Journal of the American College of Cardiology Vol. 51, No.

1, 2008
© 2008 by the American College of Cardiology Foundation ISSN 0735-1097/08/$34.00
Published by Elsevier Inc. doi:10.1016/j.jacc.2007.07.085

Atrial Fibrillation and Cardiac Surgery

Association of Atrial Nicotinamide Adenine

Dinucleotide Phosphate Oxidase Activity With the
Development of Atrial Fibrillation After Cardiac Surgery
Young M. Kim, BSC,* Hassan Kattach, MRCS,† Chandi Ratnatunga, FRCS,† Ravi Pillai, FRCS,†
Keith M. Channon, MD, FRCP* Barbara Casadei, MD, DPHIL, FRCP*
Oxford, United Kingdom

Objectives Our goal was to evaluate the role of myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase
activity and plasma markers of oxidative stress in the pathogenesis of post-operative atrial fibrillation (AF).

Background Atrial fibrillation is a common complication of cardiac surgery, leading to increased morbidity and prolonged
hospitalization. Experimental evidence suggests that oxidative stress may be involved in the pathogenesis of AF;
however, the relevance of this putative mechanism in patients undergoing cardiac surgery is unclear.

Methods We measured basal and NADPH-stimulated superoxide production in right atrial appendage samples from 170
consecutive patients undergoing conventional coronary artery bypass surgery. Plasma markers of lipid and pro-
tein oxidation (thiorbabituric acid-reactive substances, 8-isoprostane, and protein carbonyls) were also measured
in blood samples drawn from a central line before surgery and after reperfusion.

Results Patients who developed AF after surgery (42%) were older and had a significantly increased atrial NADPH oxi-
dase activity than patients who remained in sinus rhythm (SR) (in relative light units/s/␮g protein: 4.78 ⫾ 1.44
vs. 3.53 ⫾ 1.04 in SR patients, p ⬍ 0.0001). Plasma markers of lipid and protein oxidation increased signifi-
cantly after reperfusion; however, neither pre-operative nor post-operative measurements differed between pa-
tients who developed AF and those who remained in SR after surgery. Multivariate analysis identified atrial
NADPH oxidase activity as the strongest independent predictor of post-operative AF (odds ratio 2.41; 95% confi-
dence interval 1.71 to 3.40, p ⬍ 0.0001).

Conclusions Atrial NADPH oxidase activity is independently associated with an increased risk of post-operative AF, suggesting
that this oxidase system may be a key mediator of atrial oxidative stress leading to the development of AF after
cardiac surgery. (J Am Coll Cardiol 2008;51:68–74) © 2008 by the American College of Cardiology Foundation

Atrial fibrillation (AF) is a frequent complication of most
types of cardiac surgery, occurring in 35% to 50% of patients
within the first 2 to 5 post-operative days (1,2). Post-
operative AF can induce hemodynamic deterioration, in-
crease the risk of stroke, and is invariably associated with
prolonged hospitalization and a significant additional cost
to patient care (1,2). Little is known of the mechanisms
underlying post-operative AF, although emerging evidence
indicates that inflammation and oxidative stress associated
with cardiac surgery and cardiopulmonary bypass (CPB)
From the *University Department of Cardiovascular Medicine and the †Department
of Cardiothoracic Surgery, John Radcliffe Hospital, Oxford, United Kingdom. Young
Kim received a scholarship from the Wellcome Trust Cardiovascular Research
Initiative, a Clarendon Award, and the Mary Goodger Award from the University of
Oxford. Drs. Casadei and Channon are funded by the British Heart Foundation and
the Garfield Weston Trust.
Manuscript received April 23, 2007; revised manuscript received July 16, 2007,
accepted July 24, 2007.
may play an important role (3– 6). It has recently been
reported that myocardial oxidative injury leads to impaired
atrial contraction, altered myofibrillar energetics (7), and a
reduction in the atrial effective refractory period (AERP)
(8), which, in turn, increases the vulnerability to AF (9).
See page 75
Importantly, short-term treatment with high doses of anti-
oxidant and anti-inflammatory agents attenuates AERP
shortening and the vulnerability to AF in animal models of
rapid atrial pacing (8,10,11), suggesting that atrial electro-
physiological remodeling leading to AF may be prevented
by inhibiting the myocardial formation of reactive oxygen
species (ROS).
An increasing body of experimental evidence indicates
that formation of the ROS, superoxide, by cardiovascular
nicotinamide adenine dinucleotide phosphate (NADPH)
JACC Vol. 51, No. 1, 2008 Kim et al. 69
January 1/8, 2008:68–74 Atrial NADPH Oxidase Activity and Post-Operative AF

oxidases plays a critical role in the development of hyper- study. Exclusion criteria were a Abbreviations
tension, myocardial hypertrophy, and heart failure (12). We preceding history of AF or and Acronyms
have recently reported that human atrial myocytes have the other atrial arrhythmias, con-
AERP ⴝ atrial effective
capacity to produce ROS via a phagocytic-type NADPH comitant valvular disease, and refractory period
oxidase, and that NADPH-stimulated superoxide produc- the use of anti-arrhythmic AF ⴝ atrial fibrillation
tion is increased in patients with sustained or paroxysmal drugs other than beta-blockers.
CABG ⴝ coronary artery
AF (13). Nicotinamide adenine dinucleotide phosphate The demographic and clinical bypass grafting
oxidase activity is potently stimulated by cytokines (12), characteristics of the patients CPB ⴝ cardiopulmonary
suggesting that this oxidase system may be an important are shown in Table 1. Patients bypass
link between the systemic inflammatory response to cardiac were asked to withdraw treat- LVEF ⴝ left ventricular
surgery and myocardial oxidative stress. ment with aspirin 3 days before ejection fraction
Taken together, these data suggest that atrial NADPH surgery. Similar surgical and NADPH ⴝ nicotinamide
oxidase activity may be a more sensitive predictor of ROS- anesthetic protocols were fol- adenine dinucleotide
mediated myocardial electrophysiological changes leading to lowed in all patients. All phosphate

post-operative AF than plasma markers of oxidative stress. CABG procedures were per- NO ⴝ nitric oxide
To test this hypothesis, we prospectively evaluated the formed using the on-pump RAA ⴝ right atrial
relationship between ROS production in the right atrial technique. Post-operatively, appendage

myocardium, plasma markers of protein and lipid oxidation, heart rate and rhythm were RLU ⴝ relative light units
and the occurrence of post-operative AF in 170 consecutive continuously monitored for the ROS ⴝ reactive oxygen
patients who underwent first-time elective coronary artery first 48 h and at 4-h intervals species

bypass grafting (CABG) surgery. thereafter for 5 days. Post- SR ⴝ sinus rhythm
operative AF was defined as the TBARS ⴝ thiorbabituric
Methods characteristic arrhythmia last- acid-reactive substances
ing for at least 30 s. The study
One hundred seventy consecutive patients undergoing was approved by the local re-
first-time elective CABG surgery were recruited in this search ethics committee, and all patients gave written
Clinical and Demographic Characteristics informed consent.
Table 1 Clinical and Demographic Characteristics Measurements of superoxide production in right atrial
appendage (RAA) homogenates. A sample of the RAA
SR (n ⴝ 99) AF (n ⴝ 71) p Value
was obtained in all patients at the time of venous
Pre-operative
Male gender (%) 75 (76) 57 (79) 0.68
cannulation, before the initiation of CPB. Superoxide
Age (yrs) 67 ⫾ 7 69 ⫾ 7 0.04
production was measured in homogenized RAA tissue by
Hypertension, n (%) 66 (67) 49 (68) 0.92 lucigenin-enhanced chemiluminescence using a single
Diabetes mellitus, n (%) 23 (23) 20 (28) 0.52 tube luminometer (Berthold FB12, Berthold, Pforzheim,
Family history of CVD, n (%) 51 (52) 37 (51) 0.93 Germany) modified to maintain the sample temperature
History of CHF, n (%) 12 (12) 9 (13) 0.91 at 37oC, as described and validated previously (13).
Previous MI, n (%) 46 (47) 35 (49) 0.83 Briefly, basal chemiluminescence from RAA homoge-
Current smokers, n (%) 13 (13) 7 (10) 0.48 nates was measured after subtraction of the background
History of COPD, n (%) 10 (10) 7 (10) 0.96 chemiluminescence of the lucigenin (5 ␮ mol/l)-
Aspirin, n (%) 82 (84) 62 (86) 0.65
containing buffer. Further measurements were taken after
application of NADPH (100 ␮mol/l). A single investi-
Beta-blockers, n (%) 73 (74) 55 (76) 0.78
Diuretics, n (%) 23 (23) 24 (33) 0.15
Nitrates, n (%) 58 (59) 45 (62) 0.66
gator blinded to the patients’ post-operative outcome
ACEI/ARB, n (%) 62 (63) 50 (69) 0.40
performed all measurements and analyses. Three basal
CCB, n (%) 39 (40) 24 (33) 0.39 and NADPH-stimulated measurements of superoxide
Statins, n (%) 89 (91) 67 (93) 0.60 production were obtained and averaged for each patient.
Intra-operative Because it is well-established that high concentrations
CPB, min 61 ⫾ 18 62 ⫾ 25 0.72 of lucigenin ⱖ20 ␮mol/l) are subject to redox cycling, we
ACC, min 30 ⫾ 11 30 ⫾ 8 0.99 (13) have previously validated basal and NADPH-
Number of grafts 3⫾1 3⫾1 0.93 stimulated superoxide measurements in human atrial
Post-operative samples obtained by using 5 ␮mol/l lucigenin with those
Inotropic support, n (%) 40 (40) 35 (49) 0.40
obtained by using the luminol analogue L-012 (in the
Start of ACEI/ARB treatment (day) 3.6 ⫾ 2.7 3.8 ⫾ 2.8 0.75
presence of ebselen) and the superoxide dismutase-
Start of beta-blocker treatment (day) 2.5 ⫾ 1.7 3.3 ⫾ 2.7 0.05
inhibitable ferricytochrome c reduction, respectively. In
ACC ⫽ aortic clamp time; ACEI ⫽ angiotensin-converting enzyme inhibitor; AF ⫽ atrial fibrillation; both cases measurements were closely correlated with
ARB ⫽ angiotensin II receptor blocker; CCB ⫽ calcium-channel blocker; CHF ⫽ congestive heart
failure; COPD ⫽ chronic obstructive pulmonary disease; CPB ⫽ cardiopulmonary bypass time;
those obtained in parallel by lucigenin-enhanced chemi-
CVD ⫽ cardiovascular disease; MI ⫽ myocardial infarction; SR ⫽ sinus rhythm. luminescence (basal measurements: r ⫽ 0.88, p ⬍ 0.005,
70 Kim et al. JACC Vol. 51, No. 1, 2008
Atrial NADPH Oxidase Activity and Post-Operative AF January 1/8, 2008:68–74

n ⫽ 8; NADPH-stimulated: r ⫽ 0.99, p ⬍ 0.0001, n ⫽ AF was most common on the second and third post-
11). Specificity of this measurement for superoxide was operative days. No significant differences between groups
confirmed by data showing near abolition of the chemi- were found with respect to treatment, cardiovascular risk
luminescence signal in the presence of specific scavengers factors and co-morbidity, duration of CPB or aortic cross-
such as superoxide dismutase or tiron both in right atrial clamp, and number of grafted vessels (Table 1). However,
homogenates and in isolated atrial myocytes (13). patients who developed post-operative AF were slightly
Plasma markers of oxidative stress. Fasting blood samples older and had a higher basal and NADPH-stimulated
were drawn from a central line immediately after the superoxide production than those who remained in SR
induction of anesthesia and 10 min after the administration (Fig. 1). In-hospital post-operative treatment with beta-
of protamine in 124 patients. blockers was initiated in a similar percentage of patients from
Thiorbabituric acid-reactive substances (TBARS), a each group (i.e., in 63% of SR patients and in 69% of patients
marker of lipid peroxidation, were measured in plasma using who developed AF, p ⫽ 0.43); however, these agents tended to
a previously described fluorometric technique (14,15). Co- be started slightly later in patients who developed AF after
efficients of variation for within and between runs were 4% surgery (Table 1). There was no difference in the post-
and 7%, respectively. Each sample was run in triplicate. operative use of angiotensin-converting enzyme inhibitors or
Hemodilution in post-operative samples was adjusted using angiotensin II receptor blockers (i.e., these agents were initi-
a correction factor as described previously (16). Plasma ated in 67% of patients who remained in SR and 68% of
protein carbonyl levels (a marker of protein oxidation) were patients who developed AF, p ⫽ 0.41) or in the time of
evaluated using an enzyme-linked immunoadsorbent assay initiation of treatment after surgery (Table 1).
method (17) and 8-isoprostane levels using a commercial Plasma levels of TBARS and protein carbonyls increased
enzyme immunoassay kit (Cayman Chemical, Ann Arbor, significantly after reperfusion (Fig. 2), reflecting the increase
Michigan). Carbonyl content was calculated from the stan-
dard curve (prepared using oxidized and reduced bovine
serum albumin) and expressed as nmol carbonyl per mg of
immunoglobulin. Adjusted (16) total (free and esterified)
8-isoprostane levels in purified plasma samples taken after
CPB and reperfusion are given in pg/ml. All measurements
were carried out by a single investigator blinded to the
patients’ post-operative outcome.
Statistical analysis. Differences between patients who
developed AF and those who maintained sinus rhythm
(SR) in the post-operative period were evaluated with a t
test or Wilcoxon rank sum test for continuous variables
and a chi-square test for categorical variables. To identify
the subset of variables that predicted the occurrence of
post-operative AF, we entered our variables in a logistic
regression model using block entry of variables (SPSS
13.0 for Windows, SPSS Inc., Cary, North Carolina).
Criteria for entry and exclusion from this multivariate
analysis were p ⬍ 0.05 and p ⱖ 0.10, respectively;
however, variables had been shown to affect the occur-
rence of post-operative AF (i.e., use of beta-blockers or
inhibitors of the renin-angiotensin system [2] or the
activity of the cardiovascular NADPH oxidase, i.e., diabetes
mellitus [18] and treatment with statins [19]) were also entered
into the model. Only main effects (i.e., no interaction terms)
were fitted in this model. The association of atrial NADPH
oxidase activity with post-operative AF was also assessed by Figure 1 Basal and NADPH-Stimulated Superoxide Production
categorization of the patients’ cohort into NADPH oxidase
activity quartiles. Data are presented as mean ⫾ SD or as Box plots of basal and nicotinamide adenine dinucleotide phosphate (NADPH)-
stimulated superoxide production from right atrial appendage homogenates of
percent of the total (%). patients who remained in sinus rhythm (SR) (n ⫽ 99) and those who devel-
oped atrial fibrillation (AF) post-operatively (n ⫽ 71). The difference in basal
superoxide production between SR and AF patients did not quite reach statisti-
Results cal significance (p ⫽ 0.07); however, NADPH-stimulated superoxide production
was significantly greater in patients who developed AF in the post-operative
Seventy-one patients (42%) developed AF after CABG period (*p ⬍ 0.0001). RLU ⫽ relative light units.
surgery, and, as reported previously (1), the occurrence of
JACC Vol. 51, No. 1, 2008
January 1/8, 2008:68–74
Plasma Levels of TBARS and Protein Carbonyls PRE
Figure 2
and POST Cardiopulmonary Bypass and Reperfusion
Box plots of plasma levels of thiorbabituric acid-reactive substances (TBARS)
and protein carbonyl before (PRE) and after (POST) cardiopulmonary bypass
and reperfusion in patients who remained in SR (n ⫽ 71) and in those who
developed AF post-operatively (n ⫽ 53). Thiorbabituric acid-reactive substances
and protein carbonyls increased significantly after reperfusion (*p ⬍ 0.05 vs.
PRE for both); however, these markers did not differ between patients who
developed AF versus those who remained in SR. Abbreviations as in Figure 1.
in systemic oxidative stress associated with cardiac surgery.
However, neither pre-operative nor post-operative plasma
levels of TBARS or protein carbonyl differed between
patients who developed AF and those who remained in SR
after surgery (Fig. 2). Equally, the perioperative increase in
these plasma markers of oxidative stress was not signifi-
cantly associated with the occurrence of post-operative AF (p
⫽ 0.31 for TBARS and p ⫽ 0.45 for protein carbonyls).
Similar findings were obtained when post-operative plasma
levels of 8-isoprostane were compared (28.5 ⫾ 2.9 pg/ml in
patients who developed AF vs. 28.0 ⫾ 3.8 pg/ml in patients
who remained in SR, p ⫽ 0.86). There was no correlation
between myocardial basal or NADPH-stimulated superoxide
production and plasma markers of oxidative stress (not shown).
Atrial NADPH-stimulated superoxide release increased
with age (r ⫽ 0.20, p ⫽ 0.009) but was not significantly
affected by pre-operative treatment with statins or inhibitors
of the angiotensin-converting enzyme/angiotensin II recep-
tors (p ⫽ 0.36 and p ⫽ 0.81, respectively), by left ventricular
ejection fraction (LVEF) (p ⫽ 0.52), or by the diagnosis of
diabetes mellitus (in relative light units [RLU]/s/␮g: 4.23 ⫾
1.62 in patients with diabetes vs. 3.99 ⫾ 1.28 in nondiabetic
Kim et al. 71
Atrial NADPH Oxidase Activity and Post-Operative AF
patients, n ⫽ 43 and 127, respectively, p ⫽ 0.33) or history
of congestive heart failure (p ⫽ 0.89). In contrast, basal
superoxide production from the RAA increased with the
severity of left ventricular dysfunction: a) 0.064 ⫾ 0.03
RLU/s/␮g in patients with an LVEF ⬎50%; b) 0.072 ⫾
0.03 RLU/s/␮g in patients with an LVEF between 30% to
50%; and c) 0.089 ⫾ 0.05 RLU/s/␮g in patients with an
LVEF ⬍30%, n ⫽ 83, 65, and 22, respectively; p ⫽ 0.002
for a vs. c and p ⫽ 0.04 for b vs. c) and was significantly
higher in patients with a clinical history of congestive heart
failure (0.068 ⫾ 0.03 RLU/s/␮g vs. 0.086 ⫾ 0.05 RLU/
s/␮g in patients with a history of congestive heart failure,
n ⫽ 149 and 21, respectively, p ⫽ 0.02).
Multivariate analysis applied in a model-selection proce-
dure identified myocardial NADPH-stimulated superoxide
production as the most important independent predictor of
post-operative AF in our patients (odds ratio 2.41; 95%
confidence interval 1.71 to 3.40, p ⬍ 0.0001) (Table 2); age
was no longer associated with an increased risk of post-
operative AF after the addition of NADPH-stimulated
superoxide production to the model. Characterization of our
population according to quartiles of atrial NADPH-
stimulated superoxide confirmed a strong association be-
tween these measurements and the occurrence of post-
operative AF (Table 3).
Discussion
This is the first prospective study to compare the potential
value of myocardial and systemic markers of oxidative stress
in predicting the occurrence AF after CABG surgery. Our
findings clearly show that NADPH-stimulated superoxide
production in the RAA is independently associated with an
increased risk of post-operative AF. Plasma markers of
oxidative stress increased significantly after CPB and reper-
fusion; however, neither pre-operative nor post-reperfusion
levels of plasma TBARS, protein carbonyl, or 8-isoprostane
discriminated between patients who developed AF and
those who maintained SR in the post-operative period.
These findings indicate that ROS production in the atrial
myocardium may be a more accurate indicator of myocardial
oxidative stress and its functional correlates (e.g., reduction
in AERP and arrhythmogenesis) (8,10,11) than systemic
markers of oxidative stress and suggest that targeting tissue-
Multivariate Analysis
Table 2 Multivariate Analysis
OR 95% CI p Value
Age (yrs) 1.03 0.98–1.08 0.30
Diabetes (no/yes) 0.77 0.33–1.73 0.52
Post-operative beta-blockers (no/yes) 0.70 0.33–1.50 0.36
ACEI/ARB (no/yes) 0.65 0.30–1.39 0.27
Statins (no/yes) 1.78 0.50–6.44 0.38
NADPH-stimulated superoxide (RLU/s/␮g) 2.41 1.71–3.40 ⬍0.00001
Post-operative beta-blockers refers to patients who received treatment with beta-blockers before
discharge. ACEI/ARB refers to patients who received treatment with ACEI or ARB before surgery.
CI ⫽ confidence intervals; NADPH ⫽ nicotinamide adenine dinucleotide phosphate; OR ⫽ odds
ratio; other abbreviations as in Table 1.
72 Kim et al.
Atrial NADPH Oxidase Activity and Post-Operative AF
Occurrence
to Quartiles of
of Post-Operative
Atrial NADPH Oxidase
AF According
Activity
Occurrence of Post-Operative AF According
Table 3
to Quartiles of Atrial NADPH Oxidase Activity
Quartiles of NADPH Oxidase Activity
First Second Third
Post-operative AF, n (%) 5 (12) 15 (36) 23 (53)
Quartiles of NADPH oxidase activity (in RLU/s/␮g): first, 1.77 to 3.17; second, 3.18 to 3.81; third,
3.82 to 4.76; fourth, 4.80 to 9.84.
AF ⫽ atrial fibrillation; NADPH ⫽ nicotinamide adenine dinucleotide phosphate; RLU ⫽ relative
light units.
specific sources of superoxide production may prove a more
successful therapeutic strategy than systemic antioxidant
interventions.
Oxidative stress and AF. Myocardial oxidative damage is
associated with atrial electrophysiological remodeling and
higher inducibility of AF in animal models of rapid atrial
pacing, both of which are prevented by treatment with high
doses of antioxidant or anti-inflammatory agents (8,10,11).
These findings are not easily reconciled with the disappoint-
ing results of antioxidant vitamins trials in humans. Poor
patient selection and monitoring of the antioxidant effects of
treatment have been implicated in the therapeutic failure of
antioxidant agents in humans (20). However, it is equally
plausible that interventions that are at least partially effective
in reducing plasma markers of lipid and protein oxidation in
humans may not lead to a significant reduction in oxidative
injury in the tissue of interest. This hypothesis is supported
by increasing evidence indicating that both myocardial and
vascular tissue contain constitutively active and highly mod-
ulated oxidase systems and that local ROS production plays
an important role in the pathogenesis of common myocar-
dial disease states (12). Importantly, controlled clinical trials
have recently demonstrated that interventions that prevent
activation of NADPH oxidases by vasoactive hormones or
inflammatory cytokines (i.e., angiotensin II receptor block-
ers [12], statins [21], or steroids [22]) are also highly
effective in preventing the occurrence of AF in hypertensive
subjects (23) and in patients after cardiac surgery (24,25).
Sources of ROS in the human myocardium. Nicotin-
amide adenine dinucleotide phosphate oxidase–dependent
ROS production in the myocardium or vessel wall has been
implicated in the pathogenesis of a wide array of cardiovas-
cular diseases (12), including paroxysmal or chronic AF
(13). As excess ROS production may lead both to nitric
oxide (NO) scavenging (and the production of the powerful
pro-oxidant molecule peroxynitrite) and to ‘uncoupling’ of
NO synthases (which causes these enzymes to produce
superoxide rather than NO [26]), it remains unclear to
which extent the pathophysiological correlates of increased
myocardial superoxide production are due to direct oxida-
tion of key proteins substrates or to a reduction in myocar-
dial NO bioavailability.
We have previously reported that a membrane-bound
NOX2-containing NADPH oxidase is the main source of
ROS production in the human atrial myocardium and isolated
atrial myocytes (13). Here we show that atrial NADPH
JACC Vol. 51, No. 1, 2008
January 1/8, 2008:68–74
oxidase activity is an independent predictor of the occurrence
of post-operative AF. It could be argued that measurements in
RAA samples obtained after reperfusion might have reflected
perioperative myocardial oxidative injury more accurately.
Fourth p for Trend However, NADPH oxidases have not been convincingly
28 (67) ⬍0.00001
implicated with the oxidative stress that accompanies ischemia
and reperfusion (27). Indeed, our preliminary data show no
significant difference in NADPH oxidase activity in RAA
samples obtained before CPB and shortly after reperfusion in
the same cohort of patients (unpublished data, Y. M. Kim,
May 2005). In contrast, several vasoactive substances (e.g.,
angiotensin II, endothelin I) and inflammatory mediators (e.g.,
interleukin-6 and tumor necrosis factor-alpha) that are released
in response to cardiac surgery and CPB are now known to be
potent activators of cardiovascular NADPH oxidases (12).
Thus, the perioperative inflammatory response may have a
major role in stimulating atrial NADPH oxidase activity,
which, in turn, would increase myocardial oxidative stress
leading to electrophysiological changes (i.e., shortening of the
AERP [8]) that facilitate the new onset of AF in the early
post-operative period. In other words, atrial NADPH oxidase
may serve as an important link between systemic inflammation
and tissue oxidative injury. In agreement with this hypothesis,
basal superoxide measurements did not differ between patients
who maintained SR and those who developed AF after
surgery. Whereas superoxide production in response to
NADPH reflects the stimulated NADPH oxidase activity,
basal superoxide release results from the background activity of
several oxidase systems and as such is less likely to bear a
relationship with the inflammation-driven post-operative
myocardial oxidative stress.
We considered that a lower signal-to-noise ratio may
have contributed to our inability to detect significant differ-
ences in basal superoxide measurements between SR and
post-operative AF patients; however, the use of a single vial
luminometer (modified to maintain our samples at a phys-
iological temperature) increases the sensitivity of this tech-
nique and largely circumvents the problems in detecting low
levels of superoxide that are encountered when using stan-
dard plate luminometers. Our observation that basal— but
not NADPH-stimulated—atrial superoxide production in-
creased proportionally with the severity of left ventricular
dysfunction suggests that these measurements may reflect
different pathophysiological mechanisms.
It is possible that atrial stretch and remodelling may affect
the activity of NADPH oxidases, which, in turn, may
contribute to the recently reported association between
atrial volume and post-operative AF (28). Unfortunately,
because the majority of patients who have cardiac surgery in
our institution have their pre-operative assessment (e.g.,
coronary angiogram and echocardiography) elsewhere, we
did not have access to accurate echocardiographic measure-
ments of atrial volume; thus, the relationship between atrial
size, NADPH oxidase activity, and post-operative AF could
not be investigated.
JACC Vol. 51, No. 1, 2008
January 1/8, 2008:68–74
Plasma markers of lipid and protein oxidation increased
significantly shortly after reperfusion, in agreement with the
notion that cardiac surgery and CPB increase systemic
oxidative stress; however, neither marker predicted post-
operative AF. More robust indexes of lipid peroxidation,
such as the determination of urinary levels of F2-
isoprostanes by gas chromatography/mass spectrometry
(29), might have provided a more accurate assessment of
systemic oxidative stress in our patients; however, perform-
ing these complex and time-consuming measurements on a
large scale is impractical and unlikely to detect myocardial
oxidative stress as accurately as measurements of atrial
NADPH oxidase activity. In agreement with this hypoth-
esis, we found no significant correlation between measure-
ments of atrial superoxide production and plasma markers
of oxidative stress (not shown).
Conclusions
We showed that atrial NADPH oxidase activity is indepen-
dently associated with an increased risk of AF in patients
undergoing conventional CABG surgery, whereas the post-
operative rise in plasma markers of protein and lipid
oxidation bears no association with this outcome. Our data
suggest that preventing the activation of cardiovascular-
specific oxidases may be more effective in averting myocar-
dial oxidative injury and its functional consequences than
the systemic administration of antioxidant vitamins. Testing
this hypothesis may provide an important key for under-
standing the role of oxidative stress in human cardiovascular
disease.
Acknowledgments
The authors would like to thank Dr. Philip E. James and
Mrs. Joan Parton from the Wales Heart Research Institute,
Cardiff University School of Medicine for helpful discus-
sions on measurements of reactive species and oxidative
stress and for carrying out the thiorbabituric acid-reactive
substances, 8-isoprostane, and protein carbonyl measure-
ments. The authors are grateful to Professor Martin Farrall
(Department of Cardiovascular Medicine, University of
Oxford) for his invaluable help with the statistical analysis of
the data.
Reprint requests and correspondence: Dr. Barbara Casadei,
University Department of Cardiovascular Medicine, John Radcliffe
Hospital, Oxford, OX3 9DU, United Kingdom. E-mail:
barbara.casadei@cardiov.ox.ac.uk.
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