in activation of these path- ways with cTn release. Such speculation would fit with the release of cTn shown to occur in normal subjects who are rapidly atrially paced (Turer et al. 2011) and/or exposed to dobutamine (Siriwardena et al. 2012). However, other non- apoptotic mechanisms also could explain these findings.
cTn release by cell wounds
Apparently viable cardiomyocytes in Petri dishes or in animal models exchange macromolecules over their plasma mem- branes (Cooper and McNeil 2015, Demonbreun and McNally 2016). The evidence that macromolecular exchange occurs in living cardiomyocytes is based on the detection of albumin (McNeil and Khakee 1992, Clarke et al. 1995) and other extracellular macromolecules (Hoffstein et al. 1975) in the cytoplasm of apparently normal cardiomyocytes. This macro- molecular exchange over the plasma membrane is higher if the cardiomyocytes are stressed either by contraction, are stretched by external forces (Page et al. 1992, Swildens et al.
2010), are challenged by beta-adrenergic stimulation (Boutet et al.1976, Clarke et al.1995) or after limited ischaemia. The cellular uptake of albumin and other large molecules in these studies seems to occur through transient disruptions in the plasma membrane and is not due to endocytosis or albu- min entering T tubules (Clarke et al. 1995). Macromolecular exchange via holes in the plasma membrane without cardio- myocyte death is possible, in part, because the cytoplasm is
a macromolecular gel with restricted diffusion (Ellis 2001), and because dystrophin complexes stabilize the membrane by forming links between the contracting sarcomere and the extracellular matrix (Allen et al.2016) (Figure 3(B)).
However, the most important protection against cell death when the plasma membrane is injured is a Ca2þ- dependent repair system, thoroughly examined by McNeil (Terasaki et al. 1997) and discovered by Heilbrunn almost 90 years ago during his studies of echinoderm oocytes (Heilbrunn 1930). This is why cells do not expel the cyto- plasm like a balloon bursting if poked with a micropipette (Terasaki et al. 1997) or if the plasma membrane is injured by a localized laser burn (Demonbreun et al.2016). Cells are surprisingly resilient and repair membrane holes larger than 10lm2 within seconds, in a process called cell wound repair (Terasaki et al. 1997) (Figure 2(D)). If the same insults are induced in Ca2þ-free culture media, the membrane holes persist, eventually leading to cell death (McNeil and Kirchhausen 2005). The Ca2þ concentration required to elicit a cell wound response is 100 times higher than the Ca2þ concentration seen during muscle contraction in most experimental systems (Steinhardt et al. 1994, Terasaki et al.
1997). The result of the cell wound response is the formation of a Ca2þ-impermeable patch within a few seconds.
The molecular details of cell wound repair are still unclear but most data indicate that the Ca2þ-impermeable patch is formed by intracellular membrane vesicles fusing with the plasma membrane (Miyake and McNeil 1995, Terasaki et al.
1997, Andrews et al. 2015, Davenport et al. 2016), aided by MG53 (Cai et al. 2009) and dysferlin (Han and Campbell 2007), proteins able to bind to and promote the fusion of two membranes (Figure 2(D)) (McNeil2014).
Mice (Wenzel et al.2007) and humans (Han and Campbell 2007) with mutations in genes involved in cell wound repair develop dystrophies, that is contraction-induced accumula- tion of muscle injury that ultimately leads to muscle weak- ness. In addition, dystrophies with mutations in other muscle cell-stabilizing functions, such as Duchenne muscular dys- trophy, show up to 16-fold upregulation of dysferlin, MG53 and other cell wound repair genes (Waddell et al. 2011).
Mice with MG53 mutations show impaired cell wound repair in skeletal muscle (Cai et al.2009) and are more sensitive to cardiac ischaemia than normal mice (Cao et al. 2010).
Dystrophies caused by dysferlin mutations sometimes lead to cardiomyopathy and heart failure in humans (Kuru et al.
2004). Mice with dysferlin mutations display inefficient cell wound repair in cardiomyocytes and are susceptible to pres- sure overload-induced cardiomyopathy. In addition, dysferlin- mutated mice have stable cTnI elevations that are aggra- vated by exercise (Han et al. 2007). These findings indicate that cell wound repair is important in cardiomyocytes and, if impaired, can result in stable cTnI elevations, at least in mice.
Cell wounds induced by contraction likely increase under ischaemic conditions. However, if the plasma membrane injury is limited, the cardiomyocytes survive after reoxygena- tion, likely as a result of cell wound repair with accumulation of extracellular macromolecules in the cytoplasm as the only evidence of transient plasma membrane injury (Boutet et al.
1976). This can be shown by resident colloidal lanthanum in apparently healthy cardiomyocytes surrounding necrotic car- diomyocytes after experimental ischaemic injury and i.v.
injection of colloidal lanthanum complexes with a median diameter of 40 Å (Hoffstein et al. 1975, Burton et al. 1977), roughly half the size of albumin.
Under limited ischaemic conditions or limited beta-adren- ergic stimulation, elevated serum levels of myocardial injury biomarkers such as LDH, CK and cTn are observed without obvious cell death and sometimes with a reciprocal lowering of these markers in apparently living cardiomyocytes (Chiong et al.1974, Zhang et al.2008, Hickman et al.2010). Although these studies did not detect cardiomyocyte death, it remains possible that areas of cell death and in particular apoptosis could have been missed by their histological examinations.
These and similar studies of elevated cardiac injury biomarkers are therefore unable to prove that the leakage occurred from viable cardiomyocytes. However, Spieckermann group showed that 35% of all LDH can be expelled from resting cardiomyo- cytes under ischaemic challenge without finding cell death.
Taken at face value, this finding would indicate that extensive leakage of the cellular content can result from ischaemically stressed but living cardiomyocytes (Piper et al.1984, Schwartz et al.1984). However, how cytoplasmic proteins could escape in such an abundance and still allow the cardiomyocytes to survive is unclear and have, to the best of our knowledge, never been replicated. If the above is correct, then increases in cTn elevation should not be considered proof of cardiomyo- cyte cell death (Kawahara et al.2011).
In summary, it is safe to conclude that there are mecha- nisms that could allow cTn release from living cardiomyo- cytes. The extent to which cell wounds contribute to the cTn elevations we encounter in the clinic remains to be shown.
cTn release from skeletal muscle
The clinical cTn assays all use antibodies that bind epitopes specific to cardiac isoforms of TnI and TnT. The Roche high- sensitive cTnT assay does, however, react with something in skeletal muscle, not yet observed with the cTnI assays. This was first observed in patients with neuromuscular disorders and patients with myositis (Botta et al.2008, Jaffe et al.2011, Rittoo et al.2014, Valaperta et al.2016, Wens et al.2016).
A cross-reactivity with skeletal muscle extracts from con- trol subjects has also been found. When serum was supple- mented in vitro with extracts from normal human skeletal muscle such that myoglobin levels were within what can be observed after rhabdomyolysis, cTnT levels, but not cTnI lev- els, increased above the current cut-off for MI (Schmid et al.2018).
We (unpublished) and others (Valaperta et al. 2016) have observed a similar reactivity using the Roche high-sensitive cTnT assay in skeletal muscle extracts from healthy humans, pigs and rats, while the Abbott high-sensitive cTnI assay was negative in the same extracts. Therefore, the Roche high-sensi- tive cTnT assay could in theory become positive in those with chronic skeletal muscle disease or in response to rhabdo- myolysis. In these instances, as suggested by the Biomarker
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Group of the European Society of Cardiology (Thygesen et al.
2010), an alternative cTnI assay could be used to check whether the heart is involved, since most of these assays have the same ability to diagnose and exclude myocardial infarction and to define prognosis as the Roche high-sensitive cTnT assay (Shah et al. 2015, Pickering et al. 2016). Whether this strategy it is safe in these conditions has not been examined in a prospective study, but it is in use in several clinics.
cTn elevation due to decreased cTn clearance
Once cTnT and cTnI reach the circulation, they are initially cleared with a half-life of 0.5 hours in dogs and rats (Dunn et al. 2011), followed by what we have shown is a slower, apparently kidney-dependent clearance when cTnT drops to concentrations often observed in patients with sta- ble cTn elevations (Friden et al.2017) (Figure 6(B)).
It may be that this phenomenon is similar to myoglobin clearance, thoroughly studied by Sylven (Figure 5(A)). At high levels, clearance of radioactively labelled myoglobin injected in healthy controls or in patients with acute MI has a half-life of roughly 1 h, followed by a much slower phase at low myoglobin levels (Sylven 1978, Groth and Sylven 1981) (Figure 5(A)). As myoglobin is a small protein with a molecular weight of 17 kDa, it can pass through the glom- erular membrane and is cleared in part by kidney function, as found in patients with rhabdomyolysis and myoglobin- induced acute tubular renal failure.
Despite its ability to pass through the glomerular mem- brane, clearance of myoglobin at high levels observed after MI or rhabdomyolysis is not influenced by renal function (Wakabayashi et al. 1994, Lappalainen et al. 2002), whereas the low levels found in normal subjects are strongly related to renal function (Hallgren et al.1978). Myoglobin apparently undergoes predominant extra-renal clearance at high levels (Figure 6(A)) and its renal clearance (Figure 6(B)) becomes apparent only at lower levels.
Myoglobin shares this pharmacokinetic behaviour with low molecular weight heparin cleared by a fast saturable receptor-mediated endocytosis-based system (Figure 6(A)) and slow kidney-dependent clearance (Boneu et al. 1990) (Figure 6(B)). Early studies have shown that myoglobin accu- mulates in the liver and spleen (Amako et al.1963), followed by bilirubinaemia (Bywaters and Beall1998) in patients who die from massive rhabdomyolysis, indicating that receptor- mediated endocytosis in the reticuloendothelial system is responsible for the extra-renal clearance of myoglobin (Figure 6(A)). This dual clearance system is likely similar to how cTnT is cleared, at least in rats (Figure 5(B)).
For unknown reasons, most of the cTnT measured with clinical high-sensitive cTnT assays are degradation products in patients with stable cTnT elevations (Diris et al. 2003, Friden et al. 2017), and a few hours after the ischaemic event in patients with acute MI (Cardinaels et al. 2013) (Figure 6(C)).
The major degradation products are <20 kDa (Cardinaels et al.2013, Streng et al.2016, Friden et al.2017), expected to undergo free passage over the glomerular membrane.
Therefore, cTnT measured by the current high-sensitive cTnT
assay could be, to some extent, cleared by the kidneys. We have shown this in experiments where we block renal blood flow in rats. In this context, it is important to note that very little radioactive myoglobin reached the urine in Sylven’s sub- jects mentioned above, likely because tubular resorption pre- vented myoglobin from reaching the bladder (Sylven 1978).
Likewise, tubular resorption of cTn degradation products may explain the fact that, despite apparent renal clearance, cTn levels are low in urine (Ziebig et al.2003).
Assuming the above, it appears that at low cTnT levels, often found in patients with stable elevations, kidney clear- ance appears to dominate. Therefore, at steady state and everything else being the same, cTnT levels are roughly twice as high if kidney function is reduced by 50% (Bjurman et al. 2015). As many patients with kidney failure often have stable cTnT levels 10–100 times above the 99th% upper ref- erence limit (Bjurman et al. 2015), other mechanisms must also contribute to the cTnT elevations found in this patient group (Waldum and Os2013).
However, if the cTnT levels are adjusted for kidney func- tion, the clinical high-sensitive cTnT assay becomes slightly better at distinguishing between patients with and without AMI in the emergency room (Friden et al. 2017). However, cTnT levels adjusted for kidney function do not improve prognostication (Friden et al.2017).
At the higher cTnT levels often found after large AMIs, extra-renal clearance of cTnT dominates in rats and is not influenced by renal function (Friden et al. 2017), potentially explaining why clearance of cTn is similar among AMI patients with or without renal function (Ellis et al.2001). The nature of this extra-renal clearance of cTnT has not been
(A) (B) (C)
Figure 6. Possible clearance mechanisms for cTn discussed in this review (see text for details). (A) Endocytosis (B) Filtration (C) Degradation.
Figure 5. Clearance of myoglobin in humans and cTnT in rats. (A) Concentrations of125I-labeled myoglobin during 6 hours following i.v. injection in humans. Circles represent125I levels in serum and triangles represent125I lev- els in urine. Dotted lines are projections of the two kinetic profiles found (reproduced from [Sylven1978] with permission). Most of the 125I in urine did not precipitate in acid, indicating that125I in the urine was not linked to myoglobin. (B) Relative concentrations of cTnT in rats during 2 hours following i.v. injection of rat cardiac extracts. Dotted lines are projections of two kinetic profiles found (reproduced from [Fridenet al.2017] with permission).
studied but likely occurs via scavenger receptors, a loosely defined group of receptors that are able to bind to a pleth- ora of proteins and other molecules and direct them to intra- cellular degradation (Prabhudas et al. 2014) (Figure 6(A)).
Scavenger receptor clearance of LDH has been studied by Gruber in rats and rabbits (Smit et al.1987, Smit et al.1988).
Radioactive LDH is taken up by macrophages (Hayashi and Notkins 1994) in the liver and spleen after i.v. injection. If macrophages are specifically destroyed by the LDH virus, by silica nanoparticles or by other means, mice and monkeys develop stable LDH and CK elevations (Hayashi et al. 1988, Radi et al. 2011), likely due to decreased clearance. An indi- cation that this mechanism may be relevant with regard to stable elevations in humans is a recent finding, showing that stable elevations of CK and LDH correlate with genetic muta- tions in scavenger receptors (Kristjansson et al. 2016). It is therefore formally possible that stable cTnT elevations could be due to inefficient renal or extra-renal clearance and this is therefore an area of intense study.
Conclusions
Unfortunately, although current knowledge might broaden the perspective, the information in this review makes inter- pretation of cTn elevations no less complicated. First, because of the possibility that cell wounds could explain some of the release and given the data concerning altered renal clearance, cTn elevations may not be able to serve as direct proof of myocardial necrosis especially in the absence of a clear-cut reason for its release. However, early timely limited cTn increases may be the result of scattered cardio- myocyte necrosis and/or apoptosis, or theoretically even reversible cardiomyocyte injury. Prolonged cTn elevations (>24 h), however, may be due to slow washout from local myocardial injury or the delayed necroptosis following an ischaemic event in both granulation tissue and in the remote myocardium undergoing remodelling.
Disclosure statement
JM none to declare concerning the content of the manuscript. BL has received consulting fees from Roche Diagnostics, bioMerieux Clinical Diagnostics, Philips Healthcare, ThermoFischer and Fiomi Diagnostics;
and has received a research grant from bioMerieux Clinical Diagnostics and Fiomi Diagnostics. ASJ presently or in the past has consulted for most of the major diagnostic companies.
Funding
The study was funded by the Swedish Cancer Society (OH), the Swedish Heart and Lung Foundation (OH), Assar Gabrielsson Cancer Research Foundation (OH), and LUA/ALF funding at the Sahlgrenska University Hospital (OH).
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