CLINICAL PERSPECTIVE

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(Circulation. 2005;112:1266-1273.)
© 2005 American Heart Association, Inc.

Arrhythmia/Electrophysiology

Atrial Fibrillation Increases Production of Superoxide by the Left Atrium and Left Atrial Appendage

Role of the NADPH and Xanthine Oxidases

Samuel C. Dudley, Jr, MD, PhD; Nyssa E. Hoch, BS; Louise A. McCann, BS; Clegg Honeycutt, MS; Laura Diamandopoulos, MD; Tohru Fukai, MD, PhD; David G. Harrison, MD; Sergey I. Dikalov, PhD; Jonathan Langberg, MD

From the Emory University School of Medicine, Department of Medicine, Division of Cardiology (S.C.D., N.E.H., L.A.M., C.H., L.D., T.F., D.G.H., S.I.D., J.L.) and Atlanta Veterans Administration Medical Center (S.C.D.), Atlanta, Ga.

Correspondence to Dr Samuel C. Dudley, Jr, Division of Cardiology, Emory University/VAMC, 1670 Clairmont Rd (111B), Decatur, GA. E-mail sdudley{at}emory.edu

Received January 22, 2005; revision received April 1, 2005; accepted April 18, 2005.

	Abstract

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Background— Atrial fibrillation (AF) is associated with an increased risk of stroke due almost exclusively to emboli from left atrial appendage (LAA) thrombi. Recently, we reported that AF was associated with endocardial dysfunction, limited to the left atrium (LA) and LAA and manifest as reduced nitric oxide (NO^·) production and increased expression of plasminogen activator inhibitor-1. We hypothesized that reduced LAA NO^· levels observed in AF may be associated with increased superoxide (O₂^·–) production.

Methods and Results— After a week of AF induced by rapid atrial pacing in pigs, O₂^·– production from acutely isolated heart tissue was measured by 2 independent techniques, electron spin resonance and superoxide dismutase–inhibitable cytochrome C reduction assays. Compared with control animals with equivalent ventricular heart rates, basal O₂^·– production was increased 2.7-fold (P<0.01) and 3.0-fold (P<0.02) in the LA and LAA, respectively. A similar 3.0-fold (P<0.01) increase in LAA O₂^·– production was observed using a cytochrome C reduction assay. The increases could not be explained by changes in atrial total superoxide dismutase activity. Addition of either apocyanin or oxypurinol reduced LAA O₂^·–, implying that NADPH and xanthine oxidases both contributed to increased O₂^·– production in AF. Enzyme assays of atrial tissue homogenates confirmed increases in LAA NAD(P)H oxidase (P=0.04) and xanthine oxidase (P=0.01) activities. Although there were no changes in expression of the NADPH oxidase subunits, the increase in superoxide production was accompanied by an increase in GTP-loaded Rac1, an activator of the NADPH oxidase.

Conclusions— AF increased O₂^·– production in both the LA and LAA. Increased NAD(P)H oxidase and xanthine oxidase activities contributed to the observed increase in LAA O₂^·– production. This increase in O₂^·– and its reactive metabolites may contribute to the pathological consequences of AF such as thrombosis, inflammation, and tissue remodeling.

Key Words: atrium • superoxide • tachycardia

	Introduction

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Atrial fibrillation (AF) is the most common sustained arrhythmia in the elderly, affecting >5% of the population >65 years of age.¹ It is considered a progressive disease, increasing in prevalence with age and often advancing from paroxysmal to permanent. AF not only is an independent risk factor for death but also confers significant risk of morbidity from stroke associated with embolization of thrombi developed within the left atria appendage (LAA).² It is estimated that at least two thirds of strokes in patients with AF occur by this mechanism. Despite its prevalence, the cause of AF and localized LAA thrombus formation remains unclear.

Several lines of evidence suggest an association between oxidative stress and AF. Carnes et al³ have shown that AF induced by rapid pacing in dogs decreases tissue ascorbate levels and increases protein nitration, a biomarker of oxidative and nitrosative stress. Biochemical evidence of oxidation by peroxynitrite and hydroxyl (^·OH) radicals, both downstream products of O₂^·– generation, also has been demonstrated in experimental models.⁴ CABG, a procedure frequently complicated by AF, is associated with an increase in oxidized glutathione and lipid peroxidation.⁵ Angiotensin II (Ang II) stimulates O₂^·– production by NAD(P)H oxidases by activation of the type 1 receptor, and inhibition of Ang II production reduces redox stress in the vasculature. Patients with AF are known to have increased levels of ACE and Ang II receptors.^6–8 In addition, ACE inhibition has been shown to reduce the incidence of AF in a number of different settings, including after myocardial infarction⁹ and in the setting of left ventricular systolic dysfunction.¹⁰ ACE inhibitor therapy has been shown to be a negative predictor of AF after CABG, whereas postoperative withdrawal of ACE inhibitors has been associated with the development of AF.¹¹

Recently, we reported that AF caused left atrial (LA) endocardial dysfunction manifested as a 73% decrease in NO^· production and a 1.8-fold upregulation of plasminogen activator inhibitor-1 (PAI-1).¹² One possible explanation for the reduction in NO^· is increased oxidative degradation by O₂^·–. We investigated whether O₂^·– was increased as a result of AF using a pig rapid pacing model of AF.

	Methods

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Swine Model of AF
Experiments were performed under a protocol approved by the Emory and Atlanta VA Institutional Animal Care and Use committees. The protocol is identical to that previously described.¹² Pigs (Sus scrofa) weighing 25+4 kg were studied. Using a percutaneous technique, we introduced 2 hemostatic sheaths into the right femoral vein. A 4F quadripolar catheter was advanced through a sheath and positioned in the apex of the right ventricle for temporary pacing. An ablation catheter (Boston Scientific) was positioned across the AV junction, and radiofrequency energy was applied until complete AV block was produced. Next, the right internal jugular vein was isolated, and a tined, bipolar permanent pacing lead was introduced, advanced under fluoroscopic guidance, and positioned in the apex of the right ventricle. An active fixation bipolar pacing lead was introduced using comparable technique and placed in the right atrial appendage (RAA). In the AF group, the atrial lead was attached to a neurostimulator (Itrel III, Medtronic) positioned subcutaneously in the neck. The device was programmed to a rate of 600 bpm to maintain AF. A separate single-chamber pacemaker was attached to the ventricular lead and programmed to a rate of 100 bpm. In the control group, the atrial and ventricular leads were connected to a single DDD pacemaker programmed to rate of 100 bpm and an AV delay of 120 ms. In this way, both the experimental and control animals had a paced ventricular rate of 100 bpm and differed only in their atrial rhythm.

After 1 week, animals were reanesthetized. Pacing was withheld, and an ECG was performed to ensure that AF or sinus rhythm was present in the experimental and control groups, respectively. Weights obtained showed no differences among groups, suggesting that pacing had not induced heart failure. Animals were euthanized with sodium pentobarbital intravenously, and the hearts were rapidly excised. The hearts were rinsed and dissected in Krebs-HEPES buffer, and tissue was either studied immediately or quick-frozen in liquid nitrogen for subsequent enzymatic assays and Western analysis. Sections of the LAA were taken for histological examination.

Measurements of O₂^·– Using Electron Spin Resonance and Superoxide Dismutase–Inhibitable Acetylated Cytochrome C Reduction
The cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine hydrochloride (CMH; Alexis Corp) was used to examine intracellular O₂^·– production using electron spin resonance (ESR) spectroscopy.¹³ For ESR, freshly isolated heart was allowed to equilibrate in deferoxamine-chelated Krebs-HEPES solution containing CMH (0.5 mmol/L), deferoxamine (25 µmol/L), and DETC (5 µmol/L) for 90 minutes at 37°C. After the incubation period, tissue samples were transferred into 1-mL syringes filled with Krebs-HEPES solution and frozen in liquid nitrogen. Then, the samples were placed in a liquid nitrogen ESR dewar, and time scans were obtained at 77 K with a Bruker EMX spectrometer (5 G; conversion time, 655 ms; time constant, 5243 ms; modulation frequency, 100 kHz; microwave power, 10 mW; microwave frequency, 9.78 GHz; scan time, 335 seconds). Analyses of the ESR spectra peak height were used to quantify the amount of O₂^·– produced by the tissue and were compared with buffer-only control spectra or spectra in the presence of superoxide dismutase (SOD).

To detect O₂^·– using an independent technique, SOD-inhibitable cytochrome C reduction was used as previously described.¹⁴

ESR-Based Assays for NADPH and Xanthine Oxidase Activity
Cardiac tissue was cut into 1-mm cubes that were then ground into a suspension in glass VWR 0.8-mL Micro Tissue Grinders kept on ice. The tissue suspension was treated for 3 hours in 50 mmol/L PBS treated with 3 g/100 mL Chelex-100 (filtered), 0.1 mmol/L DTPA, and the protease inhibitors bestatin (2 µmol/L), leupeptin (2 µmol/L), pepstatin (1 µmol/L), and phenylmethylsulfonyl fluoride (1 mmol/L) (pH 7.4). Sample solutions (300 µL) were placed in 1.5-mL centrifuge tubes and centrifuged at 750g at 4°C for 10 minutes. Then, 250 µL supernatant was removed and placed into new 1.5-mL tubes that were centrifuged at 30 000g at 4°C for 30 minutes. All the supernatant was removed, and the membrane pellet was resuspended in 150 µL lysis buffer. Protein concentration was measured with the Bradford method.

Protein (23 or 11.5 µg) was added to 1 mmol/L 1-hydroxy-3-carboxypyrrolidine (CPH), 200 µmol/L NADPH, or 100 µmol/L hypoxanthine and 0.1 mmol/L DTPA in a total volume of 100 µL of Chelex-treated PBS. ESR spectroscopy was used for quantitative measurements of O₂^·– production. In duplicate samples, NADPH or hypoxanthine was omitted. Superoxide formation was assayed as NADPH or xanthine-dependent, SOD-inhibitable formation of 3-carboxyproxyl radical (CP). Samples were placed in 50-µL glass capillaries (Corning). Accumulation of CP was recorded using an EMX ESR spectrometer (Bruker) and a super-high-Q microwave cavity. The ESR instrumental settings were as follows: low-field component of CP ESR spectrum, 3477.20 G; microwave frequency, 9.78 GHz; microwave power, 20 mW; modulation amplitude, 2 G; conversion time, 1312 ms; time constant, 5 seconds; resolution, 512 points; and receiver gain, 1x10⁵. Substrate-dependent CP production in membranes was inhibited 95% to 98% by SOD (50 U/mL) added directly to the sample.

SOD Activity
Tissues were homogenized in 10 volumes of 50 mmol/L potassium phosphate (pH 7.4) containing 0.3 mol/L KBr and a mixture of protease inhibitors (0.5 mmol/L phenylmethylsulfonyl fluoride, 3 mmol/L diethylene-triaminepentaacetic acid, 90 mg aprotinin, 10 mg pepstatin, 10 mg chymostatin, and 10 mg leupeptin). The homogenates were then sonicated and extracted at 4°C for 30 minutes. The extracts were then centrifuged at 3000g for 15 minutes. The enzyme activity of SOD in the cell homogenates was assayed by monitoring inhibition of the rate of xanthine oxidase (XO)–mediated reduction of cytochrome C, as previously described.¹⁵

Western Blotting for NADPH Subunits
Protein samples were prepared from the LAA tissue of control and AF pigs. Frozen tissue pieces were homogenized in Laemmli buffer and boiled for 5 minutes. To measure protein concentration, a fraction of each sample was precipitated using 72% trichloroacetic acid, followed by a Lowry protein assay (Bio-Rad Laboratories). Equal amounts of protein (40 µg) were separated by electrophoresis at 120 V for 1.5 hours on a 12.5% SDS polyacrylamide gel, transferred to nitrocellulose membranes (Amersham Biosciences), and blocked for 2 hours at room temperature with 5% milk/PBS-T. Immunoblotting was performed with Nox1 and Nox4 polyclonal antibodies (1:5000, provided by Dr Harald H.H.W. Schmidt), a p22^phox monoclonal antibody (1:1500, Rockland Immunochemicals), and a gp91^phox monoclonal antibody (1:1000, BD Biosciences). Actin (1:250, Santa Cruz Biotechnology) was used to control for protein loading. Immunoreactive bands were visualized by enhanced chemiluminescence. Densitometry was performed with Quantity One software (Bio-Rad Laboratories).

Rac1 Activation Assay
Active, GTP-bound Rac1 was precipitated with a PAK-PBD–based assay (Upstate Biotechnology). LAA tissue samples (30 mg) were homogenized in 600 µL of 1x MLB (Upstate Biotechnology) with added protease inhibitors. Samples were pretreated with glutathione agarose, and 500 µL tissue lysates was used for the pull-down assay. A Bradford assay was performed to allow normalization of protein content. PAK-1 PBD agarose (10 µg) was added to each sample and incubated at 4°C for 1 hour. For positive and negative controls, samples were incubated with GTPS or GDP, respectively, before PAK-1 PBD agarose was added. Beads were collected, washed 3 times, and resuspended in 40 µL Laemmli buffer. An equal volume of samples (20 µL) was electrophoretically separated at 130 V for 1 hour on a 12.5% SDS polyacrylamide gel, transferred to nitrocellulose membranes, and evaluated for active Rac1 by Western analysis.

Statistical Analysis
Data are presented as mean±SE. Comparisons between experimental and control animals were performed by use of a Student t test for unpaired data. Multiple means were compared by a 1-way ANOVA and a post-hoc test when significance was indicated. A value of P<0.05 was considered statistically significant.

	Results

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After cessation of atrial pacing and at the time of death, all animals in the experimental group were documented to be in AF. Weights did not vary before or after pacing, suggesting that pacing did not cause significant congestive heart failure. Inspection of the atria revealed no gross evidence of thrombus formation. Histological examination by hematoxylin and eosin staining revealed 1 microthrombus in the LAA in 23 animals examined in the experimental group and no thrombi in the control animals.

Intracellular O₂^·– Production
Intracellular O₂^·– production from various parts of the heart was examined by ESR using membrane-permeable CMH as a spin probe. The reaction of CMH with O₂^·– results in formation of a nitroxide radical that yields a characteristic spectrum (Figure 1). In the control group, O₂^·– production was comparable in each of the tissues tested (Figures 1 and 2 ). The amount of O₂^·– was 18.1±2.1 (n=5), 20.2±3.1 (n=5), 27.1±3.9 (n=5), and 23.7±1.8 (n=6) pmol/mg wet weight for the LAA, LA, RAA, and right atrium (RA), respectively.

View larger version (13K): [in this window] [in a new window]   Figure 1. Measurement of O2·– by ESR. The reaction of the intracellular spin probe CMH with superoxide radical is shown, along with representative spectra from the RA and LA before and after a week of AF. The increase in spectrum peak height implies an increase in O2·– during AF in the LA but not the RA.

View larger version (10K): [in this window] [in a new window]   Figure 2. ESR measurements of O2·– production. ESR spectra with the spin probe CMH were used to measure the production of intracellular O2·– in the LAA, LA, RAA, and RA in AF (solid bars) and control (open bars) pigs. AF significantly increases intracellular O2·– production in the LA (*P<0.02) and LAA (**P<0.02).

In the experimental animals, 1 week of AF caused 2.7- and 3.0-fold increases in O₂^·– production in the LA and LAA over the control measurements, respectively (P<0.02 in each case; Figures 1 and 2). In contrast, AF did not cause significant changes in O₂^·– production in the RA or RAA. The amount of O₂^·– detected for each segment was 54.6±6.0 (n=4), 53.8±4.9 (n=6), 38.3±4.5 (n=5), and 30.2±6.0 (n=5) pmol/mg wet weight for the LAA, LA, RAA, and RA, respectively (Figure 2).

Extracellular O₂^·– Production
Extracellular O₂^·– production from various parts of the heart was examined by SOD-inhibitable cytochrome C reduction. Superoxide production (normalized to the dry weight of tissue) in control animals was comparable in the various parts of the heart that were assessed (P=0.70 by 1-way ANOVA; Figure 3). The amount of O₂^·– was 21.4±5.4 (n=21), 13.0±7.3 (n=6), 12.1±7.3 (n=6), 15.3±10.0 (n=6), and 11.5±7.2 (n=6) pmol/mg dry weight for the LAA, LA, RAA, RA, and a coronary artery segment, respectively.

View larger version (12K): [in this window] [in a new window]   Figure 3. Extracellular O2·– production from various parts of the heart measured by SOD-inhibitable cytochrome C reduction assay. Superoxide production was measured from the LAA, LA, RAA, RA, and coronary artery (CA) in AF pigs (solid bars) and compared with control pigs (open bars). Basal production of superoxide was highest in the LAA and increased 3.0-fold after 1 week of AF (*P<0.01).

With 1 week of AF in the experimental group, there was a marked increase in extracellular O₂^·– detected in the LAA. In the LAA, AF caused a 3.0-fold increase in O₂^·– production over the control measurements (P<0.01; Figure 3). Compared with control pigs, however, no changes in O₂^·– production were observed in the other segments. The amount of O₂^·– recorded for each segment was 63.9±8.0 (n=22), 5.3±5.3 (n=6), 22.9±11.2 (n=6), 17.8±11.3 (n=6), and 11.7±10.4 (n=7) pmol/mg dry weight for the LAA, LA, RAA, RA, and coronary artery, respectively.

Determination of the Source of Increased Superoxide During AF
SODs are a major counter to increased oxidative stress in the vascular endothelium.^16–18 Therefore, we sought to determine whether the increased O₂^·– production observed in AF was the result of reduced SOD activity. Measurements of total SOD activity in the LA, LAA, and aorta showed no change between control and AF animals (Figure 4). Thus, it is unlikely that the increased O₂^·– observed in the LA and LAA during AF was due to alterations in SOD activity.

View larger version (11K): [in this window] [in a new window]   Figure 4. Total SOD activity in LA, LAA, and aorta (Ao) of control and AF pigs. There were no changes in SOD activity between the control (open bars) and AF (solid bars) pigs to explain the increase in O2·– production seen with AF.

In mammalian cells, the NAD(P)H oxidases are major sources of reactive oxygen species (ROS). Therefore, we examined the role of the NAD(P)H oxidases in the increase in O₂^·– production during AF. The NAD(P)H oxidase inhibitor apocynin (100 µg/mL) reduced LAA O₂^·– production by 91%, suggesting a role of the NADPH oxidase. To investigate this further, LAA NAD(P)H oxidase activities were compared directly using membrane preparations from AF and control pigs. There was a 4.4-fold increase in NAD(P)H activity in the LAA of pigs with AF (P=0.02; Figure 5). The NAD(P)H oxidase activity was 0.4±0.1 versus 1.8±0.5 nmol O₂^·– per 1 mg tissue per 1 minute in control versus AF pigs. The LA showed a similar trend toward increased NAD(P)H oxidase activity (P=0.06).

View larger version (22K): [in this window] [in a new window]   Figure 5. Changes in NAD(P)H oxidase– and XO-mediated O2·– production as a result of AF. LA or LAA membrane preparations were made from control or AF pigs and exposed to either NADPH or hypoxanthine, substrates for the NAD(P)H oxidase or XO, respectively. There was a statistically significant increase in O2·– production in the LAA of AF pigs compared with control pigs (*P=0.02). The trend was similar in the LA (P=0.06), suggesting an upregulation of NAD(P)H oxidase activity after 1 week of AF. Although appreciably smaller overall, hypoxanthine-dependent O2·– production was increased in the LA (**P=0.04) and LAA (#P=0.01) with 1 week of AF. Spin probe CPH was used to measure O2·– production in the membrane fractions (see Methods).

Because XO is another source of O₂^·– that can be activated concomitantly with the NAD(P)H oxidase,¹⁹ we also investigated changes in XO activity caused by AF. Although the overall O₂^·– production attributable to this system was lower than that of the NAD(P)H oxidase, incubation of LAA with oxypurinol reduced O₂^·– production by 85%. Furthermore, LAA XO activity also showed a 4.4-fold increase with AF (P=0.01), rising from 0.1±0.1 nmol O₂^·– per 1 mg tissue per 1 minute in control pigs to 0.5±0.1 nmol O₂^·– per 1 mg tissue per 1 minute in AF pigs. In the case of XO, the differences between activities in the control and AF groups in the LA were also statistically significant (P=0.04).

Mechanism of Increased NAD(P)H Oxidase Activity During AF
We evaluated whether the increased LAA NADPH oxidase activity with AF was the result of increased expression of the NADPH oxidase subunits or increased oxidase activity. Western blot analysis of Nox1, Nox2 (gp91phox), Nox4, and p22phox showed no change in relative protein amounts caused by AF, suggesting that activation rather than transcriptional regulation was responsible for the increased activity (Figure 6).^20–22 The small G protein Rac1 is essential for assembly of the active NAD(P)H oxidase complex.^23–25 Therefore, we performed experiments to determine whether AF was associated with an increase in active GTP-bound Rac1. Rac1 activity assays demonstrated that AF increased active Rac1 in the LAA by 6.9-fold (P<0.05) compared with the amount in control pigs (Figure 7).

View larger version (40K): [in this window] [in a new window]   Figure 6. Effect of AF on NAD(P)H oxidase subunit protein expression. A, LAA tissue from 4 pigs with or without AF was analyzed. Proteins were separated by PAGE, transferred to nitrocellulose membranes, and probed with corresponding antibodies. B, Densitometry normalized to actin as a protein loading control showed no statistically significant differences between control and AF animals in the amounts of gp91phox, Nox1, Nox4, or p22phox, suggesting that protein expression could not explain the increase seen in NAD(P)H oxidase activity with AF.

View larger version (17K): [in this window] [in a new window]   Figure 7. Rac1 activity in the LAA. LAA tissue from 4 AF and 4 control pigs was analyzed for the presence of active, GTP-bound Rac1 precipitated with the p21-binding domain of human PAK-1 (PAK- PBD), separated by gel electrophoresis, transferred to nitrocellulose membranes, and blotted for Rac1. For positive and negative controls, samples were incubated with GTPS or GDP, respectively, before PAK- PBD agarose was added. Densitometry showed a statistically significant 6.9-fold rise in active Rac1 during AF (P<0.05).

	Discussion

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Previously, we demonstrated that AF is associated with endocardial dysfunction that may contribute to thrombus formation in the LAA.¹² We speculated that the endocardial dysfunction observed during AF might be similar to endothelial dysfunction observed during atherosclerosis. In the case of atherosclerosis, endothelial dysfunction is accompanied by increased production of O₂^·–. In the present study, we found that AF was also associated with increased intracellular O₂^·– production in the LA and LAA and with increased extracellular O₂^·– in the LAA. The increased O₂^·– production was at least in part the result of increased NAD(P)H oxidase and XO activities. Increased NAD(P)H oxidase activity could be explained by an increase in active Rac1, a required cofactor.

Both ESR and the cytochrome C assay showed similar AF-induced increases in intracellular and extracellular O₂^·– production in the LAA. In the LA, however, we found that intracellular O₂^·– was increased and extracellular O₂^·– production as assessed by the cytochrome C assay was not altered. This difference between the 2 assays might be explained by an increased sensitivity of the ESR technique. It is also possible that there are different enzymatic sources of O₂^·– in the LA and the LAA. For example, it has been shown that there are different intracellular distributions of the Nox isoforms in vascular smooth muscle cells that might affect the ability of O₂^·– to be released extracellularly.²⁶

Our data indicate that the increase in O₂^·– production is greater in the LA and LAA than in the RA or RAA, although a nonsignificant trend toward AF caused an increase in O₂^·– in the RA and RAA. Although it is possible that this trend in the RA could reach statistical significance with a larger sample size, it is clear from our data that the increase in O₂^·– production is greater in the LA than in the RA. It is uncertain why these differences between the 2 atria are present. Flow profiles, levels of oxygen tension, and mechanical influences, however, clearly vary between the LA and RA. Each of these factors could influence activation of the NAD(P)H oxidase and XO.

The increased production of ROS may be an important cause of thrombus formation in the LAA in AF, a major risk factor for stroke. In addition to possible oxidative degradation of endocardial NO^·, O₂^·– and ROS derived from O₂^·– may affect coagulation cascade components produced by the endocardium. PAI-1 and tissue factor are prothrombotic molecules that are modulated by redox stimuli.^27–31 Tissue factor activates thrombin, and elevated tissue factor expression is associated with thrombosis in atherosclerosis and sepsis.^32,33 Tissue factor expression is inhibited tissue factor endothelial NO^· production.^29–31 Upregulation of tissue factor may also be facilitated by O₂^·–.³⁴ Consistent with these ideas, we have recently reported that AF is associated with a downregulation of LAA NO^·and increases in PAI-1 and tissue factor.^12,35

Our data suggest that the NAD(P)H oxidase is responsible for at least some of the O₂^·– produced by the atria in AF. This observation may help explain the link between AF and activation of the renin-angiotensin-aldosterone system, because Ang II is a potent stimulator of the NAD(P)H oxidase. Ang II receptors are upregulated in the LA of humans with AF.⁸ ACE inhibition reduces the likelihood of AF in dog models^36–38 and in patients after myocardial infarction associated with left ventricular dysfunction.³⁹ Inhibition of renin-angiotensin signaling also reduces the incidence of AF in humans.^39,40 Although this effect may be due to myriad properties of ACE inhibitors, the NAD(P)H oxidase is potently regulated by Ang II, and a major effect of ACE inhibition or angiotensin receptor antagonism is a reduction in the activity of this enzyme, even in normal animals. Consistent with the importance of Ang II signaling in AF, we have recently shown that cardiac-directed overexpression of ACE results in a phenotype characterized by atrial enlargement and atrial fibrillation.⁴¹

The mechanism of increased NAD(P)H oxidase activity appeared to be increased enzyme activation. Membrane subunit assembly is required to form an active NAD(P)H oxidase complex.²³ Active, GTP-bound Rac1 is an important determinant of subunit assembly, and the amount of active Rac1 has been shown to correlate with NAD(P)H oxidase–dependent O₂^·– production.^24,25 Although Western blot analysis of Nox subunits showed no changes with AF, AF was associated with a 6.9-fold increase in active Rac1. Possible mechanisms of Rac1 activation include increased Ang II receptor activation, ß-receptor activation, or increased cytosolic Ca²⁺.^42–44 Although not evident, heart failure cannot be excluded as a cause for our findings.

In addition to increased NADPH oxidase activity, we also found increased enzymatic activity of XO in the LAA in AF. The XO inhibitor oxypurinol reduced O₂^·– production in these tissues. The observation that both enzyme systems contribute to increased O₂^·– production may be the result of interplay between these and other sources of ROS. For example, recent studies have shown that ROS derived from the NADPH oxidase can increase conversion of xanthine dehydrogenase to XO.¹⁹ Likewise, hydrogen peroxide produced from another source such as XO can stimulate the NADPH oxidase.

Together, these observations support a causal relationship between AF and increased ROS production, and localized oxidative stress may explain the dominant role of the LA as a source of AF.⁴⁵ It may be that oxidative stress is another mechanism, along with electrical remodeling, that contributes to the vicious cycle of "AF begetting AF."⁴⁶

In summary, we demonstrate that, in a pacing model, AF causes oxidative stress localized to the LA and LAA that is mediated partially by activation of the NAD(P)H oxidase and XO. The activation of the NAD(P)H oxidase could be explained by increased amounts of the active cofactor Rac1. These findings may help to explain the proclivity of thrombus to form in the LAA and may have implications for the pathogenesis of AF.

	Acknowledgments

 
This study was supported by National Institutes of Health (NIH) grants HL-39006 (Dr Harrison), HL-64828 (Dr Dudley), and HL-59248 (Dr Harrison); NIH Program Project grant 58000 (Dr Harrison); Department of Veterans Affairs merit grants (Drs Harrison and Dudley); an American Heart Association Established Investigator Award (Dr Dudley); and a Southeast Affiliate American Heart Association Pre-Doctoral Fellowship (Nyssa Hoch).

	References

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1. Go AS, Hylek EM, Phillips KA, Chang Y, Henault LE, Selby JV, Singer DE. Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the Anticoagulation and Risk Factors in Atrial Fibrillation (ATRIA) Study. JAMA. 2001; 285: 2370–2375.[Abstract/Free Full Text]
   
2. Fuster V, Ryden LE, Asinger RW, Cannom DS, Crijns HJ, Frye RL, Halperin JL, Kay GN, Klein WW, Levy S, McNamara RL, Prystowsky EN, Wann LS, Wyse DG, Gibbons RJ, Antman EM, Alpert JS, Faxon DP, Fuster V, Gregoratos G, Hiratzka LF, Jacobs AK, Russell RO, Smith SC, Klein WW, Alonso-Garcia A, Blomstrom-Lundqvist C, De Backer G, Flather M, Hradec J, Oto A, Parkhomenko A, Silber S, Torbicki A. ACC/AHA/ESC guidelines for the management of patients with atrial fibrillation: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the European Society of Cardiology Committee for Practice Guidelines and Policy Conferences (Committee to Develop Guidelines for the Management of Patients With Atrial Fibrillation): developed in collaboration with the North American Society of Pacing and Electrophysiology. J Am Coll Cardiol. 2001; 38: 1231–1266.[Free Full Text]
   
3. Carnes CA, Chung MK, Nakayama T, Nakayama H, Baliga RS, Piao S, Kanderian A, Pavia S, Hamlin RL, McCarthy PM, Bauer JA, Van Wagoner DR. Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation. Circ Res. 2001; 89: E32–E38.[CrossRef][Medline] [Order article via Infotrieve]
   
4. Mihm MJ, Yu F, Carnes CA, Reiser PJ, McCarthy PM, Van Wagoner DR, Bauer JA. Impaired myofibrillar energetics and oxidative injury during human atrial fibrillation. Circulation. 2001; 104: 174–180.[Abstract/Free Full Text]
   
5. De Vecchi E, Pala MG, Di Credico G, Agape V, Paolini G, Bonini PA, Grossi A, Paroni R. Relation between left ventricular function and oxidative stress in patients undergoing bypass surgery. Heart. 1998; 79: 242–247.[Abstract/Free Full Text]
   
6. Goette A, Arndt M, Rocken C, Spiess A, Staack T, Geller JC, Huth C, Ansorge S, Klein HU, Lendeckel U. Regulation of angiotensin II receptor subtypes during atrial fibrillation in humans. Circulation. 2000; 101: 2678–2681.[Abstract/Free Full Text]
   
7. Goette A, Staack T, Rocken C, Arndt M, Geller JC, Huth C, Ansorge S, Klein HU, Lendeckel U. Increased expression of extracellular signal-regulated kinase and angiotensin-converting enzyme in human atria during atrial fibrillation. J Am Coll Cardiol. 2000; 35: 1669–1677.[Abstract/Free Full Text]
   
8. Boldt A, Wetzel U, Weigl J, Garbade J, Lauschke J, Hindricks G, Kottkamp H, Gummert JF, Dhein S. Expression of angiotensin II receptors in human left and right atrial tissue in atrial fibrillation with and without underlying mitral valve disease. J Am Coll Cardiol. 2003; 42: 1785–1792.[Abstract/Free Full Text]
   
9. Vermes E, Tardif JC, Bourassa MG, Racine N, Levesque S, White M, Guerra PG, Ducharme A. Enalapril decreases the incidence of atrial fibrillation in patients with left ventricular dysfunction: insight from the Studies of Left Ventricular Dysfunction (SOLVD) trials. Circulation. 2003; 107: 2926–2931.[Abstract/Free Full Text]
   
10. Alsheikh-Ali AA, Wang PJ, Rand W, Konstam MA, Homoud MK, Link MS, Estes NA, III, Salem DN, Al Ahmad AM. Enalapril treatment and hospitalization with atrial tachyarrhythmias in patients with left ventricular dysfunction. Am Heart J. 2004; 147: 1061–1065.[CrossRef][Medline] [Order article via Infotrieve]
    
11. Mathew JP, Fontes ML, Tudor IC, Ramsay J, Duke P, Mazer CD, Barash PG, Hsu PH, Mangano DT. A multicenter risk index for atrial fibrillation after cardiac surgery. JAMA. 2004; 291: 1720–1729.[Abstract/Free Full Text]
    
12. Cai H, Li Z, Goette A, Mera F, Honeycutt C, Feterik K, Wilcox JN, Dudley SC Jr, Harrison DG, Langberg JJ. Downregulation of endocardial nitric oxide synthase expression and nitric oxide production in atrial fibrillation: potential mechanisms for atrial thrombosis and stroke. Circulation. 2002; 106: 2854–2858.[Abstract/Free Full Text]
    
13. Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid and thiols: implications for uncoupling endothelial nitric oxide synthase. J Biol Chem. 2003; 278: 22546–554.[Abstract/Free Full Text]
    
14. Fink B, Laude K, McCann L, Doughan A, Harrison DG, Dikalov S. Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol. 2004; 287: C895–C902.[Abstract/Free Full Text]
    
15. Spitz DR, Oberley LW. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal Biochem. 1989; 179: 8–18.[CrossRef][Medline] [Order article via Infotrieve]
    
16. Inoue N, Ramasamy S, Fukai T, Nerem RM, Harrison DG. Shear stress modulates expression of Cu/Zn superoxide dismutase in human aortic endothelial cells. Circ Res. 1996; 79: 32–37.[Abstract/Free Full Text]
    
17. Fukai T, Siegfried MR, Ushio-Fukai M, Cheng Y, Kojda G, Harrison DG. Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training. J Clin Invest. 2000; 105: 1631–1639.[Medline] [Order article via Infotrieve]
    
18. Woodman CR, Muller JM, Rush JW, Laughlin MH, Price EM. Flow regulation of ecNOS and Cu/Zn SOD mRNA expression in porcine coronary arterioles. Am J Physiol. 1999; 276: H1058–H1063.[Medline] [Order article via Infotrieve]
    
19. McNally JS, Davis ME, Giddens DP, Saha A, Hwang J, Dikalov S, Jo H, Harrison DG. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. Am J Physiol Heart Circ Physiol. 2003; 285: H2290–H2297.[Abstract/Free Full Text]
    
20. Byrne JA, Grieve DJ, Bendall JK, Li JM, Gove C, Lambeth JD, Cave AC, Shah AM. Contrasting roles of NADPH oxidase isoforms in pressure-overload versus angiotensin II–induced cardiac hypertrophy. Circ Res. 2003; 93: 802–805.[Abstract/Free Full Text]
    
21. Heymes C, Bendall JK, Ratajczak P, Cave AC, Samuel JL, Hasenfuss G, Shah AM. Increased myocardial NADPH oxidase activity in human heart failure. J Am Coll Cardiol. 2003; 41: 2164–2171.[Abstract/Free Full Text]
    
22. Krijnen PA, Meischl C, Hack CE, Meijer CJ, Visser CA, Roos D, Niessen HW. Increased Nox2 expression in human cardiomyocytes after acute myocardial infarction. J Clin Pathol. 2003; 56: 194–199.[Abstract/Free Full Text]
    
23. Sarfstein R, Gorzalczany Y, Mizrahi A, Berdichevsky Y, Molshanski-Mor S, Weinbaum C, Hirshberg M, Dagher MC, Pick E. Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras. J Biol Chem. 2004; 279: 16007–16016.[Abstract/Free Full Text]
    
24. Maack C, Kartes T, Kilter H, Schafers HJ, Nickenig G, Bohm M, Laufs U. Oxygen free radical release in human failing myocardium is associated with increased activity of rac1-GTPase and represents a target for statin treatment. Circulation. 2003; 108: 1567–1574.[Abstract/Free Full Text]
    
25. Price MO, Atkinson SJ, Knaus UG, Dinauer MC. Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent. J Biol Chem. 2002; 277: 19220–19228.[Abstract/Free Full Text]
    
26. Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK. Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24: 677–683.[Abstract/Free Full Text]
    
27. Swiatkowska M, Cierniewska-Cieslak A, Pawlowska Z, Cierniewski CS. Dual regulatory effects of nitric oxide on plasminogen activator inhibitor type 1 expression in endothelial cells. Eur J Biochem. 2000; 267: 1001–1007.[Medline] [Order article via Infotrieve]
    
28. Bouchie JL, Hansen H, Feener EP. Natriuretic factors and nitric oxide suppress plasminogen activator inhibitor-1 expression in vascular smooth muscle cells. Role of cGMP in the regulation of the plasminogen system. Arterioscler Thromb Vasc Biol. 1998; 18: 1771–1779.[Abstract/Free Full Text]
    
29. Kubo-Inoue M, Egashira K, Usui M, Takemoto M, Ohtani K, Katoh M, Shimokawa H, Takeshita A. Long-term inhibition of nitric oxide synthesis increases arterial thrombogenecity in rat carotid artery. Am J Physiol. 2002; 282: H1478–H1484.
    
30. Gabazza EC, Hayashi T, Ido M, Adachi Y, Suzuki K. Adenosine inhibits thrombin -induced expression of tissue factor on endothelial cells by a nitric oxide-mediated mechanism. Clin Sci (Lond). 2002; 102: 167–175.[Medline] [Order article via Infotrieve]
    
31. Yang Y, Loscalzo J. Regulation of tissue factor expression in human microvascular endothelial cells by nitric oxide. Circulation. 2000; 101: 2144–2148.[Abstract/Free Full Text]
    
32. Green J, Doughty L, Kaplan SS, Sasser H, Carcillo JA. The tissue factor and plasminogen activator inhibitor type-1 response in pediatric sepsis-induced multiple organ failure. Thromb Haemost. 2002; 87: 218–223.[Medline] [Order article via Infotrieve]
    
33. Taubman MB, Fallon JT, Schecter AD, Giesen P, Mendlowitz M, Fyfe BS, Marmur JD, Nemerson Y. Tissue factor in the pathogenesis of atherosclerosis. Thromb Haemost. 1997; 78: 200–204.[Medline] [Order article via Infotrieve]
    
34. Polack B, Pernod G, Barro C, Doussiere J. Role of oxygen radicals in tissue factor induction by endotoxin in blood monocytes. Haemostasis. 1997; 27: 193–200.[Medline] [Order article via Infotrieve]
    
35. Diamandopoulus L, Honeycutt C, Wilcox J, Langberg J, Harrison D, Dudley S Effect of atrial fibrillation on local expression of tissue factor in the left atrium and left atrial appendage. J Am Coll Cardiol. 2005; 45: 367A. Abstract.[CrossRef]
    
36. Shinagawa K, Mitamura H, Ogawa S, Nattel S. Effects of inhibiting Na ⁺/H⁺-exchange or angiotensin converting enzyme on atrial tachycardia-induced remodeling. Cardiovasc Res. 2002; 54: 438–446.[Abstract/Free Full Text]
    
37. Li D, Shinagawa K, Pang L, Leung TK, Cardin S, Wang Z, Nattel S. Effects of angiotensin-converting enzyme inhibition on the development of the atrial fibrillation substrate in dogs with ventricular tachypacing-induced congestive heart failure. Circulation. 2001; 104: 2608–2614.[Abstract/Free Full Text]
    
38. Shi Y, Li D, Tardif JC, Nattel S. Enalapril effects on atrial remodeling and atrial fibrillation in experimental congestive heart failure. Cardiovasc Res. 2002; 54: 456–461.[Abstract/Free Full Text]
    
39. Pedersen OD, Bagger H, Kober L, Torp-Pedersen C. Trandolapril reduces the incidence of atrial fibrillation after acute myocardial infarction in patients with left ventricular dysfunction. Circulation. 1999; 100: 376–380.[Abstract/Free Full Text]
    
40. Madrid AH, Bueno MG, Rebollo JM, Marin I, Pena G, Bernal E, Rodriguez A, Cano L, Cano JM, Cabeza P, Moro C. Use of irbesartan to maintain sinus rhythm in patients with long-lasting persistent atrial fibrillation: a prospective and randomized study. Circulation. 2002; 106: 331–336.[Abstract/Free Full Text]
    
41. Xiao HD, Fuchs S, Campbell DJ, Lewis W, Dudley SC Jr, Kasi VS, Hoit BD, Keshelava G, Zhao H, Capecchi MR, Bernstein KE. Mice with cardiac restricted angiotensin converting enzyme (ACE) have atrial enlargement, cardiac arrhythmia and sudden death. Am J Pathol. 2004; 165: 1019–1032.[Abstract/Free Full Text]
    
42. Wassmann S, Laufs U, Baumer AT, Muller K, Konkol C, Sauer H, Bohm M, Nickenig G. Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells: involvement of angiotensin AT1 receptor expression and Rac1 GTPase. Mol Pharmacol. 2001; 59: 646–654.[Abstract/Free Full Text]
    
43. Ito M, Adachi T, Pimentel DR, Ido Y, Colucci WS. Statins inhibit beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes via a Rac1-dependent mechanism. Circulation. 2004; 110: 412–418.[Abstract/Free Full Text]
    
44. Cook-Mills JM, Johnson JD, Deem TL, Ochi A, Wang L, Zheng Y. Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity. Biochem J. 2004; 378: 539–547.[CrossRef][Medline] [Order article via Infotrieve]
    
45. Allessie MA, Boyden PA, Camm AJ, Kleber AG, Lab MJ, Legato MJ, Rosen MR, Schwartz PJ, Spooner PM, Van Wagoner DR, Waldo AL. Pathophysiology and prevention of atrial fibrillation. Circulation. 2001; 103: 769–777.[Free Full Text]
    
46. Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation: a study in awake chronically instrumented goats. Circulation. 1995; 92: 1954–1968.[Abstract/Free Full Text]
    

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CLINICAL PERSPECTIVE

AF is a progressive disease associated with an increased risk of stroke due almost exclusively to emboli from the LAA. Although this problem has been attributed to stasis of blood in the LAA, we recently reported that AF reduces LAA endocardial NO^·production, which could be explained in part by increased superoxide production. Here, we demonstrate that, in a pacing model, AF increases production of ROS in the LA and LAA and that this increase is mediated by activation of the NAD(P)H oxidase and XO. Activation of the NAD(P)H oxidase was associated with increased amounts of the active cofactor Rac1. Because ROSs are prothrombotic, these findings may help to explain the proclivity for the clot to form in the LAA. Along with or coupled to electrical remodeling, increased radical production also may contribute to the vicious cycle of "AF begetting AF." There is evidence that therapies known to alter ROS production affect AF. For example, the NAD(P)H oxidase is activated by Ang II. In the TRACE trial, ACE inhibitor use was associated with a lower incidence of AF. In a prospective trial, losartan has been associated with a lower recurrence rate of AF after cardioversion. Results from the present study suggest that therapies designed to diminish ROS production may be useful for reducing the incidence of thromboembolism and AF.

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