Impact of Exercise Stress Testing on Diagnostic Gene

Expression in Patients With Obstructive

and Nonobstructive Coronary Artery Disease
David M. Filsoof, MDa, Robert E. Safford, MD, PhDa, Kristin Newby, MD, MHSb,
Steven Rosenberg, PhDc, Dana G. Kontras, RNa, Alice Baker, MPHc,
Olufunso W. Odunukan, MBBS, MPHa, and Gerald Fletcher, MDa,*
A blood-based gene expression test can diagnose obstructive coronary artery disease (CAD).
The test is sensitive to inammatory and immune processes associated with atherosclerosis.
Acute exercise engages short-term inammatory pathways, and exercise stress testing may
affect results of gene expression testing during the same diagnostic workup. The objective of
this study was to evaluate the effect of exercise on diagnostic gene expression testing. Ten
patients with obstructive CAD (50% stenosis) and 10 with no/minimal CAD (20% stenosis)
were identied by angiography. Blood samples for gene expression were obtained at baseline,
peak exercise, 30 to 60 minutes after testing, and 24 to 36 hours after testing. Core-lab gene
expression analysis yielded raw gene expression scores (GES) for each time point. Linear
models were used to estimate changes in GES, adjusting for CAD status and other covariates.
GES increased during peak exercise across both genders, with no signicant differences as a
function of CAD status. The overall adjusted mean GES increase at peak exercise was 0.29
(95% condence interval 0.22 to 0.36; p <0.001). GES after exercise were not signicantly
different from baseline. The change in gene expression levels during peak exercise may reect
a transient inammatory response to acute exercise that may be independent of patient
gender or CAD status. In conclusion, CAD GES increase at peak exercise testing and rapidly
return to baseline. Such may reect a transient inammatory response to acute exercise
independent of gender or extent of CAD. 2015 Elsevier Inc. All rights reserved. (Am J
Cardiol 2015;115:1346e1350)
A noninvasive, blood-based gene expression test has been
developed and validated for clinical use in evaluating the
presence and extent of obstructive CAD in symptomatic
patients.1e3 Exercise stress testing is frequently used in
clinical settings as a diagnostic procedure,4 but its effect on
diagnostic gene expression testing has not been evaluated.
Although regular exercise has anti-inammatory effects that
can reduce cardiovascular risk over time,5,6 acute exercise
has the paradoxical effect of triggering proinammatory
processes and oxidative stress.6 If exercise stress testing
precedes the blood draw for gene expression testing during
the same diagnostic workup, the gene expression results
could be affected by the proinammatory effects of acute
exercise. The impact of stress testing on gene expression may
depend on how much time elapses before the blood draw, and
it may also depend on patient-specic variables, such as
gender and degree of atherosclerosis. This study provides an
initial assessment of the effects of exercise stress testing on
diagnostic gene expression. Gene expression scores were
a
Division of Cardiovascular Diseases at Mayo Clinic, Jacksonville,
Florida; bDivision of Cardiology, Duke University, Durham, North Carolina; and cCardioDx, Redwood City, California. Manuscript received
October 22, 2014; revised manuscript received and accepted February 10,
2015.
See page 1350 for disclosure information.
*Corresponding author: Tel: (904) 953-7278; fax: (904) 953-2911.
E-mail address: etcher.gerald@mayo.edu (G. Fletcher).

0002-9149/15/$ - see front matter 2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.amjcard.2015.02.041

measured at baseline, peak exercise, and 2 time intervals after

stress testing to evaluate the magnitude and time course
of changes in gene expression. The study explores the contributions of CAD status and other clinical variables to
changes in gene expression, and it provides an exploratory
analysis of the specic genes and processes affected by acute
exercise.
Methods
This was a single-center, prospective pilot study. Patients
who had clinically indicated invasive coronary angiography
within 1 year of the study were identied and, after a discussion of risk, offered the opportunity to participate in a
clinical trial. Consenting subjects were divided into 2 study
groups. Each group had 5 men and 5 women, and the groups
were divided by the presence of obstructive (
50% stenosis
in at least 1 coronary artery) versus no/minimally obstructive CAD (20% stenosis in any coronary artery). Percent
stenosis was determined by standard visual inspection of
coronary angiographic images by the clinical angiographer.
Patients 45 to 75 years who were able to perform exercise
stress testing to >85% of maximal predicted heart rate were
eligible. Patients were ineligible for the study if they (a) had
severe CAD for which exercise testing would be unsafe;
(b) had current myocardial infarction or high-risk acute
coronary syndromes, New York Heart Association class III
to IV heart failure, severe regurgitant or stenotic valvular
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Coronary Artery Disease/Exercise Testing and Gene Expression

Table 1
Age, sex, and % stenosis on angiography
SEX/Age
F 47
F 50
F 52
F 58
F 59
F 60
F 61
F 67
F 69
F 74
M 46
M 56
M 56
M 58
M 62
M 63
M 63
M 63
M 65
M 66

%
>50%
0%
<15%
<15%
<15
>50%
0%
>75%
>50%
>50%
0%
0%
<20%
>50%
<20%
90%
90%
>50%
>50%
0%

lesions, severe left ventricular systolic dysfunction with left

ventricular ejection fraction <35%, active systemic infection,
protocol-specied rheumatologic or autoimmune conditions,
diabetes mellitus, and white blood cell count >11,000/ml;
(c) were recipients of an organ transplant; or (d) were taking
immunosuppressive therapies or chemotherapy or had major
surgery or blood transfusions within the previous 2 months.
Testing involved a 2-day process. On day 1, patients underwent exercise stress testing with a modied Bruce protocol,
and blood samples for gene expression testing were taken at 3
time points: baseline (before exercise), peak exercise during
stress testing (dened by 85% of maximal predicted heart
rate), and recovery (within 30 to 60 minutes after completion
of the stress test). Electrocardiogram (ECG), systolic and
diastolic blood pressures, and heart rate were obtained at each
time points. The presence or absence of ischemic electrocardigraphic response to exercise was determined according
to the standard stress testing criteria. On day 2, a nal blood
sample was taken for repeat gene expression testing and ECG,
blood pressure, and heart rate measurements.
For each blood sample, 2.5 cm3 of blood was drawn into
PAXgene Blood RNA tubes and shipped to the Clinical
Laboratory Improvements Amendmentsecertied laboratory
#05D1083624 (CardioDx Inc., Redwood City, California) for
gene expression testing during which RNA was extracted,
reverse transcribed, and quantied by real-time polymerase
chain reaction. Gene expression testing was quantied using
the Corus CAD (CardioDx, Inc.). Using an algorithm that
combines gender-specic gene expression components and
age functions, the assay results were converted into a raw score
(the primary metric used in the end point analyses of this
study). For clinical reporting purposes, the raw score is converted into a transformed gene expression score (GES) on a
scale of 1 to 40, as previously described.1 Higher GES are
associated with higher likelihood of obstructive CAD (dened
as
50% coronary artery stenosis per previous clinical validation studies).2,3 Changes in GES relative to baseline were

1347

measured at peak exercise, during recovery, and the day after

the exercise test. Exercise treadmill testing was conducted in
the Division of Cardiovascular Diseases at Mayo Clinic in
Jacksonville, Florida. All subjects in the nal study sample
consented to participate in the trial in accordance with the
Mayo Clinic Institutional Review Board policy.
Descriptive statistics were generated for baseline demographics, clinical characteristics, and clinical test measures.
Calculations using linear models, with the inclusion of CAD
status as a potential cofounder, were used to estimate effects of
the association between exercise and mean change in GES.
Separate models were performed for each time point of testing
(peak exercise, recovery, and delayed). Additional models
evaluated GES changes at peak exercise as a function of CAD
status and an additional covariate: baseline blood pressure,
baseline heart rate, rate pressure product, and exercise capacity. A linear model was used to evaluate the association
between changes in gene expression and the degree of electrocardigraphic ischemia. Linear models were also used to
test the association between peak exercise and changes in
expression of individual genes. All statistical analyses were
performed using R software, version 3.01, Vienna, Austria.7
The primary study end points were mean changes in GES
from baseline to peak exercise, recovery (30 to 60 minutes
after exercise), and the delayed sample (24 to 36 hours after
exercise). Changes in GES were evaluated for the study
population overall and for male and female study groups
separately. The contributions of several clinical variables to
changes in GES were also evaluated, including CAD status,
baseline heart rate and blood pressure, rate pressure product,
and exercise capacity. Secondary study end points were
changes in expression levels of the individual genes evaluated by the GES algorithm, comparing levels at peak exercise and baseline. Changes were measured for 21 of the 23
genes, omitting 2 that are used strictly for normalization.
Gene expression levels were measured by detection cutpoints during real-time polymerase chain reaction analysis.
Changes in gene expression were also evaluated for each
functional component of the GES algorithm.
Results
Tables 1 and 2 reect coronary angiogram data and
baseline GES for men and women. As previously reported,
baseline-transformed GES were higher for subjects with
obstructive CAD (mean GES 20.8, SD 7) than no/minimal CAD (mean GES 12.6, SD 10) and were higher for
men than women irrespective of CAD. Average baselinetransformed GES for men with no/minimal CAD and
obstructive CAD were 21.6 (SD 3.6) and 26.2 (SD 4.1)
and for women were 3.6 (SD 3.2) and 15.4 (SD 6.5).
There was a statistically signicant increase in GES from
baseline to peak exercise overall and when adjusted for
CAD status and gender (Table 2, Figure 1). The adjusted
mean increase in GES from baseline to peak exercise was
0.29 (95% condence interval 0.22 to 0.36; p <0.001) for
the entire study population, corresponding to a 3-point increase on the transformed GES scale (range 1 to 40).
Adjusted mean GES at both time points after exercise (30
to 60 minutes and 24 to 36 hours) were not signicantly
different from baseline levels. There was no association

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Table 2
Mean change in GES raw score during and after exercise stress testing*
Group

Overall
CAD status
Female
CAD status
Male
CAD status

20
10
10

Acute (during testing)

0.29**
0.12
0.34**
0.04
0.25**
0.21

Recovery (30-60 minutes post-testing)

(0.22, 0.36)
(-0.02, 0.27)
(0.22, 0.46)
(-0.20, 0.28)
(0.15, 0.34)
(0.02, 0.40)

-0.01 (-0.09,
-0.04 (-0.19,
-0.01 (-0.14,
-0.19 (-0.43,
-0.02 (-0.12,
0.14 (-0.10,

0.07)
0.12)
0.12)
0.06)
0.09)
0.33)

Delayed (24-36 hours post-testing)

-0.06 (-0.17,
0.14 (-0.07,
-0.07 (-0.22,
-0.02 (-0.33,
-0.06 (-0.22,
0.31 (-0.03,

0.05)
0.36)
0.09)
0.29)
0.11)
0.64)

**P <0.001.
CAD status coronary artery disease status (minimal vs. obstructive); GES gene expression score.
* Results of linear models evaluating the impact of exercise on gene expression at three time points of testing (acute, recovery, delayed), adjusting for CAD
status. The upper entry in each cell is the adjusted mean change in GES relative to baseline (mean, 95% condence interval). The lower entry is the estimated
difference in mean GES change, comparing patients with obstructive CAD vs. patients with minimal CAD. All analyses were performed using GES raw scores.

Figure 1. Change in GES raw score during and after exercise stress testing.
Figure 1 shows changes in GES raw score for each patient (n 5, each
group). Changes relative to baseline were measured at peak exercise
(acute), 30 to 60 minutes after exercise (recovery), and 24 to 36 hours after
exercise (delayed).

between severity of CAD status and overall statistically

signicant changes in mean GES at any time point.
Changes in individual gene expression levels at peak
exercise from baseline are represented in Figure 2 against
nominal (unadjusted) p values. Statistically signicant
changes (Bonferroni-Dunn adjusted p values <0.05) were
observed for 15 of 23 genes that comprise the GES algorithm
(Figure 2, Table 3). Signicant upregulation was observed for
2 genes, SLAMF7 and KLRC4, and downregulation was
observed for 13 genes. Component NKUP showed signicant
upregulation during exercise, and components NDOWN,
NEUT, and SCA1 showed signicant downregulation.
Discussion
The GES used in this study is a clinically useful tool to
gauge the presence or absence of obstructive CAD and may

Figure 2. Impact of acute exercise on gene expression. The x axis represents

the mean difference in RT-PCR values between acute exercise and baseline
for the overall population of 20 patients, adjusting for CAD status. A
positive value indicates that more PCR cycles were required to detect the
gene in the peak exercise blood samples compared with baseline, indicating
downregulation of the gene. A negative value indicates upregulation of the
gene. The y-axis is a log transformation of p-values. Genes above the
horizontal solid line demonstrated a statistically signicant change in gene
expression (p <0.05, adjusted for multiple testing). Cp cut-point in PCR
analysis; RT-PCR real-time polymerase chain reaction.

provide greater diagnostic power than traditional measures,

such as demographic factors, symptoms, medical history, risk
scores, or results of stress imaging.2e4 The diagnostic value of
gene expression testing has been previously demonstrated in 2
large multicenter validation studies, Personalized Risk Evaluation and Diagnosis in the Coronary Tree (PREDICT) and
Coronary Obstruction Detection by Molecular Personalize
Gene Expression (COMPASS).2,3 In this study, there was an
increase in GES during exercise stress testing in both genders,

Coronary Artery Disease/Exercise Testing and Gene Expression

1349

Table 3
Gene expression changes with acute exercise: By component and gene*
Algorithm term

Algorithm component

Genes

Associated cell types and processes

NUP

IL18RAP, TNFAIP6, CASP5

NDOWN

IL8RB, TNFRSF10C, TLR4, KCNE3

SCA1

S100A8, S100A12, CLEC4E

2M

NORM1

RPL28

2F

NEUT

NCF4, AQP9

NKUP

SLAMF7, KLRC4

BCELL

SPIB, CD79B

3,4

TCELL

TMC8, CD3D

Neutrophil activation and apoptosis (up-regulation)

Innate immune response to atherosclerosis
Neutrophil activation and apoptosis (down-regulation)
Innate immune response to atherosclerosis
Neutrophil activation
Innate immune response
Cellular necrosis
Lymphocyte activation
Cellular necrosis
Overall neutrophil activation
Innate immune response
Cellular necrosis
Natural killer cell activation
Innate immune response to atherosclerosis
B-cell activation
Adaptive immune response to atherosclerosis
T-cell activation
Adaptive immune response to atherosclerosis

5
6M
5/6

AF2
TSPAN
NORM2

AF289562
TSPAN16
TFCP2, HNRPF

* Components and genes in blue boldface showed statistically signicant up-regulation during exercise stress testing (P <0.05, adjusted for multiple
testing). Components and genes in green boldface italic showed statistically signicant down-regulation. GES algorithm terms and components are dened by
Elashoff et al.8

independent of CAD status, followed by a return to baseline

levels within 30 to 60 minutes after exercise testing. Because
effects of exercise stress testing on GES are transient, both
exercise stress testing and GES can be conducted during the
same diagnostic workup without affecting results of GES.
The GES used in this study uses 23 genes that are
commonly reected in CAD. Some genes in the algorithm
contribute to a higher score (higher likelihood of CAD)
through increased expression and upregulation, whereas others
contribute through decreased expression.1 For example, 2
genes related to natural killer cell activation, SLAMF7 and
KLRC4, showed upregulation during stress testing, qualitatively similar to that seen in the presence of atherosclerosis
(Table 3, Figure 2).1 These genes encode receptors that activate
natural killer cells and regulate the innate immune response to
atherosclerosis and other inammatory conditions.9,10 Three
genes associated with algorithm component NDOWN
(IL8RB, TNFRSF10C, and TLR4) showed downregulation
during stress testing, consistent with decreased expression in
obstructive CAD.1,8 These genes reect neutrophil apoptosis
and inammatory signaling responses of the innate immune
system. Most of the genes associated with components NUP,
SCA1, and NEUT showed decreased expression in this study
(Table 3). All these genes are involved in neutrophil activation
as part of the innate immune response, and they tend to show
increased expression in the presence of atherosclerosis and
chronic inammatory conditions.1,11,12 In general, exercise
stress testing was associated with a reduction in expression
of most neutrophil-associated genes and no signicant changes
in expression for B-cell and T-cell activationa pattern very
different from that associated with atherosclerosis.
Changes in GES during acute exercise stress testing may
reect physiological responses independent on the presence of

CAD. Inammation and oxidative stress increase in response

to acute exercise,5,6 which may help explain increases in gene
expression. Increased oxygen consumption during acute
exercise triggers an inammatory response characterized by
production or increased levels of reactive oxygen species,
markers of low-density lipoprotein oxidation, and proinammatory biomarkers, such as TNF, IL-1b, and CRP.5,13,14
The effects of acute exercise on gene expression may vary
depending on whether the subject is routinely active.15 Regular exercise and physical tness are associated with an
overall reduction in cardiovascular risk. This effect may be
mediated in part by long-term changes in gene expression,
contributing to a reduction in proinammatory pathways over
time.5,6,16 In effect, the inammatory response observed in
acute exercise can be reduced over time through repeated
exercise, shifting gene expression toward anti-inammatory
pathways that protect against atherosclerosis.14 For example,
CD36 gene expression is downregulated with regular exercise, which reduces the activity of monocytes that scavenge
oxidized low-density lipoprotein and protects against the
formation of atherosclerotic plaque.17 In addition, a recent
study of global blood gene expression in response to intensive
cardiovascular risk reduction, including exercise, showed
signicant changes in gene expression.18
The different responses of gene expression in acute exercise and atherosclerosis suggest that the 2 physiological
processes may engage different patterns of inammatory and
oxidative stress response. Acute exercise engages short-term
inammatory responses to oxygen depletion and muscle
injury,6 whereas atherosclerosis engages long-term processes
that contribute to and respond to the formation of plaque.6,10
However, long-term changes in inammatory processes
reduce the likelihood of plaque formation.5,6 The long-term

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The American Journal of Cardiology (www.ajconline.org)

cardioprotective effects of exercise may be mediated by

sustained changes in gene expression. These possibilities
provide a rich territory for future research on the effects of
exercise on gene expression, both short term and long term.
Larger study samples will permit more precise assessments
of the role of CAD status, the timing of exercise recovery,
and the biologic processes associated with changes in gene
expression with acute exercise.
Disclosures
This study was funded by Cardiodx. The authors have no
conicts of interest to disclose.
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