Web viewTotal word count: Address for c. orrespondence: ... Sugiyama S, Sugamura K, Nozaki T, Ohba K, Matsubara J, Sakamoto K, Nagayoshi Y, Sumida H,

Effect of coronary atherosclerotic burden and inducible myocardial ischemia on

circulating levels of high sensitivity cardiac troponin T (hs-cTnT) and N terminal

probrain natriuretic peptide (NT-proBNP) in patients with stable angina: results

from the EVINCI study.

Cardiac biomarkers and stable angina.

Chiara Caselli,a Concetta Prontera,b Rosetta Ragusa,c Riccardo Liga,d Michiel A. De

Graaf,e Valentina Lorenzoni,c Silvia Del Ry,a Daniele Rovai,a Daniela Giannessi,a

Oliver Gӓemperli,f Jeroen J. Bax,e Massimo Lombardi,b Rosa Sicari,a Jose’ Zamorano,g

Arthur J. Scholte, e Philipp A. Kaufmann,f Juhani Knuuti,h S Richard Underwood,i

Aldo Clerico, b,c Danilo Neglia, MD.a,b

a CNR, Institute of Clinical Physiology, Pisa, Italy;

b Fondazione Toscana G. Monasterio, Pisa, Italy;

c Scuola Superiore Sant’Anna, Pisa, Italy;

d Cardio-Thoracic and Vascular Department, University Hospital of Pisa, Pisa, Italy;

e Leiden University Medical Center, Leiden, The Netherlands;

f University Hospital Zurich, Zurich, Switzerland;

g University Hospital Clinico San Carlos, Madrid, Spain;

h University of Turku and Turku University Hospital, Turku, Finland;

i Imperial College London, United Kingdom

This work was supported by a grant from the European Union FP7-CP-FP506 2007

project (grant agreement no. 222915, EVINCI).

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The authors declare that they have no relationships relevant to the contents of this

paper to disclose.

Total word count:

Address for correspondence: Chiara Caselli, PhD

CNR Institute of Clinical Physiology

Area della Ricerca – Via Moruzzi, 1 56100 Pisa

Fax: 39 050 3152166

Phone: 39 050 3152019

e-mail: [email protected]

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Abstract

Background. Circulating levels of high sensitivity cardiac troponin T (hs-cTnT) and

N terminal probrain natriuretic peptide (NT-proBNP) are predictors of coronary artery

disease (CAD) and long term prognosis in patients with stable angina.

Purpose. The aim of this study was to evaluate the effect of coronary atherosclerotic

burden and inducible myocardial ischemia on cardiac release of hs-cTnT and NT-

proBNP in a contemporary European population of patients with suspected CAD.

Methods. Hs cTnT and NT-proBNP were measured in 378 patients (60.1 ±0.5 years,

229 males) with stable angina and unknown CAD enrolled in the Evaluation of

INtegrated Cardiac Imaging (EVINCI) study. All patients underwent stress imaging,

by myocardial perfusion or wall motion imaging, to detect inducible ischemia, and

coronary computed tomography angiography (CTA) to assess the presence of CAD

(>50% stenosis of at least one major coronary vessel). An individual CTA score,

expressing the coronary atherosclerotic burden, was calculated combining extent,

severity, composition, and location of plaques.

Results. In the whole population the median (IQR) value of plasma hs-cTnT was 6.17

(4.2-9.1) ng/L and of NT-proBNP was 61.66 (31.2-132.6) ng/L. In a multivariate

model, CTA score was an independent predictor of the plasma hs cTnT (coefficient

0.06, SE 0.02, p=0.0089), while ischemia was a predictors of NT-proBNP (coefficient

0.38, SE 0.12, p=0.0015). When patients were subdivided according to the

absence/presence of CAD and ischemia, hs-cTnT concentrations were significantly

increased in patients with CAD with or without inducible ischemia (p<0.005), while

only patients with CAD and ischemia showed significantly higher levels of NT-

proBNP compared with all the other groups (p<0.001).

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Conclusions. In patients with stable angina, the presence and extent of CAD is related

with increased levels of hs-cTnT also in absence of ischemia. This suggest an

ischemia-independent mechanisms of hs cTnT release linked with coronary

atherosclerosis. NT-proBNP is a strong and independent predictor of obstructive CAD

causing myocardial ischemia, thus identifying those subject at high risk of future

events, probably requiring invasive procedure.

Keywords

hs cardiac Troponin T, NT-proBNP, stable angina, coronary artery disease,

atherosclerotic burden, myocardial ischemia, cardiac imaging

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Introduction

The stable coronary artery disease (CAD) population represents a heterogeneous group

of patients for patho-physiologic substrate, clinical presentation and outcome (1).

These patients may have different morphology, severity and extent of coronary

atherosclerotic lesions and of inducible myocardial ischemia and these multiple

aspects may have independent prognostic value (2-3).

High-sensitivity cardiac troponins (hs-TnT) is commonly used in the diagnosis of

acute coronary syndromes (4-7). Recently, it has been shown that episodes of minute

troponin release, below the threshold for acute myocardial infarction, often occur in

patients with stable CAD and this has been demonstrated to predict all cause

mortality, cardiovascular mortality and heart failure (HF) (5-8). N-terminal pro brain

natriuretic peptide (NT-proBNP) is a powerful prognostic indicator in patients with

left ventricular (LV) dysfunction and HF. However, it is also a predictor of all cause

and cardiovascular mortality in patients with stable CAD without HF (9-11), Hence

both these biomarkers may be used as prognostic indicators in stable CAD (5-11).

The aim of this study was to evaluate the effect of coronary atherosclerotic burden,

assessed by CT angiography (CTA), or myocardial ischemia, assessed by stress

imaging, on cardiac release of hs-cTnT and N-terminal pro B-type natriuretic peptide

(NT-proBNP) in patients with stable CAD enrolled in the EValuation of INtegrated

Cardiac Imaging (EVINCI) (12). In the subgroup of EVINCI patients in whom

imaging fusion of CTA and myocardial perfusion imaging (MPI) was performed, the

combined effects of coronary atherosclerosis and downstream inducible ischemia on

circulating biomarkers was also evaluated.

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Materials and Methods

Population, diagnostic protocol and study design

Patients with stable chest pain or equivalent symptoms and intermediate probability of CAD were studied. These patients were enrolled at 14

European centres in the EValuation of INtegrated Cardiac Imaging for the detection and characterization of ischemic heart disease (EVINCI) study

(12). Patients with acute coronary syndrome, known CAD, left ventricular ejection fraction < 35%, significant heart valve disease, cardiomyopathy

or contraindications to stress imaging were excluded. According to the EVINCI protocol, each patient underwent CTA, stress imaging (by MPI or

WMI) and - if at least one non invasive tests was positive - invasive coronary angiography and measurement of FFR when indicated (12). Ethical

approval was provided by each participating centre and all subjects provided written informed consent.

The patients whose CTA images and plasma samples were available for core laboratory analyses were included in this study. Patients with also

available MPI studies by SPECT or PET were included in a sub-analysis. Image fusion of MPI with CCTA datasets and hybrid analysis were

performed by a dedicated core laboratory.

Blood collection and analysis

Blood samples were collected before non-invasive imaging in tubes with EDTA and then locally separated by centrifugation for 15 min at 1000 ×g.

Plasma samples were provisionally stored in a local refrigerator at - 80°C and shipped to the bio-humoral core laboratory (CNR-Institute of Clinical

Physiology, Pisa, Italy) for the final cryo-conservation in the EVINCI biological bank (13-14). Analyses of hs-cTnT and NT-proBNP were

performed at the Laboratory of Fondazione Toscana G. Monasterio (Pisa, Italy) using standard clinical laboratory procedures on automatic analyzers,

according to the recommendations made by the manufacturer (Roche Diagnostics International Ltd, Switzerland). Plasma concentrations of cTnT

were measured using the hs-cTnT method on COBAS E411 with Elecsys Troponin T hs STAT by Roche Diagnostics, as previously described in

detail (15 ).

Measurement of NT pro-BNP was performed using the electrochemical luminescence immunoassay Elecsys proBNP II by Roche Diagnostics using

monoclonal antibodies, as previously described in detail (16).

In order to complete the clinical/biohumoral profile of study patients, additional traditional biomarkers were measured using standard methods (13-

14).

Image acquisition

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Image acquisition protocols were agreed on for each technique covering patient preparation, cardiovascular stress, administration of

radiopharmaceutical or contrast medium, image acquisition and quality control. These procedures were based on best available clinical practice.

Image analysis and reporting was performed at specific core laboratories dedicated to each technique by experienced observers blinded to clinical

history and other imaging findings (12).

Coronary CTA analysis and CTA risk score

The coronary CTAs were analyzed in a core laboratory (Leiden University Medical Center, Leiden, The Netherlands) by consensus of experienced

observers blinded to any other clinical data or imaging test. First, each segment of the AHA 17-segment model was assessed for interpretability.

Segments were defined as uninterpretable in case of severe motion artefacts or low contrast resolution. Additionally, segments with a diameter ≤ 1.5

mm were excluded. Interpretable segments were evaluated for stenosis, which was then stratified into four different categories: normal if no

atherosclerosis was present, non-obstructive if the stenosis severity was <50%, obstructive for lesions with 50-70% stenosis of the coronary artery

lumen. If plaque was present, plaque composition was visually determined (calcified, mixed, non-calcified). One type of plaque composition was

assigned per segment.

In 297/376 patients, CT acquisitions for coronary artery calcium were available and the Agatston CAC score was computed according to standard

methods.

A previously established CTA risk score was derived in each patient integrating all data on the location, severity, and composition of CAD (17). In

brief, the score consists of three weight factors for each segment of the coronary tree. A stenosis severity weight factor, a stenosis location weight

factor, and a weight factor for plaque composition. All three weight factors are multiplied to calculate the segment score. The risk score for each

patient is calculated by adding all segment scores.

Non invasive stress imaging analysis

MPI and WMI were defined as abnormal if there was either an inducible perfusion abnormality or myocardial scarring. Perfusion in each of 17

segments was classified as 0 = normal, 1 = mild reduction, 2 = moderate reduction, 3 = severe reduction or 4 = absent perfusion and the segmental

scores were summed for the stress and rest images. For MPI, an inducible perfusion abnormality was defined as a summed segmental difference score

between stress and rest images ≥ 2, either from a score ≥ 1 in at least two contiguous segments or ≥ 2 in at least one segment. Scarring was defined

similarly from the summed segmental rest score. For WMI, segmental myocardial wall motion was scored at rest and during stress as normal (0),

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hypokinetic (1), akinetic (2) or dyskinetic (3). Inducible ischaemia was defined as an increase in segmental wall motion score ≥ 1 from rest to stress

in at least two contiguous segments. Scarring was defined similarly from the resting wall motion score.

Hybrid imaging

In the subgroup of 195 patients submitted to MPI by PET or SPECT with an excellent image quality, a hybrid imaging study was performed.

Individual datasets from MPI and CTA were transferred to a dedicated hybrid core laboratory blinded to clinical history and imaging findings

(University Hospital Zurich, Switzerland). Image fusion of MPI and CCTA datasets was performed on a dedicated workstation (Advantage

Workstation 4.4, GE Healthcare) using the CardIQ Fusion software package (GE Healthcare) as previously described (18). In case of H215O-PET

images, parametric myocardial blood flow (MBF) datasets, showing MBF on a segmental level, were first generated and quantitative analysis was

performed using an in-house developed software, PMOD 3.6 software package (PMOD Technologies Ltd., Zurich, Switzerland). Then hybrid

analysis was performed using an optimized alignment tool that allows projection of the MPI image on the left ventricular epicardial surface obtained

from the CTA. The 3D volume rendered fusion images allow a panoramic view of the coronary artery tree projected onto the left ventricular

myocardial perfusion territories. Images can be displayed in freely selectable angles and displayed in standard anterior, posterior, lateral, and apical

view for standardized documentation and reporting. In all patients, the image fusion procedure (including image generation and reading) was

performed.

A patient’s CTA was considered abnormal if at least one coronary artery had a diameter stenosis >50%. According to an intention-to-diagnose

strategy, any non-diagnostic segment was considered abnormal. Significant left main stem stenosis were assigned to both left anterior descending

(LAD) and left circumflex (LCX) coronary arteries. A reversible perfusion defect (ischemia) was defined as a SDS ≥2, either from a score ≥ 1 in at

least two contiguous segments or ≥ 2 in at least one segment. Myocardial scar was defined similarly as a SRS ≥2. Each perfusion defect was assigned

to one or more coronary territories according to the standardized myocardial segmentation model proposed by Cerqueria et al. (19).

All hybrid MPI/CCTA images were analysed with regard to the presence of hemodynamically significant coronary lesions. Specifically, each

abnormal myocardial segment was assigned to the pertinent vascular territory by spatial coregistration according to patients’ individual coronary

anatomy. A matched hybrid imaging finding was defined as a perfusion defect in a territory subtended by a stenotic coronary artery on CCTA. All

other combinations of pathologic findings were classified as unmatched.

Statistical analysis

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Categorical variables are presented as numbers (percentage), continuous variables as mean ± SD or median [25-75 percentile] depending on their

distribution.

Differences in continuous variables between two groups were tested using Student’s t test or Mann-Whitney test. Comparisons among groups were

performed using ANOVA analysis and Kruskall-Wallis test, Bonferroni test or Mann-Whitney test using Bonferroni correction for P-value were used

for post-hoc comparisons. Pearson’s chi-squared test was used to compare categorical data.

Univariate and multivariate linear regression were used to estimate the effect of clinical variables as well as imaging results on hs-cTnT and NT-

proBNP plasma levels. the. All models were developed considering variables with P value < 0.1 at univariate analysis and then using backward and

forward stepwise selections to build up the final models. The logarithmic transformation of continuous variables was used in linear regression

analysis.

All analyses were performed using StataCorp. 2007. Stata Statistical Software:

Release 10. College Station, TX: StataCorp LP. A 2-sided value of P <0.05 was

considered statistically significant.

Results

Baseline clinical characteristics of study population

In the whole population the median (interquartile range) value of plasma hs-cTnT was 6.17 (4.16-9.09) ng/L and of NT-proBNP was 61.66 (31.19-

132.60) ng/L.

Only 34 (9%) and 77 (20%) of patients had plasma levels of hs-cTnT and of NT-proBNP exceeding the upper limits of the normality ranges (14

pg/mL and 157 ng/L, respectively). The median values were used as cut-off points to divide patients into groups with low (< median) or high (≥

median) concentrations of hs-cTnT and NT-proBNP. Baseline characteristics of the patients are compared between groups in Table 1. Patients with

high levels of hs-cTnT were older and had higher frequency of male sex, diabetes, hypertension, anti-diabetic and anti-hypertensive treatments than

patients with low levels of hs-cTnT. Patients with high levels of NT-proBNP were also older than patients with low levels of NT-proBNP but showed

a lower frequency of male sex and diabetes and a lower LV ejection fraction. The metabolic and inflammatory profile was altered in patients with

high levels of hs-cTnT but not in those with high NT-proBNP levels.

Coronary atherosclerosis and inducible myocardial ischemia

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Results from CTA (coronary anatomy, plaque characterization and risk scores) and stress imaging (inducible myocardial ischemia) are compared

between groups of patients with high or low hs-cTnT or NT-proBNP in Table 2.

Biomarker levels were associated with the severity and the extent of CAD. Patients with high hs-cTnT as well as patients with high NT-proBNP

showed a higher frequency of obstructive coronary lesions as compared with patients with low hs-cTnT and NT-proBNP who showed a higher

frequency of normal coronary arteries. Figure 1 illustrates the increase of hs-cTnT and NT-proBNP plasma levels according to the presence of non

obstructive or obstructive coronary lesions (Figure 1A) or with the number of any coronary lesion (either obstructive or non obstructive) (Figure 1B).

The two biomarkers were differently associated with plaque types. Patients with high hs-cTnT, but not patients with high NT-proBNP, showed more

frequently calcified or mixed plaques than patients with low hs-cTnT and had a higher number of mixed plaques. CTA risk score was significantly

higher in patients with high hs-cTnT or high NT-proBNP while CAC score was significantly higher only in patients with high hs-cTnT (Table 2).

Both patients with high hs-cTnT and high NT-proBNP had higher frequency and extent of inducible ischemia (Table 2) .

Integrated effects of coronary atherosclerotic burden and inducible ischemia on circulating hs-cTnT and NT-proBNP levels

Multiple linear regression model was used to identify the independent predictors of elevated levels of hs-cTnT and NT-proBNP (Table 3). Age, male

sex and CTA risk score were independent predictors of increased hs-cTnT levels. Age, female sex, lower LVEF and presence of ischemia were

independent predictors of increased NT-proBNP levels.

Patients were subdivided in groups according to the absence or the presence of coronary atherosclerosis (CAD) and ischemia, either alone or

combined. Patients with CAD with/without ischemia showed significantly higher levels of hs-cTnT than patients without CAD, while only patients

with CAD plus ischemia had significantly higher levels of NT-proBNP as compared with all the other groups (Figure 2A).

Similar results were obtained by hybrid imaging analysis. Patients with a perfusion defect in a territory subtended by a stenotic coronary artery on

CTA (macthed) were compared with patients with all other combinations of pathologic findings (unmatched) and with normals. As compared with

normals, NT-proBNP levels were significantly increased only in patients with matched findings while hs-cTnT levels were significantly elevated in

both patients with matched and mismatched findings. (Figure 2B).

LVEF and NT-proBNP were linearly correlated in the whole population (p = 0.002). Interestingly, only patients with coronary atherosclerosis and

inducible myocardial ischemia had significantly decreased LVEF values as compared with all the other patient groups (Figure 3).

Discussion

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The present study examines the relative effects of coronary atherosclerotic burden and

inducible myocardial ischemia on circulating levels of hs-cTnT and NT-proBNP in

patients with stable CAD . The main findings of the present study can be summarized

as follows:

1. circulating levels of hs-cTnT are related with presence and extent of coronary

lesions as well as coronary plaque composition; in addition the global coronary

atherosclerotic burden, as assessed by CTA risk score, is a predictor of hs-cTnT

plasma levels independently of inducible myocardial ischemia;

2. circulating levels of NT-proBNP are mainly related with the presence of

functionally relevant coronary disease causing myocardial ischemia.

Cardiac troponins are the marker of choice for the detection of myocardial injury and

the diagnosis of myocardial infarction, as recommended by the most recent guidelines

(20-21). Over the past 10 years cTn assays have been improved in analytical

sensitivity and precision thereby allowing the measurement of cTn in nearly all

healthy subjects (22-23) and patients with stable CAD (5-6). In stable CAD

release of cTnT has been linked to the extent of coronary atherosclerosis (4-6) while

the relationship with transient inducible myocardial ischemia is debated (24-29).

In this study, an association of plasma hs-cTnT with several features of coronary

atherosclerosis as well as the presence of myocardial ischemia was observed.

However, only the global atherosclerotic burden, as assessed by CTA risk score, and

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not myocardial ischemia, was an independent predictor of hs-cTnT levels at

multivariate analysis. Patients with coronary atherosclerosis showed significantly

higher levels of hs-cTnT compared with patients without, independently of the

presence of inducible myocardial ischemia (Figure 3).

The mechanisms of hs-cTnT elevation in patients with stable CAD, who had no

occurrence of acute events with myocyte necrosis, are not fully understood.

Consistently with the present findings, it has been hypothesized that at the site of

atherosclerotic lesions, dislodging and erosion processes could cause micro-

embolization of atherosclerotic and thrombotic material into the microcirculation (4,

30-31). Plaques more prone to erosion have a different composition than more stable

plaques. It has been very recently reported that chronic clinically silent rupture of non-

calcified plaque with subsequent microembolisation may be a potential source of

troponin elevation (32). These findings are in line with results from this study, in

which patients with higher coronary atherosclerotic burden and mixed plaques showed

higher levels of hs-cTnT than patients without.

The phenomenon of microembolization can cause myocardial damage and troponin

release. Mechanisms of progressive myocardial damage involve tumor necrosis factor

alpha (TNF-alpha) and nitric oxide (NO) (33-35) . It has been observed in an animal

model of coronary microembolization that an increase in TNF-alpha is associated with

modification of myofibrillar proteins, such as tropomyosin (36). TNF-alpha binds to

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its receptors to activate a cascade of caspases leading to apoptosis (37-38).

Microembolization also causes excessive NO and oxidation (34). Excessive NO may

also trigger apoptotic pathways of cardiomyocytes (38). Importantly, in vitro studies

have demonstrated that, during the apoptotic process, caspase-3 activation results in

cleavage of cTn and subsequent release (4, 38-39). It remains to be established how

cardiomyocytes could release cTnT. Potential mechanisms beyond rupture of plasma

membrane have been suggested such as increased cellular wall permeability due to

myocardial stretch or ischemia and formation and active secretion of membranous

blebs from cardiac cells (24). Taking into account all these data, it is possible to

hypothesize that elevated hs-cTnT levels observed in patients with stable CAD may

be the result of microembolization activating the apoptotic program within cardiac

myocytes. This could explain why hs-cTnT levels are associated with the extent and

composition of atherosclerotic plaques in the present study. Whether this could be also

the basis of the association of hs-cTnT with cardiovascular mortality in patients with

stable CAD (5-6) remains to be demonstrated.

In patients with HF, elevation of NT-proBNP is proportional to the extent of

ventricular dysfunction (40). However elevation of this biomarker may be found in

patients without HF and without overt LV dysfunction. There are a few data

demonstrating the association of NT-proBNP circulating levels with coronary

atherosclerosis and myocardial ischemia in patients with stable CAD (41-43). It has

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been found that NT-proBNP plasma levels were elevated in patients with obstructive

coronary artery lesions (defined as a diameter stenosis >70%) and proportional to the

number of affected vessels. Most importantly, experimental (44-45) studies have

provided evidence that myocardial ischemia may cause per se an increased expression

and secretion of natriuretic peptides by ventricular myocytes, triggering the release

and induction of de novo synthesis of BNP. In clinical studies (41, 43, 46-48)

measurement of plasma levels of NT-proBNP and BNP can distinguish patients with

and without inducible ischemia with a high degree of accuracy even after adjustment

for LV function (43). In this study, both coronary atherosclerotic burden and presence

of inducible myocardial ischemia were predictors of higher NT-proBNP plasma levels

in patients with stable CAD. However, at variance with hs-cTnT, only the presence of

inducible myocardial ischemia was an independent predictor of higher NT-proBNP.

Patients with obstructive coronary lesions and inducible ischemia had significantly

higher values of NT-proBNP than all the other patients (Figure 2). Moreover, NT-

proBNP levels were not associated with the characteristics of coronary plaques.

Therefore, it is conceivable that the elevation of NT-proBNP in this study reflects the

chronic and cumulative effects of repetitive myocardial ischemia due to obstructive

CAD. As a matter of fact, despite patients were enrolled after exclusion of subject with

LV systolic dysfunction (LVEF < 45%), LVEF and NT-proBNP were linearly

correlated in the whole population and lower LVEF was an independent predictor of

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higher NT-proBNP values. Interestingly, only patients with obstructive coronary

disease and inducible myocardial ischemia had both significantly increased NT-

proBNP and signicantly decreased LVEF values as compared with all the other patient

groups (Figure 3). Increased release of BNP from the cardiac myocytes may be

consequent to the direct effects of repetitive ischemia on the cardiac myocytes

resulting in an increased gene expression (44-45) as well as a consequence of

subclinical ventricular dysfunction and mechanical myocardial stretching secondary

to myocardial stunning (49). Whichever the mechanism, our results underline the

relevance of circulating NT-proBNP as a marker of chronic ischemic coronary disease

and are in agreement with the known prognostic role of this biomarker in patients with

stable CAD (9-10).

A schematic representation of the proposed mechanisms explaining the increased

levels of hs-cTnT and NT-proBNP in patients with stable CAD is reported in Figure 4.

Conclusions

The present study provides evidence that circulating hs-cTnT and NT-proBNP levels,

even if in the normal range, may provide different and complementary information on

the presence and severity of CAD in patients with stable angina. Relative increase in

circulating hs-cTnT is a marker of the coronary atherosclerotic process being related

with the global atherosclerotic burden and with the type of atherosclerotic plaques. On

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the other hand, relative increase in circulating NT-proBNP is mainly associated with

inducible myocardial ischemia due to more severe coronary lesions and expresses a

subclinical LV dysfunction possibly consequent to the chronic ischemic process.

According to these results, the two biomarkers may have a relevant clinical role in the

screening process of patients with stable angina. Further studies are needed to define

whether these biomarkers can improve the accuracy of clinical and bio-humoral

models used to predict the presence, severity of CAD and outcome in patients with

stable angina. In particular, the combined use of hs-cTnT and NT-proBNP may be a

powerful tool to stratify these subjects prior to further clinical investigation and to

target personalized treatment.

As a future perspective, more information should be obtained to extend our present

understanding of patho-physiological mechanisms explaining the effects of the

different patterns of atherosclerotic disease on cardiac structure and endocrine

function. Methodological improvements could help in this direction by providing more

specific methods to distinguish intact non-degraded protein chain or proteolytic

troponin degradation products (50) as well as different active peptide BNP forms

instead of inactive peptide NT-proBNP (51).

Acknowledgements

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Roche Diagnostics International Ltd (Switzerland) kindly supplied all reagents and

calibrators used in the study for the measurement of hs-cTnT and NT-proBNP plasma

levels in the EVINCI population.

17

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Figure Legends

Figure 1.  Relation of hs-cTnT and NT-proBNP with extent and severity of CAD.

Box plots represent hs-cTnT and NT-proBNP levels stratified per CAD severity (A)

and number of plaques (B).

Figure 2. Effect of coronary atherosclerosis and inducible myocardial ischemia

on hs-cTnT and NT-proBNP plasma levels.

Circulating levels of hs-cTnT and NT-proBNP in patients subdivided according to: A)

the absence of both CAD and ischemia (“Normals” group), the presence of only CAD

(CAD), of only ischemia (Ischemia), and of both of them (CAD plus Ischemia) (A); B)

the presence of a perfusion defect in a territory subtended by a stenotic coronary artery

on CTA (matched), of all other combinations of pathologic findings (unmatched) at

hybrid imaging analysis.

Figure 3. Effect of coronary atherosclerosis and inducible myocardial ischemia

on LV function.

Figure 4. Schematic model illustrating the suggested mechanisms of hs-cTnT and

NT-proBNP increasing plasma levels in patients with stable angina.

At the site of the atherosclerotic lesions, dislodging and erosion processes could cause

micro-embolization of atherosclerotic and thrombotic material into the

microcirculation. This phenomenon can cause myocardial damage triggering the

30

apoptotic process with consequently release of hs-cTnT from cardiac myocytes into

peripheral circulation. Vice versa, increase in circulating NT-proBNP is mainly

associated with inducible myocardial ischemia due to more severe coronary lesions

and expresses a subclinical LV dysfunction possibly consequent to the chronic

ischemic process. These mechanisms could be the basis of the association of hs-cTnT

and NT-proBNP with cardiovascular mortality in patients with stable CAD.

31

Table 1. Baseline clinical and biohumoral characteristics.

Clinical variables

Low

hs cTnT

(n=188)

High

hs cTnT

(n=188)

P

value

Low

NT-proBNP

(n=188)

High

NT-proBNP

(n=188)

P

value

Demographics

Age, yr 57.7 ± 0.6 62.5 ± 0.6 <0.0001 57.4 ± 0.7 62.8 ± 0.6 <0.0001

Male sex 95 (42) 131 (58) 0.0001 128 (56.6) 98 (43.4) 0.0016

CV risk factors

Family history of CAD 70 (45.3) 58 (54.7) ns 70 (54.7) 58 (45.3) ns

Diabetes mellitus 30 (33.7) 59 (66.3) 0.0004 53 (59.5) 36 (40.5) 0.0392

Hypertension 101 (45.9) 124 (55.1) 0.0155 107 (47.6) 118 (52.4) ns

Hypercholesterolemia 116 (53) 103 (47) ns 111 (50.7) 108 (49.3) ns

Obesity 35 (44.9) 43 (55.1) ns 38 (48.7) 40 (51.3) ns

Smoking 48 (55.3) 42 (46.7) ns 49 (54.4) 41 (45.6) ns

LV Function

LVEF, % 60 [55-67] 60 [55-65] ns 62 [58.5-67] 60 [50-65] <0.0001

Medications

Beta-blockers 74 (49.3) 76 (50.7) ns 51 (34) 99 (66) <0.0001

Calcium antagonists 17 (38.6) 27 (61.4) ns 19 (43.2) 25 (56.8) ns

ARBs/ACE Inhibitors 60 (37.7) 99 (62.3) <0.0001 72 (42.3) 87 (54.7) ns

Diuretics 25 (39) 39 (61) ns 31 (48.4) 33 (51.6) ns

Nitrates 16 (43.2) 21 (56.8) ns 16 (43.2) 21 (50.8) ns

Anti-thrombotics 104 (46.4) 120 (53.6) ns 100 (44.6) 124 (55.4) 0.0117

Oral antidiabetics/Insulin 23 (31.5) 50 (68.5) 0.0004 40 (54.8) 33 (45.2) ns

Statins 99 (50.3) 98 (49.7) ns 93 (44.7) 104 (55.3) ns

Bio-humoral variables

Creatinine, mg/dL 0.82 [0.68-0.97] 0.86 [0.75-1.02] 0.0113 0.85 [0.72-1.00] 0.83 [0.73-0.98] ns

Glucose, mg/dL 101 [90.8-114] 105.0 [93-126] 0.0033 104 [102-120] 102 [92-115.3] ns

32

Total cholesterol, mg/dL 183 [147.3-220] 177[142.3-209.5] ns 185[1473.3-216] 171[142-214.8] ns

LDL cholesterol, mg/dL 103 [78-136] 99 [75-128] ns 107 [78-131.5] 96 [75-128.2] ns

HDL cholesterol, mg/dL 52 [41-65] 47 [39.3-58] 0.0087 49 [41-59] 50 [40-63] ns

Triglycerides, mg/dL 98 [70.3-147.5] 109 [77.3-156] 0.0440 113 [77.3-163] 98 [71.3-128.5] 0.0048

hs-CRP, mg/dL 0.13 [0.07-0.30] 0.19 [0.09-0.38] 0.0427 0.14 [0.07-0.36] 0.18 [0.09-0.34] ns

CK-MB, mg/dL 1.4 [1.0-2.1] 1.9 [1.3-2.9] <0.0001 1.5 [1.0-2.4] 1.7 [1.2-2.5] ns

hs-cTnT, ng/L 4.2 [3.0-5.2] 9.09 [7.3-12.6] <0.0001 5.67 [4.0-8.3] 6.75 [4.5-9.6] 0.0122

NT-proBNP, ng/L 55.3[28.05-106.3] 68.0 [36.5-168.3] <0.0001 31.2 [16.1-49.1] 132.6[87.2-206.4] <0.0001

Continuous variables are presented as mean ± standard error or median [25-75 percentile], categorical variables as absolute N and (%). ARB = Angiotensin Receptor Blockers; ACE = Angiotensin Converting Enzyme.

33

Table 2. Coronary imaging and inducible ischemia.

Low

hs cTnT

(n=188)

High

hs cTnT

(n=188)

P

value

Low

NT-proBNP

(n=188)

High

NT-proBNP

(n=188)

P

value

Coronary anatomy

Normals 80 (71.4%) 32 (28.6%)

<0.0001

67 (59.8%) 45 (40.2%)

0.0004

Patients with non-obstructive

CAD (<50%)65 (46.1%) 76 (53.9%) 77 (54.6%) 64 (45.4%)

Patients with obstructive

CAD (≥50%)43 (35%) 80 (65%) 44 (35.8%) 79 (64.2%)

Coronary plaques

Patients with calcified plaque 57 (41.9%) 79 (58.1%) 0.0182 66 (49.1%) 70 (50.1%) ns

Patients with mixed plaque 87 (39.2%) 135 (60.8%) <0.0001 103 (46.4%) 119 (53.6%) ns

Patients with non-calcified plaque 47 (44.3%) 59 (55.7%) ns 52 (49.1%) 54 (50.9%) ns

N. of calcified plaques 0.7 ± 0.12 1 ± 0.13 ns 0.9 ± 0.1 0.8 ± 0.1 ns

N. of mixed plaques 1.7 ± 0.19 3.3 ± 0.25 <0.0001 2.2 ± 0.22 2.8 ± 0.25 ns

N. of non-calcified plaques 0.4 ± 0.06 0.5 ± 0.07 ns 0.4 ± 0.06 0.5 ± 0.07 ns

Risk scores

CTA risk score 4.0 [0.0-15.0] 12.9 [5.6-23.1] <0.0001 7.2 [0.0-16.6] 11.8 [1.7-22.3] 0.0084

CAC score (n= 297) 6 [0-127] 78 [6-423] <0.0001 31 [0-202] 46 [0-273] ns

Myocardial ischemia

Patients with inducible ischemia

(any modality)37 (37.7%) 61 (62.3%) 0.0046 34 (34.7%) 64 (65.3%) 0.0004

Patients with inducible ischemia

(MPI, n=298)33 (37.1%) 56 (62.9%) 0.0209 29 (32.6%) 60 (67.4%) 0.0003

SDS at MPI (n=298) 2.76 ± 0.57 4.10 ± 0.65 0.0274 1.79 ± 0.39 5.06 ± 0.74 <0.0001

Continuous variables are presented as mean ± standard error or median [25-75 percentile], categorical variables as absolute N and (%).

34

Table 3. Predictive factors of hs cTnT and NT-proBNP plasma levels at

multivariate analyses.

Variables hs cTnT * NT-proBNP **

Coefficient (SE) P Coefficient (SE) P

Age 0.011 (0.003) 0.0016 0.036 (0.007) <0.0001

Sex 0.278 (0.057) <0.0001 - 0.391 (0.109) 0.0004

LVEF --- --- - 0.993 (0.349) 0.0047

CTA Risk Score 0.060 (0.023) 0.0089 --- ---

Presence of Ischemia --- --- 0.384 (0.120) 0.0015

* adjusted for medications significantly associated with hs cTnT at univariate analysis, including ACE inhibitors/ARBs and insulin/oral anti-diabetics.** adjusted for medications significantly associated with NT-proBNP at univariate analysis, including beta-blockers, ACE inhibitors/ARBs, anti-thrombotics, and statins. ARB = Angiotensin Receptor Blockers; ACE = Angiotensin Converting Enzyme.

35