Background NOX2, the catalytic subunit of NADPH oxidase, is suggested to play a role in favouring the occurrence of atrial fibrillation (AF) after cardiac surgery via formation of reactive oxidant species. However, its role in spontaneous AF is still unclear.
Objective To define the role of NOX2 and isoprostanes, a marker of oxidative stress, in the different settings of AF.
Methods The study was performed on 174 patients with AF (82 with paroxysmal/persistent AF and 92 with permanent AF) and 90 controls matched for sex, age and atherosclerotic risk factors. Urinary isoprostanes and serum levels of soluble NOX2-derived peptide (sNOX2-dp) were measured in each patient.
Results Urinary isoprostanes and sNOX2-dp concentrations were significantly higher in patients with paroxysmal/persistent AF than in those with permanent AF and controls. Compared with controls, patients with permanent AF showed a weak increase in sNOX2-dp and no difference in isoprostanes. Multivariable analyses demonstrated that baseline values of sNOX2-dp and urinary isoprostanes were independently associated with the type of AF (paroxysmal/persistent vs permanent; β=−224, p=0.007 and β=−231, p=0.005, respectively). A significant correlation between sNOX2-dp levels and urinary excretion of isoprostanes was also detected (R=0.707, p<0.001).
Conclusions This study provides evidence that NOX2 is upregulated only in patients with paroxysmal/persistent AF and is responsible for overproduction of isoprostanes. This finding warrants further study to see if inhibition of NOX2 may reduce the risk of paroxysmal/persistent AF.
- Atrial fibrillation
- oxidative stress
- urinary F(2)-isoprostanes
- endothelial function
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Atrial fibrillation (AF) is the most common cardiac arrhythmia that is associated with a high risk of cardiovascular events such as ischaemic stroke and increased morbidity and mortality.1 The mechanisms underlying the initiation and perpetuation of AF are not well understood, but emerging evidence indicates a link between AF and inflammation and suggests a role for inflammation in eliciting oxidative stress.2–5 Experimental models of atrial electrophysiological stimulation were associated with enhanced oxidative stress and antioxidant treatment was able to decrease the vulnerability to AF.6 Previous studies in animal models showed that NADPH oxidase is responsible for the increased production of reactive oxygen species (ROS) in the left atrium appendage in AF.7 Studies in humans corroborated this hypothesis, showing that atrial myocytes from the right atrial appendage produce an abundant amount of ROS via NADPH oxidase activation8 and that antioxidant treatment reduces the occurrence of AF in patients undergoing cardiac surgery.9 ,10 Moreover, it seems that the sources of ROS vary with the duration of AF. Thus, the right atrium of patients who develop postoperative AF shows upregulation of NOX2 and p22phox subunits of NADPH oxidase while permanent AF is associated with upregulation of mitochondrial oxidases and uncoupled NOS activity.11
These findings suggest that oxidative stress may be implicated in the occurrence of AF secondary to cardiac surgery. However, it remains to be established if oxidative stress also occurs in patients who spontaneously experience paroxysmal/persistent AF. We have recently developed a method which measures the activity of NOX2, the catalytic subunit of NADPH oxidase, in serum.12 This assay reflects prevalently NOX2 activation from circulating cells such as platelets and leucocytes and represents a useful approach to investigating its regulation ex vivo.
Based on this, we sought to investigate the relationship between oxidative stress and different AF types (paroxysmal-persistent vs chronic AF) by measuring serum levels of NOX2 and urinary excretion of isoprostanes, a reliable marker of oxidative stress.13
Patients and controls
One hundred and seventy-four consecutive patients (112 men, 62 women, age 73.1±7.3 years) referred to our Division of I Clinica Medica as inpatients or outpatients for AF were recruited between October 2007 and August 2010. The patients were divided into two groups: those with paroxysmal (n=73)/persistent (n=9) AF (50 men, 32 women, age 73.4±7.3 years) and those with permanent AF (62 men, 30 women, age 72.8±7.4 years).
Patients were excluded if they had rheumatic AF, severe congestive heart failure (New York Heart Association functional class 4) or the presence of prosthetic valves; acute myocardial infarction or unstable angina during the previous month; had undergone carotid endoarterectomy or coronary or peripheral revascularisation procedures during the previous 6 months; acquired or congenital valvular disease (except mitral valve prolapsed or mitral annulus calcification). We also excluded patients with inflammatory or neoplastic diseases, thyroid dysfunction or those treated long term with corticosteroids. Patients with atrial flutter or other atrial tachyarrhythmias were also excluded. Furthermore, subjects were excluded from the study if they had liver or pancreatic insufficiency, serious renal disorders (serum creatinine >2.8 mg/dl) or if they were current smokers or were taking antioxidant vitamins.
Ninety control subjects frequency matched for age, sex and cardiovascular risk profile (hypertension, diabetes mellitus, hypercholesterolaemia, smoking habit, history of major cardiovascular events) were recruited from our metabolic outpatient clinic between October 2007 and August 2010.
At entry, each patient's medical history was taken and he/she underwent a physical examination. The following diagnostic procedures were also performed: routine blood laboratory tests, baseline 12-lead ECG, M-mode and two-dimensional echocardiography with echocolor Doppler.
Paroxysmal AF was defined as AF lasting more than 30 s with spontaneous termination. Persistent AF was defined as AF lasting more than 7 days and requiring either pharmacological therapy or electrical cardioversion for termination. AF that was refractory to cardioversion or that was allowed to continue was classified as permanent. Hypertension was defined as arterial pressure values >140/90 mm Hg or if patients were on treatment with antihypertensive drugs according to the 2007 European Society of Hypertension/European Society of Cardiology (ESH/ESC) guidelines.14 Diabetes was diagnosed according to the American Diabetes Association definition.15 Hypercholesterolaemia was defined as previously described.16
Blood sampling and soluble NOX2 detection
After overnight fasting (12 h) and supine rest for at least 10 min, blood samples were collected in vacutainers (Vacutainer Systems, Plymouth, UK) and centrifuged at 300 g for 10 min to obtain supernatant which was stored at −80°C until use. Serum levels of soluble NOX2-derived peptide (sNOX2-dp) were detected by ELISA as previously described.12 The peptide was recognised by a specific monoclonal antibody against the amino acidic sequence (224–268) of the extra membrane portion of NOX2. Values were expressed as pg/ml; intra-assay and inter-assay coefficients of variation were 5.2% and 6%, respectively.
Isoprostanes: urinary 8-iso-PGF2α assays
Urinary 8-isoprostaglandin F2α (8-iso-PGF2α) was measured by a previously described and validated EIA assay method.17 Ten ml urine was extracted on a C-18 SPE column; the purification was tested for recovery by adding a radioactive tracer (tritiated 8-iso-PGF2α) (Cayman Chemical Company, Ann Arbor, Michigan, USA). The eluates were dried under nitrogen, recovered with 1 ml of buffer and assayed in a 8-iso-PGF2α specific EIA kit (Cayman Chemical). Urinary 8-iso-PGF2α concentration was corrected for recovery and creatinine excretion. Values are expressed as pg/mg of creatinine. Intra-assay and inter-assay coefficients of variation were 2.1% and 4.5%, respectively.
Categorical variables were reported as counts (percentage) and continuous variables as means±SD unless otherwise indicated. Independence of categorical variables was tested by χ2 test. Normal distribution of parameters was assessed by the Kolmogorov–Smirnov test.
The Student t test or one-way ANOVA with post hoc Bonferroni test was used as appropriate to assess the distribution of variables between the study groups. Bivariate analysis was performed with Pearson product moment correlation analysis. Appropriate non-parametric tests (Mann–Whitney U test and Spearman rank correlation test) were used for all other variables.
Multivariable regression analyses were performed in order to investigate the relationship between baseline values of sNOX2-dp (or urinary isoprostanes) and the AF type (paroxysmal/persistent vs permanent) in AF patients and were determined in a forward stepwise variable selection procedure. Age, gender, hypertension, diabetes mellitus, hypercholesterolaemia, smoking habit, history of stroke, history of myocardial infarction, heart failure, ejection fraction, left atrium diameter, use of antiplatelets, oral anticoagulants and statins were considered possible confounding variables. Only variables with values of p<0.10 in the univariate analysis were candidates for the multivariable model.
Only p values <0.05 were regarded as statistically significant. All tests were two-tailed and analyses were performed using computer software packages (IBM SPSS V.19.0, SPSS Inc).
Sample size determination
We calculated that 69 patients per group were required to have a 90% chance of detecting as significant at the 5% level a mean±SD difference of 5±9 pg/ml in sNOX2-dp between groups.
The clinical characteristics of the patients included in the study are shown in table 1. Patients with AF and controls had a similar distribution of cardiovascular risk factors. A history of myocardial infarction and heart failure was more common in patients with permanent AF than in those with paroxysmal/persistent AF and controls; ejection fraction was lower in patients with permanent AF. Left atrium diameter was higher in patients with permanent AF than in those with paroxysmal/persistent AF. Oral anticoagulant therapy was more common in patients with AF; conversely, antiplatelet therapy was more common in controls.
Analysis of oxidative stress showed different behaviour between the three groups. Compared with patients with permanent AF and controls, those with paroxysmal/persistent AF had higher values of NOX2 (table 1, figure 1A). Compared with controls, patients with permanent AF showed a weak but significant increase in NOX2 levels.
Similar results were obtained for urinary isoprostanes. Thus, compared with patients with permanent AF and controls, those with persistent AF had higher urinary excretion of isoprostanes (table 1, figure 1B). No significant difference in urinary excretion of isoprostanes was found between patients with permanent AF and controls.
Correlation analysis showed a significant correlation between NOX2 activity and urinary excretion of isoprostanes (figure 2; R=0.707, p<0.001).
Multivariable analyses adjusted for left atrium diameter, ejection fraction, heart failure, history of stroke and myocardial infarction demonstrated that baseline values of sNOX2-dp and urinary isoprostanes were independently associated with AF type (paroxysmal/persistent vs permanent) in AF patients (β=−224, p=0.007 and β=−231, p=0.005, respectively).
This study provides evidence that, in patients with AF, oxidative stress assessed by NOX2 activity and urinary isoprostanes is increased only in patients with paroxysmal/persistent AF.
Oxidative stress has been hypothesised to be implicated in favouring AF. Thus, experimental models of atrial pacing showed an association between markers of oxidative stress such as peroxynitrite and AF,6 and a reduced risk of AF in animals with low formation of ROS.18
The relationship between oxidative stress and AF has also been studied in patients with AF. Systemic markers of oxidative stress have been detected in 40 patients with persistent/permanent AF.19 Kim et al8 demonstrated that NOX2, the catalytic subunit of NADPH oxidase, is the main source of oxidative stress in human atrial myocytes and is increased in the right atrial appendage of patients with a history of AF. The enhanced activity of NOX2 seemed to be independent of its expression but could be attributable to post-translational modifications of the oxidase subunits.8 The same group substantiated these findings in patients undergoing cardiac surgery, and identified NADPH oxidase-dependent superoxide anion production in the right atrial appendage as the most important independent predictor of AF in patients who developed AF after cardiac surgery.20 Of note, the increase in oxidative stress was dependent on different sources of ROS according to the type of AF.11 Thus, while NADPH oxidase was upregulated in patients who developed spontaneous but reversible AF after surgery, other sources of ROS such as uncoupled eNOS and mitochondrial oxidases were implicated in upregulating oxidative stress in patients with permanent AF.11 Our study supports and extends these findings, showing that the behaviour of NADPH oxidase is related to the type of AF. Unlike this previous study, we compared the behaviour of NOX2 in patients who spontaneously developed paroxysmal/persistent AF with patients with permanent AF. NOX2 was found to be upregulated in patients with paroxysmal/persistent AF compared with those with permanent AF and controls. Consistent with NOX2 upregulation was the behavior of urinary isoprostanes which was increased only in patients with paroxysmal/persistent AF.
A previous study from our group showed a close association between NOX2 activity and production of isoprostanes in humans.21 Thus, in patients with hereditary deficiency of NOX2, urinary excretion of isoprostanes was significantly lowered than in controls.21 Such a relationship was reinforced by the significant increase in urinary excretion of isoprostanes in patients with NOX2 upregulation, suggesting that NOX2 is a major determinant of isoprostane formation. The increase in isoprostanes in patients with paroxysmal/persistent AF and its association with NOX2 upregulation are in agreement with these findings and suggest that, in this setting, NOX2 upregulation may be responsible for the enhanced formation of isoprostanes.
Changes in NOX2 and isoprostanes seem to be peculiar to patients with paroxysmal/persistent AF as patients with permanent AF had similar values of isoprostanes and a modest increase in NOX2 compared with controls.
Multiple regression analysis supported this result with both markers of oxidative stress being significantly associated with paroxysmal/persistent AF, suggesting a potential cause-effect relationship between them. However, this hypothesis must be investigated further by determining whether NOX2 inhibition actually prevents the occurrence of AF. We cannot exclude the possibility that the upregulation of NOX2 simply reflects the inflammatory status which often complicates the clinical course of AF.
In conclusion, we found a significant association between NOX2 regulation and paroxysmal/permanent AF, suggesting NOX2 activation as a potential trigger of this cardiac arrhythmia. An interventional study with inhibitors of NOX2 is needed to support the causative role of NOX2 in favouring the occurrence of AF.
Disclosures All authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Funding This study was supported by a grant from Sapienza-University of Rome to FV (Ateneo Federato 2009).
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided bySapienza University Ethics Committee, Rome, Italy.
Provenance and peer review Not commissioned; internally peer reviewed.