Frontiers in Laboratory Medicine xxx (2017) xxx–xxx

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Frontiers in Laboratory Medicine

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Release of cardiac troponin from healthy and damaged myocardium

Alan H.B. Wu
Department of Laboratory Medicine, University of California, San Francisco, California, United States

a r t i c l e i n f o a b s t r a c t

Article history: Cardiac troponins T and I are proteins released into serum after cardiac injury, and are the standard
Received 25 July 2017 biomarkers for patients presenting to the emergency department with a suspicion of acute myocardial
Received in revised form 29 August 2017 infarction (AMI). Cardiac troponin that appears in blood within a few hours is due to release from the
Accepted 25 September 2017
cytosolic pool. A sustained irreversible release over the ensuing days is due to the degradation of the
Available online xxxx
myofibrils, although recent data have challenged this concept. The analytical sensitivity for troponin
assays have significantly improved since the initial release of commercial troponin assays over 20years
Keywords:
ago. As a result, the specificity of troponin for AMI has steadily declined, with abnormal concentrations
Troponin
Demand ischemia
seen in many non-cardiac diseases such as renal failure, sepsis, pulmonary embolism, and cardiac injury
Blebs after chemotherapy such as with trastuzumab and doxorubicin. There are many theories as to how tro-
Integrins ponin is released into blood from patients with reversible myocardial ischemia and from patients with
Catecholamines cardiac damage that is not related to ischemia. These theories include release of free subunit release
Stress through bleb formation, transient imbalance of oxygen supply and demand such as what occurs with
Chemotherapy acute vasospasm of coronary vessels, pulmonary embolism with right heart damage, apoptosis, acute car-
Heart failure diac stress leading to release of catecholamines and integrins, myocardial stretching, inflammation, and
Apoptosis
release of degraded troponin peptides. The mechanisms for these etiologies are reviewed.
Ó 2017 Chinese Research Hospital Association. Production and hosting by Elsevier B.V. on behalf of KeAi.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Biomarker release after myocardial injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Evidence for reversible myocardial ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Reversible injury after pulmonary embolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
The role of apoptosis in reversible cardiac injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Release of troponin from autoantibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Stress-induced release of cardiac troponin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Myocardial injury following prolonged exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Exercise-induced release of skeletal muscle troponin as a model for cardiac injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Mechanism of cardiac damage after chemotherapy with trastuzumab and doxorubicin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Release of troponin through myocardial stretching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Inflammation as a mechanism of cardiac troponin release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Release of troponin fragments into the circulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Disclosures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

E-mail address: alan.wu@ucsf.edu

https://doi.org/10.1016/j.flm.2017.09.003
2542-3649/Ó 2017 Chinese Research Hospital Association. Production and hosting by Elsevier B.V. on behalf of KeAi.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Wu A.H.B. Release of cardiac troponin from healthy and damaged myocardium Frontiers in Laboratory Medicine (2017),
https://doi.org/10.1016/j.flm.2017.09.003
2 A.H.B. Wu / Frontiers in Laboratory Medicine xxx (2017) xxx–xxx

Introduction
Troponin is a complex of three regulatory proteins that is bound
to tropomyosin and actin, the thin filament of striated muscle. Tro-
ponin C binds to calcium, troponin I inhibits actin-activated myo-
sin Mg+2ATPase, and troponin T is the tropomyosin binding
protein.1 Cardiac troponin C has complete amino acid homology
with skeletal muscle troponin C and is therefore not used as a car-
diac biomarker. Both troponin T and I exist in low concentrations
within the cytoplasm.
The Task Force for Universal Definition of Myocardial Infarction
has defined several types of myocardial infarction based on the
underlying etiology.2 Under this designation, Type I AMI is charac-
terized by the rupture of a coronary artery plaque with the resul-
tant formation of a thrombus. Type II AMI is characterized by
vasospasm or endothelial dysfunction, fixed atherosclerosis with
a supply versus demand imbalance, or a supply versus demand
imbalance alone. International cardiology and laboratory medicine
guidelines have recommended the use of cardiac troponin (cTnT or
cTnI) as the standard biomarker for diagnosis of acute myocardial
infarction.2,3 Currently, these markers cannot be used to classify
the type of AMI present.
and metabolies appear in blood with the first few minutes after
reversible ischemia. It has been long thought that proteins are
not released into the circulation until there is permanent damage
to the myocytes. Given the compensatory mechanisms to preserve
vital myocardial functions, irreversible injury does not occur for an
hour or more. Once released from the cells, macromolecules ini-
tially inter the lymphatic system and gradual passage to the circu-
lation, unless there is restoration of coronary artery blood flow
through reperfusion, in which case there may be more direct
access.
The smaller the protein the earlier that they appear in blood.
Thus the order of appearance of cytoplasmic proteins used as car-
diac biomarkers are: myoglobin, troponin, creatine kinase, and lac-
tate dehydrogenase. The proteins that are entirely cytosolic in
origin, exhibit a mono-phasic release pattern. Proteins such as
myosin light chains are found exclusively in the thin or thick fila-
ment of muscle have a delayed release due to the gradual break-
down of the sarcomere. Troponin has both cytosolic and
structural distributions, therefore following injury, the appearance
in blood exhibits a biphasic release pattern. Since the clearance of
free troponin once in blood is only 2h,3 the prolonged appearance
of troponin of 57days.

Biomarker release after myocardial injury Evidence for reversible myocardial ischemia

Prolonged myocardial ischemia leads to an oxygen deficit and
irreversible myocardial necrosis. A deficit in the delivery of blood
to myocardial tissues, either due to increased demand or the pres-
ence of a ruptured coronary artery plaque causes myocardial ische-
mia which initiates a cascade of molecular and cellular events.4
Early oxygen deficits lead to a transition from aerobic metabolism
as a source of ATP production, to the less efficient anaerobic path-
way. In order to reduce ATP utilization, the myocyte shuts down
the ATP-dependent sodiumpotassium membrane pump. The
reduced or lack of coronary artery blood flow lead to the accumu-
lation of metabolites such as lactate. These low molecular weight
substances are able to pass through the interstitial space directly
into the vascular space. The reduced or lack of coronary artery
blood flow also leads to the accumulation of metabolites such as
lactate and phosphate. By bypassing the interstitium, these ions
The release of cardiac troponin into the circulation does not
always follow the classic biphasic pattern as described above. In
cases where the initial appearance of troponin is followed by a
rapid decline back to baseline concentrations with 24h suggest
the absence of permanent myofibril damage. When early genera-
tions of troponin assays were used, it could be argued that the ana-
lytical sensitivity was insufficient to track the steady decline of
troponin following minor myocardial injury (Fig. 1A). With the
release of highly sensitive troponin assays, it can be confirmed that
the rapid clearance is not the result of assay insensitivity. There-
fore, for patients who exhibit a rapid appearance and decline in
cardiac troponin, e.g., over 24h, is evidence of reversible myocar-
dial ischemia (i.e., no evidence of structural damage as it would
be indicated with the release of intact troponin complexes, Fig. 1B).
Permanent irreversible damage would show the biphasic release

A. Injury indeterminant B. Reversible injury C. Irreversible injury

50 50 o 50
0 o
o
cutoff
40 concentraon 40 40
o o
Hs-cTnT, ng/L
cTnT, ng/L

Hs-cTnT, ng/L

30 30 30
??
o
20 20 20 o
o cutoff
o cutoff
10 o concentraon 10 o concentraon
o 10

0 0 0
0 12 h 0 12 0 12 24 36 48 h
Time Time Time

Fig. 1. Transient increase in cardiac troponin in a patient who presents with chest pain. A. Injury indeterminate. Use of a conventional troponin assay with a cutoff of 40ng/L.
A peak positive result occurs on the second sample. The subsequent sample 4h later is below the cutoff, i.e., the actual result cannot be accurately measured. B. Reversible
injury. The third sample is near the cutoff concentration. C. Irreversible injury. Use of a high-sensitive troponin assay with a cutoff of 13ng/L on the same patient. The third
and subsequent samples are above the cutoff concentration.

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 A.H.B. Wu / Frontiers in Laboratory Medicine xxx (2017) xxx–xxx 3

A. B. C.

40 40 40

o
30 30 30
o PCI:
90%
20 20 occlusion 20
Hs-cTnT, ng/L

o RCV
o
10 10 10
o o
o o
o
0 0 0
Aer, 2 h
24 h aer

24 h aer

24 h aer
before

before
Aer, 2 h

before
Aer, 2 h
57 y female 54 y male male aer PCI
Fig. 2. Transient release and clearance of troponin in the context of demand ischemia. A, B. Troponin concentrations before, immediately after and one day after a 50-km
bicycle ride in two middle-aged individuals. C. The same individual from Panel B undergoes another bicycle ride a few months after percutaneous coronary angioplasty with
stent placement. Used with permission from Aw et al. 2015;446:68.

A. individual underwent another bicycle ride a few months later. Tro-

B. Bleb
formation
Bleb release
and rupture
Fig. 3. A. Release of troponin through irreversible myocyte injury. B. Release of
troponin through bleb formation following reversible myocyte injury. Used with
permission from Hickman et al. 2010;411(45):318323.
(Fig. 1C). This concept was illustrated in a report by Aw et al.,
where two middle-aged individuals (one male, one female) under-
went a 50km bicycle ride (Fig. 2A and B).5 Using a high-sensitive
troponin assay, both individuals exhibited a significant increase
after the exercise that returned to near baseline concentrations
with 24h. One of these individuals underwent an elective angio-
gram which revealed the presence of an occlusion in the right coro-
nary artery. After stent placement in the affected artery, this
ponin concentrations did not increase this time (Fig. 2C), leading
the authors to suggest that the release in troponin in these two
individuals was reversible and due to demand ischemia. It may
be possible that both individuals suffered a silent but minor Type
II AMI immediately after the exercise, but in the absence of symp-
toms to fulfil the criteria for AMI diagnosis, this conclusion could
not be rendered.
Studies conducted in animal models and cardiac myocytes have
shown that during the initial stages of myocardial ischemia, blebs
are formed. These subcellular structures contain cytoplasmic pro-
teins that are released into blood upon rupture of these blebs.6 This
concept has been well established in liver models of reversible
anoxic injury. Fig. 3A shows the bi-phasic release of troponin fol-
lowing irreversible injury. The initial phase is due to release from
the cytoplasmic pool. This is followed by the slow degradation of
the sarcomere and release of complexed troponin. Fig. 3B shows
a mono-phasic release of troponin through bleb formation. Only
cytoplasmic forms of troponin are contained within these blebs
which lyse upon release into the circulation. Bleb formation may
explain how troponin can appear in blood in the absence of cell
necrosis.
The concept that the late appearing troponin is due to slow
degradation of myofibrils has been recently challenged by Starn-
berg et al.,7 at least for cTnT. They performed in vitro studies of
cTnT from human cardiac tissue and found that over 80% of the
cTnT, including the sacromeric complexes, can be extracted from
human cardiac tissue using large volumes of human sera. Since
there was no effect of varying the serum to cardiac tissue ratio
for the extraction of non-structural cytoplasmic proteins (CK-MB
and myoglobin), they concluded that the binding of the troponin
complex to tropomyosin is not as tight as previously thought. This
may explain why coronary artery reperfusion is associated with an
accelerated washout of troponin from damaged myocytes com-
pared to non-reperfusion, which has been known for many years.8
One might conclude from these observations that the absence of
the prolonged increase in troponin after the onset of injury is not
evidence of reversible injury but rather of increased extraction

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4 A.H.B. Wu / Frontiers in Laboratory Medicine xxx (2017) xxx–xxx
from the contractile pool with good blood flow through the area
where the heart was damaged. These data do not explain absence
of the prolonged release of the other 20% non-extractable troponin
if the injury is entirely irreversible.
Reversible injury after pulmonary embolus
Many clinical studies have shown that cardiac troponin is
released after acute pulmonary embolism (PE). The exact etiology
for troponin rise in blood is unknown but could be due to direct
myocardial injury to the right ventricles from to the increased
pressure that occurs with blockage of the pulmonary artery or
one of its major branches.9 This causes hypoxia, hypoperfusion,
and reduced coronary perfusion.10 The release kinetics of troponin
following may be different from myocardial damage from the left
ventricles. Muller-Bardorff et al. showed that troponin can be
cleared within 24h to suggest that the amount of troponin released
is milder, and that it may originate from the cytosolic pool of the
myocyte.11 This is due in part to the lower troponin tissue content
in the right ventricle compared to the left for cTnT and cTnI
(roughly 2030% lower).12 Troponin is not released in patients with
deep vein thrombosis, often the precipitating condition that pro-
duces a PE. This is because cardiac troponin is not present in
non-cardiac vascular tissue.
The role of apoptosis in reversible cardiac injury
Apoptosis is defined as programmed cell death and is part of the
normal cycle of cells and tissues. It is a highly regulated process
that is initiated through cell stress or signals received from other
cells. Once initiated, enzymes such as caspases become active to
indiscriminately degrade the proteins that make up each cell.
There are several analytical means by which apoptotic cells can
be detected including electron microscopy, flow cytometry, and
the TUNEL assay. The TUNEL assay makes use of terminal deoxynu-
cleotidyl transferase and fluorescence to identify the 3hydroxyl
termini of DNA damaged as part of the apoptosis process.13
The importance of apoptosis as a mechanism to release tro-
ponin into blood has been demonstrated by several investigators.
In the study by Weil et al. an ischemia model was induced in a
swine model by inserting and inflating a balloon catheter within
the second diagonal branch of the left anterior descending (LAD)
artery for 10min.14 Upon deflation, histopathology was conducted
one hour after reperfusion and examined for evidence of cell necro-
sis and apoptosis using the TUNEL assay. The authors found that
troponin was released after restoration of blood flow, with no evi-
dence of myocardial damage. However, they found a sixfold higher
numbers of TUNEL positive cardiomyocytes in the LAD region,
compared to a remote non-ischemic region. This report concluded
that apoptosis is a mechanism to release troponin during myocar-
dial ischemia.
Release of troponin from autoantibodies
The development of autoantibodies directed against cardiac tro-
ponin may be another means by which there can be cardiac injury.
The hypothesis is that in some patients, the release of troponin
after myocardial injury illicits an autoimmune response by the
host. The presence of these antibodies can cause myocarditis.15
In a clinical study, patients suffering myocardial infarction have
less myocardial recovery if they demonstrated high titers to cTnI-
antibodies, compared to those with no antibodies.16 Autoantibod-
ies only form in a minority of patients with AMI. To provide further
evidence of this hypothesis, when patients are treated with
immunoadsorption to remove these specific auto-antibodies, the
Please cite this article in press as: Wu A.H.B. Release of cardiac troponin from healthy and damaged myocardium Frontiers in Laboratory Medicine (2017),
https://doi.org/10.1016/j.flm.2017.09.003
ejection fraction improved in several patients with dilated car-
diomyopathy.17 In an animal model, injection of anti-cTnI antibod-
ies from mouse T cells into healthy mice caused myocardial
dysfunction.18 These animals developed severe inflammation with
increased concentrations of chemokines.
Stress-induced release of cardiac troponin
Stress is a mechanism for disease progression in the patho-
physiology of many diseases. This adaptive response may explain
how troponin is released in patients with Tako-Tsubo cardiomy-
opathy.19 These are cases of cardiac injury that occur when there
is significant emotional stress and not associated with myocardial
ischemia. The release of catecholamines as a defense mechanism
against stress has been implicated in initiating cardiac myocardial
damage.20 There can be other medical conditions that release
adrenaline and noradrenalin and are associated with cardiac
injury. Drugs such as cocaine block the reuptake of catecholami-
nes at preganglionic sympathetic nerve endings and is toxic to
the heart.21 Cocaine also causes vasospasm, so if the recipient
has any underlying atherosclerotic disease, it can cause a type II
AMI. Cases of cocaine-induced rhabdomyolysis have also been
described and thought to be related to arterial vessel
constriction.22
The induction of myocardial stress through treadmill exercise
or pharmacologically is often used as a diagnostic procedure to
determine if cardiovascular disease is present. The objective is to
determine if an individuals cardiac blood flow is sufficient to meet
the oxygen demands during exertion. The test is stopped if the
patient becomes intolerant such as the development of chest pain,
or if there is evidence for myocardial ischemia on the electrocar-
diogram, echocardiogram, or nuclear imaging. The cardiac troponin
concentrations in serum when measured using assays with con-
ventional sensitivity assays are within normal limits signifying
the absence of irreversible cardiac injury. With the use of high sen-
sitivity assays, Sabatine et al. demonstrated an increase in cTnI in
the absence of ischemia, as determined by nuclear perfusion imag-
ing.23 The authors concluded that stress can produced a transient
myocardial ischemia without any permanent damage. These
results were not duplicated with a high sensitivity cTnT assay
was used after patients underwent thallium stress testing.24 But
in a study of stress induced by atrial pacing, cTnT was found to
be increased in the coronary sinus and peripheral blood of subjects
without angiographic correlates of myocardial ischemia.25
Myocardial injury following prolonged exercise
For myocardial infarction, coronary artery occlusion is the pre-
cipitating event for stress and would therefore not be a good model
for the influence of stress on biomarker release. Instead, long dis-
tance running on hard pavement from healthy individuals who
are trained for marathon events is a model for stress-induced
myocardial injury in subjects who are healthy and fit on a cardio-
vascular basis. Biomarkers such as creatine kinase-MB isoenzyme,
myoglobin, and cardiac troponin release have been conducted on
individuals completing endurance events.26,27 The release of CK-
MB and myoglobin do not offer any insights as to myocardial injury
because these proteins are found in abundance within the skeletal
muscle and are released in all participants. While the appearance
of cardiac troponin is from the myocardium, it is important to rec-
ognize if the release is due to structural damage to the heart or
reversible cardiac injury due to stress.
Advanced nuclear and radiographic imaging can be used to
address this question. In the study by Mousavi et al. blood was
taken in 14 individuals after running a marathon.28 All had
 A.H.B. Wu / Frontiers in Laboratory Medicine xxx (2017) xxx–xxx
increases in CK, myoglobin, and cTnT after the race. Echocardiogra-
phy and cardiac magnetic resonance imaging was conducted
within 3days of completing the event. With procedures demon-
strated a transient right ventricular systolic change, and persistent
left and right ventricular diastolic abnormality. However, the
gadolinium-enhanced imaging showed no evidence of myocardial
edema suggesting no true myocardial necrosis. These authors con-
cluded that stress to the heart is sufficient to release troponin
without the production of permanent cardiac injury.
In a study of non-elite participants of a marathon, Neilan et al.
examined echocardiographic measures after a race.29 They con-
cluded that the lack of left ventricular wall motion abnormality
and a different release pattern suggested a non-ischemic etiology
for release of troponin into the blood of these runners. Also, these
participants had longer finishing times (>4h), sufficient to establish
the onset of irreversible injury, unlike those of elite runners (23h
finishing times).
Exercise-induced release of skeletal muscle troponin as a model
for cardiac injury
In addition to the cardiac isoforms, there are two separate
skeletal troponin I isoforms (sTnI), one each for slow-twitch and
fast-twitch fibers.30 These have different amino acid sequences
enabling the creation of specific immunoassays for these proteins.
Controlled studies of sTnI release following after exercise have
been conducted with the results compared to release of cTnI after
cardiac damage. Inducing cardiac injury in healthy humans on an
experimental basis is not ethical due to the potential for long-
term health consequences. In the study by Sorichter et al., sTnI
were measured in subjects undergoing level or downhill running,
high-force eccentric contractions, or high-force isokinetic contrac-
tions.31 Results were compared to other skeletal muscle biomark-
ers, creatine kinase and myoglobin (cytosolic proteins), and
myosin light chains (structural protein). These investigators
demonstrated that sTnI was increased early after injury, with only
a short delay compared to myoglobin, a slightly smaller molecular
weight protein (molecular weight 16.7kDa). Moreover, there was
only a minor secondary rise in sTnI. In contrast, there was a delay
in the release of the other markers, CK, due to its larger size (85
kDa), and myosin light chain, due to the gradual breakdown of
the myofibrils (there is no cytosolic pool). They concluded that
additional release modes must be involved to explain the early
release of sTnI, given that the cytosolic component is a small frac-
tion (34%) of the total sTnI content. They suggested an increase in
intracellular calcium which could active calpains, non-lysosomal
proteolytic enzymes that can breakdown muscle fibers. Whether
or not this occurs in the human myocardium after injury is
unknown.
Mechanism of cardiac damage after chemotherapy with
trastuzumab and doxorubicin
It has been known for many years that use of certain
chemotherapy agents such as trastuzumab and doxorubicin are
associated with development of heart failure and release of cardiac
troponin.32 There is a pattern of consistent elevations in the blood
of patients treated with chemotherapeutics33 in contrast to the
acute rise and fall of biomarkers seen after AMI. The initial use of
these agents is associated with reversible myocardial injury with
no changes in left ventricular dysfunction as measured by the
echocardiogram. Over time, heart failure occurs which may lead
to a reduction in the amount of drug that patient can tolerate,
and thereby decrease the effectiveness of the patients treatment.
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5
There have been few theories on the mechanism of troponin
release following chemotherapy. One involves the human epider-
mal growth factor receptor (HER), which are transmembrane onco-
gene receptors that help regulate cell survival. For women with
breast cancer, overexpression of HER-2, however, is linked to poor
prognosis. Trastuzumab is a monoclonal antibody that binds to
HER-2, blocking its signaling pathways, and leads to improved
breast cancer survival.34 HER-2 is also important in cardiomyocyte
survival. Through the aid of neuregulin-1 (cell adhesion molecule),
HER-2 dimerizes with HER-4 leading to activation of signaling
pathways that stabilizes myofibril structures, inhibits apoptosis,
and decreasing the production of reactive oxygen species. Binding
trastuzumab to HER-2 blocks dimerization to HER-4, thereby shift-
ing the ratio of antiapoptotic proteins towards proapoptotic pro-
teins. When doxorubicin is added to the anti-cancer regimen, it
increases oxidative stress and indirectly inhibits neuregulin-1
activity. As documented in the sections above, increased stress
and apoptosis releases troponin in the absence of myocardial
ischemia.
Release of troponin through myocardial stretching
Patients with chronic heart failure undergo myocardial remod-
eling that involves stretching of the myocytes. This stimulates the
release of atrial natriuretic peptide (ANP) and B-type natriuretic
peptide (BNP) from the atrium and ventricles of the heart, respec-
tively, as a compensatory mechanism for reduced cardiac output
that is observed with myocardial enlargement. In addition to these
hormones, cardiac troponin is released with cardiac damage, and
has been shown to be risk predictor of future adverse cardiac
events.35
The mechanism for release of troponin following myocardial
stretching is poorly understood. One theory is the mediation by
the release of integrins. The integrins are transmembrane glyco-
protein receptors that provides a link between the intracellular
with the extracellular space. Together with other signaling mole-
cules, the integrins stimulate myocardial stretching. Hessel et al.
performed a study whereby cultured myocytes were treated by
the pentapeptide, Gly-Arg-Gly-Asp-Ser, known to induce integrin
secretion.36 They found that significantly a higher level of intact
cTnI release relative to control (phosphate-buffered saline and
the reverse peptapetide, Ser-Asp-Gly-Arg-Gly, known to not stim-
ulate integrin release). There was no increase in lactate dehydroge-
nase, a marker of irreversibility, in the treated group versus
controls. Nuclear staining also showed no difference in the amount
of necrotic cells present, when compared to controls. In an animal
model of isolated rat hearts, Feng et al. also showed that myocar-
dial stretching can release troponin in the absence of irreversible
myocardial ischemia.37 In this latter study, mechanical straining
resulted in in situ degradation of the troponin protein. This may
facilitate its release into blood (see next section).
Inflammation as a mechanism of cardiac troponin release
The presence of inflammation has been suggested as an under-
lying etiology of troponin release from the heart. This stems from
observations in patients with sepsis who have increased concen-
trations in troponin in the absence of myocardial infarction. Sys-
temic inflammation is a consistent etiologic factor in sepsis. For
example, in one study of sepsis, 73% individuals without an AMI
or possible AMI had increased concentrations of cTnT.38 Similar
results have been seen with cTnI.39 It is unclear how inflammation
induces troponin release. In an editorial, Wu postulated that tro-
ponin may be caused by reversible myocardial ischemia based on
the observation that troponin can be cleared from the circulation
6 A.H.B. Wu / Frontiers in Laboratory Medicine xxx (2017) xxx–xxx
T Troponin T
C C
I I
ternary binary
complex complex
Fig. 4. Antibody recognition for various troponin forms. T=cTnT, molecular weight 35 kda, I=cTnI, molecular weight 24kDa, C=cTnC, molecular weight 18kDa. N=amino-
terminus, C=carboxy-terminus. The antibody binding sites are in the mid region of the molecules. There can be multiple antibodies used in the assay. The troponin subunits
are not drawn to represent the actual 3-dimensional structure.
of septic patients within 24h.40 Reversibility was also suggested by
the absence of lactic acid, a hallmark for irreversible damage.
The role of inflammation as a means for troponin release has
been challenged by others. In a study of cardiovascular magnetic
resonance study following endurance exercise, inflammation was
not suggested as the cause for troponin release.41 In this study,
gadolinium imaging revealed no differences in ventricular vol-
umes, stroke volume and ejection fractions and did not fulfill crite-
rial for myocardial inflammation. Given this data, it may be
possible that an imbalance between oxygen supply and demand
causes troponin release in sepsis, not inflammation. This may be
the consequence of fever, respiratory failure, microcirculation dys-
function, and hypotension.10 Other conditions associated with
acute inflammation such as hypersensitivity reaction, chemical
injury or irradiation exposures are not associated with an increase
in cardiac troponin unless there is direct or indirect cardiac
involvement.
Release of troponin fragments into the circulation
An attractive theory that might explain the release of troponin
following reversible ischemia stems from the observation that
ischemia can produce degradation of free troponin T and I subunits
in situ. In the study by McDonough et al., isolated rat hearts were
subjected to variable amounts of ischemia followed by reperfu-
sion.42 Rat cTnI became progressively degraded from both the
amino- and carboxy-terminus ends of the protein. The size of cTnI
went from 24kDa to 15 and 16kDa fragments. cTnT underwent a
similar degradation from 35kDa to 33, 27 and 25kDa fragments.
Smaller molecular weight fragments originating from the cyto-
plasm may be better able to traverse across cell membranes that
have become leaky but not irreparably damaged.
Studies conducted on human blood after AMI have demon-
strated the existence of these fragments.43,44 Katrukha et al.
showed that cTnT is degraded from a 35kDa protein to a 29kDa
fragment in serum but not in plasma.45 They showed that the con-
version was catalyzed by thrombin, which is activated during clot
formation. Mingels et al. showed that even smaller cTnT fragments
(<18kDa) are seen in blood of patients with end-stage renal dis-
ease.46 These observations occurring in vivo may and may not have
relevance regarding the mechanism of troponin release into the
blood, but rather, how they are cleared. Commercial troponin
assays that are directed to the central part of the molecule (for
either cTnT or cTnI) will detect these fragments, as illustrated in
Fig. 4. Under this approach, most of the central portion of the tro-
ponin fragments will be detected.47 In patients with end stage
renal disease, retention of these immune-reactive fragments lead
to increased concentrations.48
Please cite this article in press as: Wu A.H.B. Release of cardiac troponin from healthy and damaged myocardium Frontiers in Laboratory Medicine (2017),
https://doi.org/10.1016/j.flm.2017.09.003
N C 35 kDa
I I Troponin C
C 18 kDa
Troponin I
I 24 kDa
free immunoreactive N C
Troponin I
troponin I fragments I degradation
<24 k
Summary
Although troponin has been used as routine clinical biomarker
for diagnosis and risk stratification for acute coronary syndromes,
how they are released is the ripe subject for future research inves-
tigations. As explored in this work, it is very likely that there are
different mechanisms for release depending on the underlying
physiologic and pathophysiologic conditions. Some have argued
how troponin appears in blood is strictly an academic exercise
with no medical significance.49,50 This may be true because the
current commercial assays are designed to measure as many forms
of troponin that exists in blood (complexed, free, and immunoreac-
tive fragments) as possible. If knowledge of the pathophysiology of
troponin is known more precisely, a next generation troponin
assay could be constructed that are directed towards specific tro-
ponin epitopes according to their release pattern, in order to pro-
vide differential information relative to the total troponin assays
(T or I). For example, if free forms are released in the absence of
complexes, this could indicate either an early release following
myocardial necrosis, or reversible release in the absence of struc-
tural damage. Constructing assays that recognize only a portion
of the total amount of troponin released will not be simple given
that the same epitopes for the free subforms are also present in
many of the complexed and degradation forms (see Fig. 4). Mass
spectrometry may prove to be useful given that the exact peptide
structures can be targeted. This technique, however, is not amend-
able for rapid analysis.
Disclosures
The author have no conflicts of interest.
Funding
This work was partially funded by a grant from the American
Heart Association.
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