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Circulating HtrA2 after primary percutaneous coronary intervention in ST-

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Article in International Journal of Cardiology · December 2017

DOI: 10.1016/j.ijcard.2017.08.022

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 International Journal of Cardiology 243 (2017) 485–491

Contents lists available at ScienceDirect

International Journal of Cardiology

journal homepage: www.elsevier.com/locate/ijcard

Circulating HtrA2 as a novel biomarker for mitochondrial induced

cardiomyocyte apoptosis and ischemia-reperfusion injury in
ST-segment elevation myocardial infarction
M. Hortmann a,⁎,1, S. Robinson a,b,1, M. Mohr a,1, D. Haenel a,1, M. Mauler a,c,1, D. Stallmann a,1, J. Reinoehl a,1,
D. Duerschmied a,1, K. Peter b,d,1, C. Bode a,1, I. Ahrens a,e,1
a
Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Germany
b
Department of Medicine, Monash University, Melbourne, Australia
c
Faculty of Biology, University of Freiburg, Germany
d
Baker IDI Heart and Diabetes Institute, Melbourne, Australia
e
Augustinerinnen Hospital, Academic Teaching Hospital University of Cologne, Cologne, Germany

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

Article history: Background: Ischemia-reperfusion (I/R) injury in ST-segment elevation myocardial infarction (STEMI) signifi-
Received 25 November 2016 cantly contributes to overall myocardial damage. As a consequence of I/R injury in the heart, the high-
Received in revised form 15 May 2017 temperature requirement protein A2 (HtrA2) is released from the mitochondrial intermembrane space of
Accepted 20 May 2017 cardiomyocytes to the cytoplasm, whereupon it induces apoptosis.
Available online 24 May 2017
Methods: Serum was obtained from STEMI (n = 37), non–ST-segment elevation myocardial infarction (NSTEMI)
(n = 20), stable coronary artery disease (CAD) (n = 17) and patients with CAD excluded (n = 9). In STEMI, I/R
Keywords:
ST-segment elevation myocardial infarction
injury was assessed via measurement of ST-segment resolution.
Apoptosis Results: HtrA2 was significantly increased in STEMI compared to NSTEMI, stable CAD and patients with CAD ex-
Ischemia-reperfusion injury cluded (981.3 (IQR: 543.5–1526.2) pg/mL vs. 494.5 (IQR: 413.8–607) pg/mL vs. 291 (IQR: 239–458.5) pg/mL vs.
Mitochondria 692.2 (IQR: 276.6–964.7) pg/mL; p ≤ 0.0001). STEMI patients with HtrA2 level of at least the median or above had
HtrA2 a higher peak creatine kinase (CK) (p = 0.0002) and cardiac troponin T levels (cTnT) (p = 0.0019). Significantly
more STEMI patients with HtrA2 levels of at least the median or above were identified as I/R injury (87% vs.
42%; p b 0.0001). Serum HtrA2 demonstrated a superior area under a curve in a receiver operating character-
istic analysis for predicting I/R injury compared to CK, creatine kinase myocardial-band (CK-MB) and cTnT
levels (AUC = 0.7105 vs. AUC = 0.5632 vs. AUC = 0.5660 vs. AUC = 0.5407 respectively).
Conclusion: HtrA2 shows promise as a novel potential biomarker for mitochondrial-induced cardiomyocyte
apoptosis and may help to identify I/R injury after STEMI.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction In the coronary vasculature I/R injury causes microvascular dysfunc-

The extent of myocardial damage in patients with acute ST-segment
elevation myocardial infarction (STEMI) depends on both the ische-
mic time to reperfusion as well as an injury induced by ischemia-
reperfusion (I/R) resulting in a complex cascade of cellular myocardial
and coronary microvascular reactions [1,2]. Coronary microvascular
damage and myocardial I/R injury are connected, but the causal rela-
tionship is unclear.
⁎ Corresponding author at: Department of Cardiology and Angiology I, Heart Centre
Freiburg University, Faculty of Medicine, University of Freiburg, Hugstetter Street 55,
79106 Freiburg, Germany.
E-mail address: marcus.hortmann@universitaets-herzzentrum.de (M. Hortmann).
1
This author takes responsibility for all aspects of the reliability and freedom from bias
of the data presented and their discussed interpretation.
tion, increased permeability, microembolization, impaired vasomotion
and stasis leading to capillary destruction [1]. Despite restoration of epi-
cardial blood flow through percutaneous coronary intervention (PCI)
impaired myocardial blood flow is often clinically observed. The no-
reflow phenomenon is the most severe form of microvascular I/R injury
[3]. Despite the progress in PCI, one third of STEMI patients still fail
to show complete reperfusion due to I/R injury and no-reflow [4,5].
These patients exhibit poorer outcomes [6,7]. Failure of adequate ST
segment resolution usually reflects no-reflow [8].
Cardiomyocyte necrosis is a result of uncoordinated pathophysio-
logical mechanisms during ischemia, which is accelerated during I/R
injury [9].
Cellular calcium overload, digestion of the cytoskeleton and sarco-
lemma by calpains and reactive oxygen species (ROS) all contribute to

http://dx.doi.org/10.1016/j.ijcard.2017.05.088
0167-5273/© 2017 Elsevier B.V. All rights reserved.
486 M. Hortmann et al. / International Journal of Cardiology 243 (2017) 485–491
necrotic cell death [1]. The loss of cardiomyocytes is not only a conse-
quence of necrosis but also of regulated cell death (apoptosis). Opening
of the mitochondrial permeability transition pore (MPTP) is pivotal
for the initiation of both forms [1]. Ischemia causes necrosis, whereas
apoptosis is thought to be the main mechanism of myocardial cell
death in I/R injury. However, this has only been demonstrated in a
murine model of myocardial infarction and I/R injury [10]. Overall, the
quantitative contribution of apoptosis and necrosis to myocardial cell
death is not clear but mitochondria are known to play a decisive role
in both processes [1].
Mitochondria function as central regulators of apoptosis [11].
Excessive reactive oxygen species (ROS), Bcl-2 proteins and dysregu-
lated calcium promote mitochondrial membrane permeabilisation
during reperfusion and cause the release of apoptotic factors into the
cytosol [12]. As a consequence of I/R injury in the heart, the high-
temperature requirement protein A2 (HtrA2) is released from the
mitochondria intermembrane space of cardiomyocytes to the cytosol
together with cytochrome c, whereupon it induces protease activity-
dependent apoptosis mediated via caspases [13,14]. In aging rats the
expression and leakage of HtrA2 is increased and enhances I/R injury
[15]. Cytosolic HtrA2 has also demonstrated functional importance
in rat hearts, whereby inhibition of HtrA2 using UCF-101 resulted in
amelioration of heart dysfunction following I/R injury in vivo [16]. In
a recent publication cardiac specific overexpression of HtrA2 in a
mouse model induced myocardial apoptosis and cardiac dysfunction
[17]. Cytochrome c is currently a promising biomarker for I/R injury
[18,19]. To our knowledge, it is currently not known whether HtrA2
is also released into the circulation. Hence, circulating HtrA2 might
also show potential as a novel biomarker for mitochondrial-induced
I/R injury and consequent cellular apoptosis. If validated, HtrA2 might
have a number of utilities, including being able to measure the efficacy
of I/R targeted therapeutics and also allowing the identification of pa-
tients with I/R injury after STEMI, giving a better idea of prognosis,
thereby directing further treatment. Establishing a valid marker for
I/R injury is particularly interesting because currently validated bio-
markers in myocardial infarction, such as troponins and CK-MB, are
markers of myocardial cell lysis, but they are not able to specifically as-
sess myocardial damage induced by mitochondrial-associated I/R injury
and apoptosis.
The aim of this study was to determine if HtrA2 is detectable and
elevated in the serum of patients with STEMI after PCI, compared to pa-
tients with non–ST-segment elevation myocardial infarction (NSTEMI),
stable coronary artery disease (CAD) and a control group who had CAD
excluded by coronary angiogram. STEMI is known to be associated with
severe ischemia and when PCI is instituted, there is abrupt reperfusion,
theoretically resulting in greater I/R injury compared to the other
groups studied in this report. We also assessed if any correlation exists
between an increase in HtrA2 and other important clinical variables.
We further hypothesized that HtrA2 is able to predict reperfusion injury
assessed via measurement of ST-segment resolution or lack thereof on
electrocardiogram [5,8,20].
2. Methods
2.1. Patient population
In this single-center prospective study, peripheral venous blood was obtained consec-
utively from 37 STEMI patients, 20 NSTEMI patients, 17 patients with stable CAD and
9 patients with CAD excluded who presented to the emergency department with
cardiac-suspected chest pain between September 2013 and August 2015.
Patients with STEMI as evidenced by ST-elevation N0.1 mV in at least two contiguous
leads were included in this study.
Major exclusion criteria included previous severe heart failure (i.e. known LVEF b30%
prior to qualifying infarct) and cardiogenic shock.
The final study population consisted of 83 patients.
The protocol of this study conforms to the ethical guidelines of the 1975 Declaration
of Helsinki and was henceforth approved by the institutional ethical committee of
Universitätsklinikum Freiburg (Ethics number 287/12). Written informed consent was
obtained from all patients.
2.2. Patient characteristics
All patient clinical characteristics were obtained retrospectively from the hospital's
electronic database. Pharmacologic treatment before and after PCI was conducted ac-
cording to the European Society of Cardiology (ESC) Guidelines [21,22]. Maximum levels
of all laboratory values, including serum creatinine, creatine kinase (CK), creatine kinase
myocardial-band (CK-MB), cardiac troponin-T (cTnT) and C-reactive protein (CRP) within
24 h of the ischemic event were recorded and used for statistical analysis. The left ventric-
ular ejection fraction (LVEF %) for each patient was measured soon after angiography
using 2-dimensional echocardiography (Simpson method).
2.3. Primary percutaneous coronary intervention (pPCI)
pPCI was performed with standard catheters. All STEMI and NSTEMI patients received
aspirin (minimum of 250 mg) and an ADP receptor blocker (prasugrel 60 mg, ticagrelor
180 mg or clopidogrel 600 mg). Unfractionated heparin (5000 U) was administered
prior to angiography. Eptifibatide (GPIIb/IIIa-blocker) was given at the discretion of the
interventional cardiologist.
2.4. HtrA2 in patient serum
Peripheral venous blood was obtained 4.75 (IQR: 3–7) h after PCI. Serum was obtained,
left to clot at room temperature for 30 min, before being centrifuged for 10 min at
2000 rpm. Serum samples were aliquoted and stored at −20 °C. The circulating level of
HtrA2 was measured by a commercially available human ELISA immunoassay for HtrA2/
Omi (Human HtrA2/Omi Quantikine® ELISA Kit, R&D Systems). The ELISA was performed
on samples in duplicates according to the manufacturer's instructions. Some samples ran-
domly underwent a second ELISA in duplicate to investigate for inter-assay variability. All
samples and standards were analyzed on a microplate reader set at a wavelength correc-
tion of 540 nm. HtrA2 concentrations were interpolated by comparison of the optical den-
sities to the standard curve. The lower detection limit of the assay was 31.25 pg/mL.
2.5. Ischemia-reperfusion injury
Analysis for I/R injury was performed by analyzing ECGs within the STEMI group be-
fore and 2.25 (IQR: 1.5–3.5) h after PCI. A decrease in ST-elevation of a minimum of 70%
was set as the cut-off to regard a patient as being adequately reperfused [5,8,20,23].
Patients within the STEMI group could then be subdivided into adequately reperfused or
reperfusion-injury groups according to this ECG criterion. 8 of the STEMI patients had to
be excluded from analysis, due to lack of required ECGs in the hospital database.
2.6. Statistical analysis
The primary objective of this study was the analysis of HtrA2 levels between STEMI
patients, NSTEMI patients, patients with stable CAD, and patients with CAD excluded. A
further aim was to correlate HtrA2 levels with the likelihood of adequate reperfusion
after PCI in the STEMI group. On this basis, a total of 83 patients were enrolled in this
study for analysis. Continuous variables are presented as mean ± standard deviation
(SD) if found to follow a Gaussian distribution according to the D'Agosstino-Pearson
omnibus normality test, or as median ± lower and upper quartiles if found to follow a
non-Gaussian distribution. Continuous patient data were compared using a one-way
ANOVA if found to follow a Gaussian distribution with Bonferroni's correction for multiple
comparisons, otherwise data underwent a Kruskal-Wallis test with Dunn's correction.
Categorical patient characteristics are presented as percentages, and differences between
patient groups were compared using a Chi-square analysis.
HtrA2 levels between all groups were analyzed using a Kruskal-Wallis test with
Dunn's correction for multiple comparisons and are presented as dot plots showing the
median and interquartile range (IQR).
The HtrA2 median concentration was calculated within the STEMI group and patients
within this group underwent further subdivision according to whether they presented
with HtrA2 levels above or equal to the calculated median (n = 19) or below the median
(n = 18). Patient characteristics were analyzed between these two STEMI groups, with
continuous data being analyzed via a Student's t-test if found to follow a Gaussian distri-
bution, otherwise data underwent a Mann-Whitney test.
Patients within the STEMI group further underwent a correlation analysis to analyze
for significant correlations between HtrA2 levels and peak CK levels and other clinical cor-
relates. Correlations were analyzed by a Pearson correlation test for parametric data or by
a Spearman test for non-parametric data.
HtrA2 levels between STEMI patients deemed to have suffered ischemia-reperfusion
injury compared to those who reached adequate reperfusion were compared using a
Mann-Whitney test and are presented as dot plots showing the median and interquartile
range (IQR). The STEMI group with HtrA2 above or equal to median levels and the group
with HtrA2 below median levels were also analyzed for the percentage of patients consid-
ered to be adequately reperfused and compared via a Chi-square analysis.
Finally, HtrA2 levels, peak CK-levels, peak CK-MB levels and peak cTnT levels within the
STEMI group all underwent a receiver-operating characteristic (ROC) analysis, analyzing for
the sensitivity and specificity of each marker for diagnosing ischemia-reperfusion injury
according to the aforementioned ECG criterion.
A p-value of ≤0.05 was considered statistically significant for all analyses except for
the analysis of correlation. This particular analysis involved multiple comparisons, thus
 M. Hortmann et al. / International Journal of Cardiology 243 (2017) 485–491 487

requiring correction by Bonferroni's method in order to avoid spurious positive correlates.
This set the new statistically significant p-value at ≤0.0056 for the correlation analysis.
All analyses were performed using Graph Pad Prism v5.0 (GraphPad Software, Inc., La
Jolla, CA).
3. Results
3.1. Patient characteristics
The average age across the entire patient population was 67 years.
The STEMI population was composed of 78% men, in comparison to
75% men in NSTEMIs, 71% in the stable CAD group and 44% men in
the CAD excluded group. Table 1 presents the baseline characteristics
across all patient groups. Significant differences were found in the
number of patients with diagnosed diabetes mellitus and dyslipidemia,
with patients within the stable CAD group displaying a higher preva-
lence of both conditions. There were also significant differences in
medications, with patients within the STEMI and NSTEMI groups
being the only patients to have been given prasugrel, ticagrelor and
eptifibatide. Patients within the stable CAD group were more likely
to have been on a statin. Finally, there were some statistically signifi-
cant differences in laboratory values, with patients in the STEMI group
having significantly increased levels of both creatine kinase (CK) and
cardiac troponin T (cTnT) at peak within 24 h of the ischemic event.
There were no other statistically significant differences between the
patient groups.
The STEMI patient group was further subdivided into patients with
HtrA2 levels above or equal to the median HtrA2 level (n = 19), and
patients with HtrA2 levels below the median level (n = 18). Baseline
characteristics were further compared for significant differences be-
tween these two groups. This is displayed in Table 2. Amongst these
two groups, the STEMI patients with HtrA2 levels above or equal to
the median suffered from significantly more anterior infarctions and
displayed significantly higher levels of C-reactive protein (CRP), creatine
kinase (CK) and cardiac troponin T (cTnT) at peak within 24 h of the
ischemic event. There were no other statistically significant differences.
3.2. HtrA2 levels
HtrA2 levels were detectable in serum across all patients 4.75 (IQR:
3–7) h after PCI. HtrA2 levels ranged between 85.5 and 5306.1 pg/mL,
with a median value of 581.9 pg/mL (IQR: 404.5–1011.8).
Multiple comparisons (Fig. 1) showed that HtrA2 levels were sig-
nificantly increased in STEMI patients compared to NSTEMI patients,
patients with stable CAD and patients with CAD excluded (981.3

Table 1
Main patient characteristics.

Characteristic All STEMI NSTEMI Stable CAD CAD excluded p-Value

Age 67 ± 12.8 65 ± 13.7 68.5 ± 14.5 67 ± 11.4 71 ± 8.1 0.2840

Men 73% 78% 75% 71% 44% 0.0663
DM 22% 8% 30% 41% 22% 0.0003***
HTN 61% 62% 40% 65% 67% 0.1395
Dyslipidemia 33% 19% 25% 71% 33% 0.0001****
Smokers 40% 38% 35% 47% 22% 0.0760
Family history 32% 22% 36% 35 22% 0.1756
Overweight 22% 22% 15% 24% 11% 0.1558

Number of diseased vessels

1 Excl. stable CAD – 30% 35% 20% – – 0.1665
2 Excl. stable CAD – 33% 32% 35% – – 0.1665
3 Excl. stable CAD – 37% 32% 45% – – 0.1665

Infarct related coronary

LAD Excl. stable CAD – 72% 76% 65% – – 0.1246
LCA Excl. stable CAD –5% 6% 5% – – 0.1246
RCA Excl. stable CAD – 67% 57% 85% – – 0.1246
Anterior infarction Excl. stable CAD – 46% 54% 43% – – 0.3875
Prasugrel 24% 49% 5% 0% 0% 0.0001****
Ticagrelor 29% 30% 65% 0% 0% 0.0001****
Eptifibatide 9% 22% 0% 0% 0% 0.0001****
Rivaroxaban 7% 4% 5% 12% 11% 0.1221
Statin 43% 43% 30% 71% 33% 0.0017**
LVEF 47% ± 8% 47% ± 6% 47% ± 8% 45% ± 11% 55% ± 4% 0.1158
Serum creatinine (mg/dL) 0.96 (0.8–1.17) 0.955 (0.77–1.155) 1.05 (0.87–1.415) 0.93 (0.85–1.01) 0.83 (0.745–1.0725) 0.4659
CK Peak (ng/dL) 1151 ± 1533 2049 ± 1756 439 ± 444 111 ± 47 112 ± 70 0.0001****
cTnT Peak (ng/dL) 0.445 (0.1305–3.4875) 1.29 (0.181–5.14) 0.373 (0.23–0.725) 0.004 (0.0035–0.0085) – 0.0019**
Time blood taken for HtrA2 analysis 4.75 (3–7) 4.75 (3–8.25) 4.75 (2.5–6.125) 4.75 (2.5–6) 5 (4–5.5) 0.5400
after PCI (hours)
CRP (mg/l) 7 (3–41) 7 (3–30.75) 17 (5.75–49) 3 (3–5.25) 3 (3–9.5) 0.3958

Values are presented as mean ± SD; median value (25th to 75th percentile range) or %.
DM: Diabetes mellitus.
HTN: Hypertension.
LAD: Left anterior descending coronary artery.
LCA: Left circumflex coronary artery.
RCA: Right coronary artery.
LVEF: Left ventricular ejection fraction.
CK: Creatine kinase.
cTnT: Cardiac troponin T.
HtrA2: High temperature requirement protein A2.
PCI: Percutaneous coronary intervention.
CRP: C-reactive protein.
A p-value of ≤0.05 was considered statistically significant.
488 M. Hortmann et al. / International Journal of Cardiology 243 (2017) 485–491

Table 2
Main patient characteristics in STEMI groups.

Characteristic All STEMI Htra2 ≥ Median Htra2 b Median p-Value

Age 65 ± 13.7 64.5 ± 11.7 62 ± 14.6 0.5836

Men 78% 84% 72% 0.5216
DM 8% 5% 11% 0.1957
HTN 62% 58% 67% 0.5691
Dyslipidemia 19% 16% 22% 0.3833
Smokers 38% 47% 27% 0.0774
Family history 22% 21% 22% 1.0000
Overweight 22% 21% 22% 1.0000

Number of diseased vessels

1 35% 32% 39% 0.5830
2 32% 37% 28% 0.5830
3 32% 32% 33% 0.5830

Infarct related coronary

LAD 76% 74% 78% 0.1643
LCA 6% 16% 6% 0.1643
RCA 57% 63% 50% 0.1643
Other 57% 53% 61%
Anterior infarction 54% 68% 39% 0.0301*
Aspirin 95% 100% 89% 0.6121
Clopidogrel 14% 11% 17% 0.3184
Prasugrel 49% 53% 44% 0.4608
Ticagrelor 30% 32% 28% 0.6619
Eptifibatide 22% 26% 17% 0.2412
Rivaroxaban 4% 5% 6% 1.0000
Statin 43% 53% 33% 0.0926
LVEF 47% ± 6% 46% ± 7% 47.5% ± 6.2% 0.5666
Serum creatinine (mg/dL) 0.955 (0.77–1.155) 0.97 (0.88–1.1175) 0.935 (0.7525–1.0725) 0.5677
CK Peak (ng/dL) 2049 ± 1756 2872 ± 1814 1290 ± 1364 0.00024**
cTnT peak (ng/dL) 1.29 (0.181–5.14) 5.14 (2.28–6.37) 2.81 (0.3435–5.625) 0.0090**
CK-MB peak (ng/dL) 222 (144.5–493) 241.5 (158.25–510.5) 301 (159.75–403) 0.9818
Time blood taken for HTRA2 analysis after PCI (hours) 4.75 (3–8.25) 7 (3–8.75) 4 (3.25–8.25) 0.8034
CRP (mg/l) 7 (3–30.75) 17 (7.25–70.75) 4 (3−10) 0.0469*

Values are presented as mean ± SD; median value (25th to 75th percentile range) or %.
DM: Diabetes mellitus.
HTN: Hypertension.
LAD: Left anterior descending coronary artery.
LCA: Left circumflex coronary artery.
RCA: Right coronary artery.
LVEF: Left ventricular ejection fraction.
CK: Creatine kinase.
cTnT: Cardiac troponin T.
HtrA2: High temperature requirement protein A2.
PCI: Percutaneous coronary intervention.
CRP: C-reactive protein.
A p-value of ≤0.05 was considered statistically significant.

(IQR: 543.5–1526.2) pg/mL vs. 494.5 (IQR: 413.8–607) pg/mL vs. 291
(IQR: 239–458.5) pg/mL vs. 692.2 (IQR: 276.6–964.7) pg/mL; p ≤ 0.0001).
3.3. Ischemia-reperfusion injury and HtrA2 levels
Patients who were deemed to have suffered from I/R injury after
pPCI displayed higher levels of HtrA2 in comparison to those deemed
to have achieved adequate reperfusion; however, this difference
did not reach statistical significance, rather showing a trend (1098
(IQR: 614.9–1776.2) pg/mL vs. 566.6 (IQR: 489.7–751.6) pg/mL;
p = 0.0694). This is demonstrated graphically in Fig. 2. Table 3 demon-
strates that significantly more patients who had HtrA2 levels above
or equal to the median level (n = 15) were identified as inadequately
reperfused by ST-resolution in comparison to patients who had HtrA2
levels below the median level (n = 14) (13% vs. 87%; p ≤ 0.0001).
Furthermore, as shown in Fig. 3, HtrA2 levels exhibited superior sensi-
tivity and specificity for diagnosing ischemia-reperfusion injury as
analyzed by the area under the curve in a ROC analysis compared
to peak levels of validated diagnostic markers such as CK, CK-MB
and cTnT (AUC = 0.7105 vs. AUC = 0.5632 vs. AUC = 0.5660 vs.
AUC = 0.5407).
3.4. Correlation analysis
After Bonferroni's correction for multiple comparisons (Supplemen-
tary table 1), the only clinical characteristics that demonstrated a signif-
icant correlation with HtrA2 levels were peak CK levels and peak cTnT
levels within 24 h of the ischemic event (Supplementary Fig. 1). Peak
CK levels only exhibited significant correlation with HtrA2 levels after
Bonferroni's correction.
4. Discussion
In this study we investigated for the first time if HtrA2 is detectable
and elevated in the serum of patients with STEMI after PCI compared
to patients with NSTEMI, stable CAD and a control group who had
CAD excluded by coronary angiogram, in order to validate HtrA2 as a
potentially novel biomarker for mitochondrial reperfusion-associated
cardiomyocyte apoptosis and severe I/R injury. We assessed if HtrA2
correlates with clinical variables and markers of cell lysis (CK, CK-MB
and cTnT). We further investigated whether HtrA2 can predict I/R injury
in STEMI patients after PCI, via measurement of ST-segment resolution
on electrocardiogram pre- and 2.25 (IQR: 1.5–3.5) hours post-PCI.
 M. Hortmann et al. / International Journal of Cardiology 243 (2017) 485–491 489

8000 Table 3
**** Percentage of patients who achieved adequate reperfusion according to ECG compared to
those who suffered ischemia-reperfusion injury according to whether HtrA2 levels were
above/equal or below the median.
*
≥Median (n = 15) bMedian (n = 14)
6000 Adequate reperfusion 13% 57%
Reperfusion injury 87%*** 42%

A p-value of ≤0.05 was considered statistically significant.

HtrA2 (pg/mL)

p b 0.0001.

4000
further cell death and negates the interventional benefit. The death of
cardiomyocytes during STEMI results from both necrosis and apoptosis.
Troponins and CK-MB are routinely used in clinical practice and are
validated biomarkers of cell lysis, necrosis and overall myocardial
2000 damage to estimate infarct size. Myocardial damage induced by mito-
chondrial I/R injury and induction of apoptosis cannot be monitored
due to a current lack in specific biomarkers. Therefore, I/R injury by ap-
optosis cannot be quantified. Cytochrome c, a mitochondrial apoptotic-
associated protein, has shown promise to evaluate I/R injury [18,19].
0 HtrA2 is another pro-apoptotic-associated protein localised in the inter-
membrane space of mitochondria in cardiomyocytes and released into
)

9)
)

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20
37

17

the cytosol to induce apoptosis when I/R injury occurs [13].

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In our study we found that serum HtrA2 was significantly increased

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in patients with STEMI after PCI compared to NSTEMI, stable CAD and
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CAD-excluded patients. STEMI patients also showed a trend towards

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being increased in patients who were identified as inadequately re-

Fig. 1. HtrA2 levels differ between patients with STEMI, NSTEMI, CAD and CAD excluded. perfused. Furthermore, patients with HtrA2 levels above or equal to
Data are presented as dot plots with median and interquartile range. the median demonstrated a significantly greater likelihood towards in-
adequate reperfusion as assessed by ST-resolution. In addition, serum
HtrA2 demonstrated a superior area under the curve in a receiver oper-
In this current day and age, the outcome after STEMI has been re- ating characteristic analysis for predicting reperfusion injury compared
markably improved by medications and PCI. Despite advancements to CK, CK-MB and cTnT. HtrA2 levels of at least the median had a signif-
in treatment, more than one third of STEMI patients do not show icantly higher peak CK and cTnT, and levels also correlated significantly
complete reperfusion due to severe I/R injury [1,4]. I/R injury causes with HtrA2.
The correlation with CK might reflect a correlation with larger infarct
size. However in this study echocardiography did not reveal a difference
in ejection fraction indicating that there was at least not a clinically
8000 relevant difference in infarction size.
The cellular mechanisms by which HtrA2 is released into the circula-
tion need to be further explored. CK and cTnT are released when cell
membrane rupture occurs during the necrotic process. HtrA2 could
6000 also reach the circulating blood via cell rupture during necrosis. To the
best of our knowledge, it is currently not known whether HtrA2 is also
HtrA2 (pg/mL)

released into the circulation from apoptotic cells. In the literature is

has been demonstrated that cytochrome c is released from both apopto-
4000 tic and necrotic cells [24]. However, this has neither been demonstrated
for cardiomyocytes nor HtrA2. HtrA2's correlation with CK and cTnT
might indicate that necrosis induces the simultaneous release of these
markers, albeit it is possible that the mechanism by which HtrA2 is re-
2000 leased into the circulation is also a result of apoptosis. However, it re-
mains to be confirmed whether HtrA2 levels are not elevated before
significant reperfusion injury occurs (i.e. before PCI), when necrosis
markers are known to be released. Furthermore, the half-life of HtrA2
0
in the circulation has yet to be investigated. Additionally, it is still un-
)
)

known whether circulating free HtrA2 induces downstream cellular

19
10

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and molecular effects.

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ry
n

It is interesting to note that in our study HtrA2 levels were signifi-

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ju
us

In

cantly raised in STEMI patients but not in NSTEMI patients, whereas

rf

on
e

si
ep

myocardial necrosis markers such as cTnT and CK were elevated in

fu
R

er
e

both groups. Given that I/R injury has been found to typically arise in pa-
ep
at
qu

tients with acute STEMI, this gives a stronger indication that HtrA2
de
A

might be a marker specific to I/R injury [5,8,20].

Fig. 2. HtrA2 levels differ between STEMI patients who were deemed to achieve adequate
HtrA2’s validity as a possible biomarker for mitochondrial-induced
reperfusion versus those who suffered ischemia-reperfusion injury. Data are presented as myocardial apoptosis presents a number of potential clinical applica-
dot plots with median and interquartile range. tions. For instance, HtrA2 might be valuable as a potential biomarker
490 M. Hortmann et al. / International Journal of Cardiology 243 (2017) 485–491
Fig. 3. ROC analyses comparing sensitivity and specificity for diagnosing ischemic-reperfusion injury for cardiac markers HtrA2, CK, CK-MB and cTnT levels at peak within 24 h of
angiography
HtrA2: High temperature requirement protein A2
CK: Creatine kinase
CK-MB: Creatine kinase myocardial-band
cTnT: Cardiac troponin T.
for apoptosis induced by reperfusion injury and could therefore be com-
bined with cell lysis markers to estimate the final infarct size. Further-
more, HtrA2's association with impaired myocardial reperfusion might
help to identify patients at risk for adverse events. However, the value
of HtrA2 as a biomarker needs to be confirmed in larger studies. HtrA2
could also be helpful in the identification of cardio-protective drugs
and the efficacy these medications have on reducing I/R injury.
It remains to be assessed if targeting reperfusion injury in the era of
early PCI has any clinical benefit, and, indeed, large randomized studies
have yet to demonstrate any favorable effect on outcome [25]. To trans-
late pharmacological interventions into clinical practice has been diffi-
cult [3]. Although four drugs (brain natriuretic peptide, exenatide,
metoprolol, and esmolol) have been shown to reduce the size of infarct,
clinical studies with outcome as primary endpoint are missing [1,26].
Mitochondria have been the primary target for cardioprotection against
I/R injury. In a small pilot trial, cyclosporine, which inhibits the opening
of mitochondrial permeability-transition pores occurring during re-
perfusion injury, has been associated with a smaller infarct size [27].
However, this could not be proven nor translated into better clinical
outcomes in larger clinical trials [23,28]. Furthermore, treatment with
the mitochondrial stabilising peptide elamipretide was not associated
with a decrease in myocardial infarct size in STEMI patients [29].
Besides pharmacological intervention, conditioning procedures
have been shown to be the most powerful cardioprotective interven-
tions in animal experiments [30]. Although proof-of-concept studies
currently show promise, the translation into clinical daily practice is dif-
ficult and still subject to on-going research due to the uncertainty on the
efficacy of conditioning procedures.
If mitochondria targeting interventions in the future were shown to
be beneficial, HtrA2 might be useful for monitoring these beneficial
effects.
Furthermore, preventing HtrA2 release in STEMI may decrease in-
farct size and improve patient outcomes, as has been demonstrated in
animal models [16]. In a recent publication cardiac specific overexpres-
sion of HtrA2 in a mouse model induced myocardial apoptosis and car-
diac dysfunction [17]. The authors concluded that strategies directly
inhibiting HtrA2 or its cytosolic translocation from mitochondria
might protect against heart injury. This supports the hypothesis of
HtrA2's important contribution to I/R injury and its use as a possible
target to prevent it.
Ultimately, HtrA2 has shown promise as a potential biomarker
for identifying patients with I/R microvascular injury and no-reflow. In
our study, patients with HtrA2 levels of at least the median were signif-
icantly associated with compromised resolution of ST-segment eleva-
tion post PCI, which might have clinical utility given that this lack of
ST-segment resolution has been significantly associated with early
mortality [31]. Incomplete resolution of ST-segment elevation evaluated
on standard 12-lead ECG in STEMI after PCI has also been shown to be a
powerful prognostic marker for 90-day prognosis, death, development
of heart failure and shock [20]. Therefore, HtrA2 might also show
value as a prognostic marker; however, this must be validated in future
studies.
5. Limitations
The results of this study support our hypothesis. However, our study
has some limitations. The clinical characteristics were obtained retro-
spectively and hence our results may be subject to bias and con-
founders. Furthermore, this study was limited by the relatively small
sample size of patients with STEMI and not powered to identify signifi-
cant HtrA2 levels. This may also explain why HtrA2 levels did not reach
statistical significance in its association with reperfusion injury assessed
by ECG. HtrA2 might also show value as a novel prognostic marker after
STEMI. However, the sample size was too small to analyze in-hospital
mortality. Furthermore, patients were not followed up to assess for
long-term mortality or morbidity.
A strength of the study is that blood was drawn at similar time points
between patient groups, making them comparable. However, a time
course study analyzing the biokinetics of circulating HtrA2 is yet to be
performed.
There is currently no role for HtrA2 in distinguishing the different
entities of acute coronary syndrome. High values above the median of
the STEMI group might help to identify severe I/R injury.
Hence, HtrA2 may be a promising novel biomarker for mitochondrial-
induced apoptosis and severe ischemia-reperfusion injury in the heart.
However, the clinical value is speculative and more work is required to
understand the potential role of HtrA2 as a biomarker [32,33]. Further-
more, there is no proof that HtrA2 is a marker of I/R injury and apopto-
sis, and it might simply be a marker of severe necrosis and cell lysis. In
addition, the contribution of apoptosis to myocardial cell death in I/R
injury is not clear and needs to be assessed.
A further limitation to our study is that we only used the surrogate
parameter of ST-elevation resolution to identify I/R injury and micro-
vascular dysfunction, whereas other more visually direct markers,
such as TIMI myocardial perfusion grade, contrast echocardiography
and cardiovascular magnetic resonance imaging are currently in prac-
tice. Future studies should include these methods and correlate HtrA2
specifically to microvascular I/R injury.
Ultimately, our findings need to be verified in studies with larger pa-
tient cohorts and further investigation into the physiology of HtrA2
must be undertaken.
 M. Hortmann et al. / International Journal of Cardiology 243 (2017) 485–491
6. Conclusions
To the best of our knowledge, we have described for the first time
significantly increased serum levels of HtrA2 in patients with STEMI
compared to NSTEMI and stable CAD. HtrA2 correlated significantly
with the peak levels of cell lysis biomarkers (CK and cTnT). HtrA2
serum levels also demonstrated a trend in being correlated to adequacy
of reperfusion post-PCI assessed via measurement of ST-segment reso-
lution, and exhibited superior sensitivity and specificity for predicting
reperfusion injury compared to other biomarkers.
Consequently, HtrA2 shows promise as a potential biomarker for the
identification of ischemia-reperfusion injury and cardiomyocyte apo-
ptosis, which has a number of potential clinical applications, including
both diagnostic and prognostic utility, as well as possibly exhibiting
value for analyzing the efficacy of anti-apoptotic therapeutics. Our
data warrants further validation in larger clinical trials.
Conflict of interest
The authors report no relationships that could be construed as a con-
flict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ijcard.2017.05.088.
References
[1] G. Heusch, B.J. Gersh, The pathophysiology of acute myocardial infarction and strat-
egies of protection beyond reperfusion: a continual challenge, Eur. Heart J. 38
(2017) 774–784.
[2] D.M. Yellon, D.J. Hausenloy, Myocardial reperfusion injury, N. Engl. J. Med. 357
(2007) 1121–1135.
[3] G. Heusch, The coronary circulation as a target of cardioprotection, Circ. Res. 118
(2016) 1643–1658.
[4] M.J. Claeys, J. Bosmans, L. Veenstra, P. Jorens, H. De Raedt, C.J. Vrints, Determinants
and prognostic implications of persistent ST-segment elevation after primary angio-
plasty for acute myocardial infarction: importance of microvascular reperfusion
injury on clinical outcome, Circulation 99 (1999) 1972–1977.
[5] L. De Roeck, S. Vandamme, B.R. Everaert, V. Hoymans, S. Haine, T. Vandendriessche,
et al., Adiponectin and ischemia-reperfusion injury in ST segment elevation myocar-
dial infarction, Eur. Heart J. Acute Cardiovasc. Care 5 (2016) 71–76.
[6] L. Bolognese, N. Carrabba, G. Parodi, G.M. Santoro, P. Buonamici, G. Cerisano, et al.,
Impact of microvascular dysfunction on left ventricular remodeling and long-term
clinical outcome after primary coronary angioplasty for acute myocardial infarction,
Circulation 109 (2004) 1121–1126.
[7] I. Eitel, S. de Waha, J. Wohrle, G. Fuernau, P. Lurz, M. Pauschinger, et al., Comprehen-
sive prognosis assessment by CMR imaging after ST-segment elevation myocardial
infarction, J. Am. Coll. Cardiol. 64 (2014) 1217–1226.
[8] L.J. Feldman, P. Coste, A. Furber, P. Dupouy, M.S. Slama, J.P. Monassier, et al., Incomplete
resolution of ST-segment elevation is a marker of transient microcirculatory dysfunc-
tion after stenting for acute myocardial infarction, Circulation 107 (2003) 2684–2689.
[9] B. Ibanez, G. Heusch, M. Ovize, F. Van de Werf, Evolving therapies for myocardial
ischemia/reperfusion injury, J. Am. Coll. Cardiol. 65 (2015) 1454–1471.
[10] S. Hashmi, S. Al-Salam, Acute myocardial infarction and myocardial ischemia-
reperfusion injury: a comparison, Int. J. Clin. Exp. Pathol. 8 (2015) 8786–8796.
[11] A.B. Gustafsson, R.A. Gottlieb, Heart mitochondria: gates of life and death, Cardiovasc.
Res. 77 (2008) 334–343.
[12] K.A. Webster, Mitochondrial membrane permeabilization and cell death during
myocardial infarction: roles of calcium and reactive oxygen species, Future Cardiol.
8 (2012) 863–884.
View publication stats
491
[13] M.S. Bhuiyan, K. Fukunaga, Activation of HtrA2, a mitochondrial serine protease
mediates apoptosis: current knowledge on HtrA2 mediated myocardial ischemia/
reperfusion injury, Cardiovasc. Ther. 26 (2008) 224–232.
[14] H.R. Liu, E. Gao, A. Hu, L. Tao, Y. Qu, P. Most, et al., Role of Omi/HtrA2 in apoptotic cell
death after myocardial ischemia and reperfusion, Circulation 111 (2005) 90–96.
[15] K. Wang, J. Zhang, J. Liu, J. Tian, Y. Wu, X. Wang, et al., Variations in the protein level
of Omi/HtrA2 in the heart of aged rats may contribute to the increased susceptibility
of cardiomyocytes to ischemia/reperfusion injury and cell death: Omi/HtrA2 and
aged heart injury, Age (Dordr.) 35 (2013) 733–746.
[16] M.S. Bhuiyan, K. Fukunaga, Inhibition of HtrA2/Omi ameliorates heart dysfunction
following ischemia/reperfusion injury in rat heart in vivo, Eur. J. Pharmacol. 557
(2007) 168–177.
[17] K. Wang, Y. Yuan, X. Liu, W.B. Lau, L. Zuo, X. Wang, et al., Cardiac specific overexpres-
sion of mitochondrial Omi/HtrA2 induces myocardial apoptosis and cardiac dys-
function, Sci. Rep. 6 (2016) 37927.
[18] Z.B. Liu, X.H. Fu, G. Wei, J.L. Gao, Cytochrome c release in acute myocardial infarction
predicts poor prognosis and myocardial reperfusion on contrast-enhanced magnetic
resonance imaging, Coron. Artery Dis. 25 (2014) 66–72.
[19] G. Marenzi, M. Giorgio, M. Trinei, M. Moltrasio, P. Ravagnani, D. Cardinale, et al.,
Circulating cytochrome c as potential biomarker of impaired reperfusion in ST-
segment elevation acute myocardial infarction, Am. J. Cardiol. 106 (2010) 1443–1449.
[20] C.E. Buller, Y. Fu, K.W. Mahaffey, T.G. Todaro, P. Adams, C.M. Westerhout, et al., ST-
segment recovery and outcome after primary percutaneous coronary intervention
for ST-elevation myocardial infarction: insights from the assessment of Pexelizumab
in acute myocardial infarction (APEX-AMI) trial, Circulation 118 (2008) 1335–1346.
[21] P.G. Steg, S.K. James, D. Atar, L.P. Badano, C. Blomstrom-Lundqvist, M.A. Borger, et al.,
ESC guidelines for the management of acute myocardial infarction in patients
presenting with ST-segment elevation, Eur. Heart J. 33 (2012) 2569–2619.
[22] C.W. Hamm, J.P. Bassand, S. Agewall, J. Bax, E. Boersma, H. Bueno, et al., ESC
guidelines for the management of acute coronary syndromes in patients presenting
without persistent ST-segment elevation: the task force for the management
of acute coronary syndromes (ACS) in patients presenting without persistent
ST-segment elevation of the European Society of Cardiology (ESC), Eur. Heart J. 32
(2011) 2999–3054.
[23] F. Ottani, R. Latini, L. Staszewsky, L. La Vecchia, N. Locuratolo, M. Sicuro, et al.,
Cyclosporine a in Reperfused myocardial infarction: the multicenter, controlled,
open-label CYCLE trial, J. Am. Coll. Cardiol. 67 (2016) 365–374.
[24] R. Jemmerson, B. LaPlante, A. Treeful, Release of intact, monomeric cytochrome c
from apoptotic and necrotic cells, Cell Death Differ. 9 (2002) 538–548.
[25] J.T. Lonborg, Targeting reperfusion injury in the era of primary percutaneous coro-
nary intervention: hope or hype? Heart 101 (2015) 1612–1618.
[26] D.J. Hausenloy, H.E. Botker, T. Engstrom, D. Erlinge, G. Heusch, B. Ibanez, et al.,
Targeting reperfusion injury in patients with ST-segment elevation myocardial in-
farction: trials and tribulations, Eur. Heart J. 38 (2017) 935–941.
[27] C. Piot, P. Croisille, P. Staat, H. Thibault, G. Rioufol, N. Mewton, et al., Effect of cyclo-
sporine on reperfusion injury in acute myocardial infarction, N. Engl. J. Med. 359
(2008) 473–481.
[28] T.T. Cung, O. Morel, G. Cayla, G. Rioufol, D. Garcia-Dorado, D. Angoulvant, et al.,
Cyclosporine before PCI in patients with acute myocardial infarction, N. Engl. J.
Med. 373 (2015) 1021–1031.
[29] C.M. Gibson, R.P. Giugliano, R.A. Kloner, C. Bode, M. Tendera, A. Janosi, et al.,
EMBRACE STEMI study: a phase 2a trial to evaluate the safety, tolerability, and effi-
cacy of intravenous MTP-131 on reperfusion injury in patients undergoing primary
percutaneous coronary intervention, Eur. Heart J. 37 (2016) 1296–1303.
[30] G. Heusch, T. Rassaf, Time to give up on Cardioprotection? A critical appraisal of
clinical studies on ischemic pre-, post-, and remote conditioning, Circ. Res. 119
(2016) 676–695.
[31] R. Schroder, K. Wegscheider, K. Schroder, R. Dissmann, W. Meyer-Sabellek, Extent of
early ST segment elevation resolution: a strong predictor of outcome in patients
with acute myocardial infarction and a sensitive measure to compare thrombolytic
regimens. A substudy of the International Joint Efficacy Comparison of Thrombolytics
(INJECT) trial, J. Am. Coll. Cardiol. 26 (1995) 1657–1664.
[32] J.O. Baker, R. Tyther, C. Liebetrau, J. Clark, R. Howarth, T. Patterson, et al., Cardiac
myosin-binding protein C: a potential early biomarker of myocardial injury, Basic
Res. Cardiol. 110 (2015) 23.
[33] E. Giannitsis, H.A. Katus, A long way to translation: will cMyC survive? Basic Res.
Cardiol. 110 (2015) 24.