PRECLINICAL STUDY
Role of Rac1 GTPase Activation in Atrial Fibrillation
Oliver Adam, MD*,
Gregg Frost*,
Florian Custodis, MD*,
Mark A. Sussman, PhD ,
Hans-Joachim Schäfers, MD ,
Michael Böhm, MD* and
Ulrich Laufs, MD*,*
* Klinik für Innere Medizin III, Kardiologie, Angiologie und internistische Intensivmedizin, Universitätsklinikum des Saarlandes, Homburg/Saar, Germany
Abteilung für Thorax- und Herz- Gefäßchirurgie, Universitätsklinikum des Saarlandes, Homburg/Saar, Germany
San Diego State University, SDSU Heart Institute and Department of Biology, San Diego, California.
Manuscript received January 9, 2007;
revised manuscript received February 15, 2007,
accepted March 5, 2007.
* Reprint requests and correspondence: Dr. Ulrich Laufs, Klinik für Innere Medizin III Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, 66424 Homburg/Saar, Germany. (Email: ulrich{at}laufs.com).
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Abstract
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Objectives: We aimed to study the role of Rac1 GTPase in atrial fibrillation (AF).
Background: The signal transduction associated with AF is incompletely understood. We hypothesized that activation of Rac1 GTPase contributes to the pathogenesis of AF via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and production of reactive oxygen species.
Methods: Old mice with cardiac-specific overexpression of constitutively active V12Rac1 (RacET) were compared with wild-type (WT) and WT undergoing transaortic constriction (TAC). In addition, samples of human left atrial appendages were analyzed in patients with sinus rhythm (SR) compared with patients with permanent AF matched for atrial diameter.
Results: At age 16 months, 75% of RacET but no WT or TAC mice showed AF. Treatment of RacET with statins for 10 months did not alter weight or fibrosis of atria or ventricles but decreased cardiac Rac1 and NADPH oxidase activity and reduced the incidence of AF by 50%. The left atria of patients with AF showed increased fibrosis, up-regulation of NADPH oxidase activity, a 4-fold increase of Rac1 total protein and membrane expression as well as up-regulation of Rac1 activity.
Conclusions: Chronic cardiac overexpression of Rac1 represents a novel mouse model for AF. Rac1 GTPase contributes to the pathogenesis of AF and identifies a target for the prevention and treatment of AF.
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Abbreviations and Acronyms
| | AF = atrial fibrillation | | ECG = electrocardiography | | LA = left atrium | | LV = left ventricle | | NADPH = nicotinamide adenine dinucleotide phosphate | | RacET = transgenic mice with cardiac overexpression of Rac1 GTPase | | SR = sinus rhythm | | TAC = transverse aortic constriction | | WT = wild-type mice |
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Atrial fibrillation (AF) is the most common arrhythmia (1). The prevalence of AF is estimated at 0.4% of the general population, increasing with age to >6% in those over 80 years. Atrial fibrillation is associated with many forms of cardiac pathology, including coronary artery disease, heart failure, arterial hypertension, valvular disease, and cardiac surgery, but a substantial proportion of patients with AF have no detectable heart disease. Hemodynamic impairment and thromboembolic events related to AF result in significant morbidity, mortality, and cost. Atrial fibrillation is associated with atrial structural remodeling due to interstitial fibrosis. Other factors potentially involved in the induction or maintenance of AF include focal inflammation, endothelial/endocardial dysfunction, ischemia, and autonomic nervous system activity, but the detailed signal transduction leading to AF is incompletely understood (1,2).
Recent evidence shows that increased atrial oxidative stress might play an important role in inducing and maintaining AF in animal models and humans (3–5). Atrial fibrillation induced by rapid atrial pacing in pigs is characterized by increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and superoxide production in the left atrium (LA) (5). Right human atrial appendages of patients with AF exhibit higher levels of the oxidative markers 3-nitrotyrosine and protein carbonyls compared with patients with sinus rhythm (SR) (4). In the left ventricle (LV), myocardial oxidative stress is mediated, in part, by increased activity of the superoxide producing nicotinamide NADPH oxidase. The Rho GTPase Rac1 regulates NADPH oxidase activity and is critical for generating oxidative stress and producing cardiac LV hypertrophy (6–9). However, the role of Rac1 GTPase in atrial myocardium and during AF is not known.
We hypothesized that activation of Rac1 GTPase might contribute to the pathogenesis of AF via activation of superoxide producing NADPH oxidase. To address this hypothesis we studied a transgenic mouse model of cardiac-specific overexpression of Rac1 and analyzed left atrial myocardium from patients with AF.
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Methods
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Animal studies.
The characteristics of transgenic mice with cardiac overexpression of constitutively active (V12) Rac1 under the control of the alpha-myosin heavy chain (MHC) promoter (RacET) and wild-type control subjects (WT) have been described previously (10). Mice were fed with normal chow (ssniff, Soest, Germany) or normal chow supplemented with 0.4 mg/d of commercially available rosuvastatin (Crestor, AstraZeneca, London, United Kingdom). The study was not funded by the pharmaceutical industry. Rosuvastatin was chosen because of previous experience with this drug in mice (6). Heart rate, regularity, and presence of P waves were documented by 2 electrocardiography (ECG) recordings (Schwarzer CU 12 system, Picker, Munich, Germany) on the day before and on the day of sacrifice. Transthoracic echocardiography (GE Systems Vivid 5 scanner, 13 MHz transducer; Waukesha, Wisconsin) was performed in all mice. Transverse aortic constriction (TAC) or sham operation was performed at age 10 weeks in male animals as described (6). Pulmonary and hepatic congestion were defined as wet weight – dry weight. The study was approved by the animal ethics committee of the Universität des Saarlandes and is in accord with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH pub. no. 85-23, revised 1996).
Human left atrial tissue.
Tissue samples of the left atrial appendage of patients undergoing mitral valve surgery were analyzed in 7 consecutive patients with SR and 8 patients with permanent AF (documented by ECG for >3 months) matched for atrial diameter, left ventricular function, and medication. The patients did not receive drugs at least 12 h before surgery. Standardized echocardiography of all patients was obtained before surgery. Myocardial tissue was immediately frozen and stored at –80°C. The study of human myocardial tissue was approved by the ethics committee of the Ärztekammer des Saarlandes (no. 131/00).
NADPH oxidase activity.
The NADPH oxidase activity was measured by a lucigenin-enhanced chemiluminescence assay as described (6).
Western analysis.
For membrane preparation, tissue lysates were pelleted by low-speed centrifugation and lysed in hypotonic medium buffer (containing protease inhibitors [in µg/ml] phenylmethylsulfonyl fluoride 10, leupeptin 2, pepstatin A 2, and aprotinin 2) followed by 30 strokes of a glas-teflon homogenizer on ice. Nuclei were removed from the homogenate by centrifugation at 95 g (Biofuge pico; Heraeus, Kendro, France, r = 7 cm, 1,100 rpm) for 5 min at 4°C. The low-speed supernatant was then pelleted by centrifugation at 100,000 g (36,000 rpm, Beckman Ti-60.1, r = 6.9 cm) at 4°C in a Beckman L8-M preparative ultracentrifuge (Beckman Instruments, Inc., Palo Alto, California). Separation of cytosolic and membrane proteins was controlled by Western blotting with GAPDH (6C5, sc-32233, Santa Cruz Biotechnology, Santa Cruz, California). Immunoblotting was performed with Anti-Rac1 (Upstate, Waltham, Massachusetts; clone 23A8), ß-Actin (1:250 dilution, Santa Cruz Biotechnology), and goat anti-rabbit or goat anti-mouse secondary antibody (1:4,000 dilution, Sigma, St. Louis, Missouri) and an enhanced chemiluminescence kit (Amersham Biosciences, Pittsburgh, Pennsylvania) followed by densitometry (6,7,11). Expression was calculated with a standard curve generated with recombinant Rac1 protein (Upstate).
Rac1 GST–p21-activated kinase pull-down assay.
Tissue was homogenized and resuspended in magnesium-containing lysis buffer (25 mmol/l HEPES [pH 7.5]; 150 mmol/l sodium chloride; 1% Igepal CA-630; 10% glycerol; 25 mmol/l sodium fluoride; 10 mmol/l magnesium chloride; 1 mmol/l EDTA; 1 mmol/l sodium orthonvanadate; 10 µg/ml leupeptin; 10 µg/ml aprotinin) and centrifuged at 78 g (Biofuge pico Heraeus, r = 7 cm, 1,000 rpm) for 5 min at 4°C. Equal amounts of supernatant protein were incubated with 10 µl of agarose labeled p21-activated kinase (PAK)-1 fusion protein (Upstate) at 4°C for 60 min. Beads were washed 3 times with magnesium-containing lysis buffer, eluted in Laemmli buffer (60 mmol/l Tris [pH 6.8], 2% sodium dodecylsulfate, 10% glycerin, 0.1% bromphenol blue) and analyzed for bound Rac1 in relation to total Rac1 content by Western blotting.
Histologic analysis.
Ten-micrometer cryosections were fixed in acetone, and total collagen was stained with 0.1% Sirius Red F3BA (Polysciences, Warrington, Pennsylvania) in a saturated picric acid solution for 15 min followed by washing in aqua dest and rapid dehydration in 100% alcohol and xylene. For morphometric analysis, all sections were examined under a Nikon E600 microscope (Nikon, Tokyo, Japan). Lucia Measurement (Nikon, Duesseldorf, Germany) version 4.6 software was used for quantification of interstial fibrosis.
Statistical analysis.
Band intensities were analyzed by densitometry. All values are expressed as mean ± SEM. Unpaired Student t tests and analyses of variance for multiple comparisons were applied. Post hoc comparisons were performed with the Bonferroni test. The significance of the correlation of Rac1 activity with NADPH oxidase activity was calculated with the Pearson’s chi-square test. The SPSS (SPSS Inc., Chicago, Illinois) software, version 12.0, was used. Differences were considered significant at p < 0.05.
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Results
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AF in mice over-expressing Rac1 in the heart.
To test the role of Rac1 in the pathogenesis of AF, we studied the long-term effect of overexpression of Rac1 in mice with cardiac overexpression of constitutively active (V12) Rac1 under the control of the alpha-MHC promoter (RacET) (n = 10 to 15) (10). These mice show 2 distinct cardiomyopathic phenotypes: a lethal dilated phenotype associated with neonatal activation of the transgene; and a cardiac hypertrophy with survival comparable to WT, which was studied here. Concentric hypertrophy was evident in 3-week-old RacET ventricles (10). Hypertrophic characteristics diminish as the mice age, but echocardiography at age 16 months showed a persistent concentric hypertrophy compared with WT and the ratio of heart weight/tibia length was increased (Table 1, Fig. 1). Functionally, RacET exhibited decreased fractional shortening compared with WT, but pulmonary and hepatic congestion as indicators of symptomatic heart failure were not significantly increased (Table 1, Fig. 1). Because the aim of the study was to characterize the role of Rac1 in AF, a control group of WT mice was subjected to TAC to differentiate the effect of pressure-induced ventricular hypertrophy and secondary Rac1 activation on the atrial phenotype from the primary cardiac over-expression of Rac1 in RacET mice. At the age of 16 months, TAC mice showed concentric hypertrophy, decreased fractional shortening and increased pulmonary congestion compared with WT (Table 1, Fig. 1). In the atria, a differential effect of long-term pressure-induced ventricular hypertrophy and transgenic cardiac Rac1 over-expression on morphology and function was observed: the RacET mice showed significantly greater atria in relation to heart weight and tibia length compared with both WT and TAC (Table 1, Fig. 1G). Importantly, ECG revealed that 75% of 16-month-old RacET mice had AF, but all WT and TAC mice showed SR.
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Figure 1 RacET Mice Develop Atrial Dilatation and Atrial Fibrillation
(A) Representative cross sections of the left ventricle (LV), and (B) M-mode echocardiographic images of the LV of wild-type (WT), transgenic mice with cardiac overexpression of Rac1 GTPase (RacET)16, and transaortic constriction (TAC)16 mice. Quantification of measurements of (C) fractional shortening (FS); (D) diastolic interventricular septum (IVSd); (E) diastolic left posterior wall (LPWd); and (F) left ventricular end-diastolic diameter (LVDd); n = 6, 5 separate measurements/animal. (G) Ratio of weight of atria to total heart and tibia length. (H) Incidence of atrial fibrillation (AF), and (I) representative electrocardiography recording. n = 10, *p < 0.05 compared with WT.
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Increased atrial collagen and NADPH oxidase activity in RacET mice.
Interstitial collagen content quantitated by Sirius red staining was significantly increased in the atria of RacET (44 ± 1% of area) compared with WT (19 ± 5%, p < 0.05). The extent of interstitial fibrosis in TAC atria (31 ± 6%) was in between WT and RacET but did not significantly differ from either group (Fig. 2). In the LV, both RacET and TAC mice showed elevated collagen content compared with the control group (WT 9 ± 2%, RacET 29 ± 3%, p < 0.005 vs. WT; TAC 24 ± 4%, p < 0.05 vs. WT). In all mice, atrial collagen content exceeded ventricular collagen.
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Figure 2 RacET Mice Exhibit Increased Atrial Fibrosis and NADPH-Oxidase Activity
(A) Representative Sirius red staining (10- and 100-fold magnification) in the left atrium (LA) and the LV in WT, RacET16, and TAC16. (B) Quantification of interstitial fibrosis in the LA and (C) the LV. n = 10, *p < 0.05 versus WT, #p < 0.05 versus TAC16. (D) Atrial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity at baseline and after exposure to angiotensin II (Ang), 1 µmol/l, 10 min, in WT, RacET16, and TAC16; n = 6, *p < 0.05 versus WT, #p < 0.05 versus TAC16. Abbreviations as in Figure 1.
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Short term TAC has been shown to increase myocardial NADPH oxidase activity (6,9). Old TAC mice (16 months) displayed equal basal NADPH oxidase activity compared with WT control subjects but showed 2-fold greater NADPH oxidase activity upon stimulation with angiotensin II, 10 µmol/l, 10 min. In contrast, RacET mice exhibited markedly increased basal as well as angiotensin II stimulated NADPH oxidase activity (443 ± 213% and 571 ± 109%, respectively, p < 0.05) (Fig. 2D).
Reduction of Rac1 activity by statin decreases AF.
Oral treatment with rosuvastatin (0.4 mg/d) was initiated on the day before TAC. At the age of 10 months, atria of RacET were moderately enlarged compared with WT (19.4 ± 1.3 mg vs. 10.1 ± 1 mg) but considerably smaller compared with 16-month-old RacET (41.2 ± 8.1 mg) (Tables 1 and 2). Treatment of RacET with statin did not alter the weight of atria or ventricles (n = 10 to 15/group) (Table 2). As expected in the model, cardiac Rac1 activity was strongly increased by approximately 30-fold in RacET compared with WT. Despite transgenic expression of constitutively active V12Rac1, statin treatment reduced Rac1 activity to 66 ± 3.5% of vehicle treated RacET (p < 0.05) (Fig. 3A). The Rac1 total protein expression was not changed, however, Rac1 membrane expression was decreased to 76 ± 9% (Figs. 3B and 3C). Echocardiography showed a small but significant increase of fractional shortening and decreased LV wall thickness and slightly increased left ventricular end-diastolic diameters in the statin-treated group (Figs. 3D to 3H). Reduction of Rac1 activity by statin treatment was associated with a reduced incidence of AF by approximately 50% (20% in RacET + statin vs. 44% AF in RacET, p < 0.05) (Fig. 4A). Atrial collagen content was not reduced in the statin group (Figs. 4C and 4D). Statin treatment inhibited basal NADPH oxidase activity in 10-month-old RacET mice (215 ± 37% vs. 75 ± 12% of WT control, p < 0.05) as well as angiotensin II–induced NADPH oxidase activity (654 ± 160% vs. 186 ± 21% of WT control, p < 0.05) (Fig. 4E). Rac1 activity directly correlated with stimulated NADPH oxidase activity (R = 0.84, p < 0.05).
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Figure 3 Statin Treatment Reduces Rac1 Activity in RacET Mice
(A) Rac1-GTPase activity determined by GST–p21-activated kinase (PAK) pull-down assays in WT, 10-month-old RacET mice (RacET10), and RacET10 treated with rosuvastatin 0.4 mg/d orally for 10 months (RacET10 + statin), n = 8, *p < 0.05 versus RacET10. (B) Representative Western blots and (C) quantification of Rac1 GTPase expression (total protein, membrane, and cytsolic fraction) related to ß-actin in RacET10 and RacET10 + statin, n = 6, *p < 0.05 versus RacET10. (D) Representative M-mode echocardiographic images of the LV of RacET10 and RacET10 + statin. Quantification of measurements of (E) FS; (F) IVSd; (G) LPWd; and (H) LVDd; n = 8, 5 to 8 separate measurements/animal; *p < 0.05. Abbreviations as in Figure 1.
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Figure 4 Statin Treatment Reduces AF and NADPH-Oxidase Activity in RacET Mice
(A) Incidence of AF and (B) representative electrocardiography recording in RacET10 and RacET10 + statin, n = 10 to 15. (C) Representative Sirius red staining (10- and 100-fold magnification) and (D) quantification of interstitial fibrosis in the LA, n = 10 to 15, p = NS. (E) Atrial NADPH oxidase activity at baseline and after exposure to Ang, 1 µmol/l, 10 min, in RacET10 and RacET10 + statin; n = 8, *p < 0.05 versus untreated RacET10 and #p < 0.05 versus RacET10 + Ang. Abbreviations as in Figure 1.
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Increased collagen and NADPH-oxidase activity in human left atria in AF.
To test the role of Rac1 GTPase in human AF, tissue samples of the left atrial appendage of patients undergoing mitral valve surgery were analyzed in 7 consecutive patients with SR and 8 patients with permanent AF matched for atrial diameter, left ventricular function, and medication. Patient characteristics are summarized in Table 3. The size of the LA was measured before surgery by echocardiography and did not differ between groups (52.7 ± 4 mm vs. 52.14 ± 3.7 mm). Interstitial fibrosis was determined by Sirius red staining. In the LA of patients with AF, collagen content was significantly higher compared with SR (14.9 ± 2.1% vs. 8.5 ± 1.3%, p < 0.05) (Fig. 5). In unstimulated LA tissue, the activity of the superoxide-producing NADPH-oxidase did not differ between SR and AF (Fig. 5C). However, after pre-incubation with phorbol-12-myristate13-acetate (PMA), 1 µmol/l, 10 min, LA from patients with AF showed a >4-fold up-regulation of NADPH oxidase activity compared with atria from SR patients stimulated with PMA (p < 0.01).
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Figure 5 Correlation of AF With Rac1 in Human LA
(A) Representative Sirius red staining (10- and 100-fold magnification) and (B) quantification of interstitial fibrosis in LA of patients with sinus rhythm (SR) or AF who underwent cardiac surgery, n = 7 to 8, *p = 0.02. (C) NADPH oxidase activity determined in unstimulated LA of patients in SR or AF and after treatment with Ang, 1 µmol/l, 10 min, and phorbol-12-myristate13-acetate (PMA), 1 µmol/l, 10 min; n = 7 to 8, *p < 0.01. (D) Representative Western blots of Rac1 GTPase expression (total protein, membrane and cystolic fraction and Rac1-PAK pull down). (E) Quantification of Rac1 total protein expression related to ß-actin, *p < 0.01; (F) membrane content of Rac1, *p < 0.01; (G) Rac1-GTPase activity determined by GST-PAK pull-down assays, *p = 0.03; n = 7 to 8. Abbreviations as in Figure 1.
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Activation of Rac1 GTPase in human left atria in AF.
Animal experiments have shown that Rac1 GTPase is a regulator of NADPH oxidase activity in the LV. Expression of atrial Rac1 is not known. Therefore, Rac1 expression and activity was assessed in human LA. Western analysis showed an increase of Rac1 total protein expression in AF to 361 ± 47% (p < 0.001) compared with SR (Figs. 5D, 5E, 5F, and 5G). The cell membrane is the main site of Rac1 function. The membrane content of Rac1 was up-regulated to 428 ± 146% in LA from patients with AF (p < 0.05) compared with SR. The cytosolic expression of Rac1 was unchanged. With an affinity binding assay with the Cdc42 and Rac1 interactive binding domain from PAK1B as pull-down probe for Rac1-GTP, we investigated Rac1-GTPase activity. Figure 5G indicates that Rac1-GTPase activity/protein expression was up-regulated to 362 ± 98% in AF compared with SR (p < 0.05). The Rac1 activity of the human left atria directly correlated with their PMA-stimulated NADPH oxidase activity (R = 0.55, p < 0.05).
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Discussion
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In summary, the study shows that the small G protein Rac1 GTPase contributes to the signal transduction during AF. Mice with chronic activation of Rac1 in the heart developed AF with age—representing a novel mouse model for chronic AF. Neither old WT mice nor old mice with left ventricular hypertrophy and secondary atrial enlargement after TAC develop AF. Statin treatment, which reduces Rac1 activity, was able to decrease the incidence of AF. Analysis of human left atrial myocardium revealed that Rac1 GTPase and NADPH oxidase activity are up-regulated in patients with AF compared with patients in SR with equal atrial diameters.
Rac1 GTPase has been shown to participate in signalling pathways of left ventricular hypertrophy (10–12). Rac1 is necessary for the activation of the NADPH oxidase complex, a major source of myocardial superoxide production (12). In AF, oxidative damage contributes to altered myofibrillar energetics and contractile dysfunction (4). An animal model of rapid atrial pacing in pigs demonstrated that a NADPH oxidase mediates increased superoxide production in the LA (5). In isolated myocytes from human right atrial appendages, NADPH oxidase and, to a lesser extent, dysfunctional nitric oxide synthase were shown to contribute to superoxide production in AF (13). The NADPH oxidase-derived ROS might have several pathologic effects in the atrial myocardium including oxidative degradation of endocardial nitric oxide, local activation of coagulation cascade components and prothrombotic molecules such as PAI-1 and tissue factor facilitating–thrombus formation, induction of fibrosis, inflammatory responses, and alteration of ion channel function (3–5,13). To test whether Rac1 activation would contribute to the pathogenesis of AF, we studied old mice with cardiac-specific transgenesis of constitutively active V12Rac1 (10). The RacET mice showed increased NADPH oxidase activity. The main novel observation was that RacET mice develop AF. At the ages of 10 months and 16 months, respectively, 44% and 75% of RacET displayed AF on ECG. This parallels the natural history of AF, which is characterized by an increased prevalence with age (1). Aging of RacET mice was associated with increased size of the atria but not the ventricles. Left ventricular weight was greater in 10-month-old compared with 16-month-old RacET, demonstrating an effect of the transgen in the atria. This observation is supported by control experiments characterizing the long-term effects of pressure-induced left ventricular hypertrophy. Transaortic constriction results in Rac1 and NADPH oxidase activation; however, the extent of this secondary Rac1 activation is significantly smaller compared with V12Rac transgenesis (6,9). Importantly, despite old age, continuous aortic banding, LV hypertrophy and fibrosis, secondary atrial enlargement, symptoms of heart failure, and increased activity of Rac1 and NADPH oxidase in the ventricle, not a single TAC mouse developed AF. The atria of 16-month-old TAC mice, all of which showed SR, were larger than the atria of 10-month-old RacET mice that exhibited AF in 44%. Statin treatment reduced the incidence of AF but did not reduce atrial size or the extent of fibrosis. These data suggest that in RacET mice atrial Rac1 transgenicity significantly contributed to the development of a substrate of AF that is at least in part independent of atrial dilatation and fibrosis.
The RacET mice show that altered Rac1-mediated signal transduction contributes to the pathogenesis of AF. However, AF itself produces changes in atrial function and structure that are likely to alter Rac1 function (14). The RacET model does not provide information regarding the extent to which the perpetuation of AF is dependent on Rac1 activity. We speculate that the basis for the Rac1-mediated atrial phenotype is likely to be a combinatorial effect of postnatal development, transgene expression, and reactive signalling mechanisms. The creation of a mouse model of conditional cardiac Rac1 transgene expression could help to overcome some of these limitations of the RacET mice.
Several mouse models of increased susceptibility for the induction (e.g., by pacing) of atrial arrhythmias have been reported (15–18). The RacET mice are unique compared with these models, because they spontaneously develop AF, no other arrhythmia was recorded, and the AF was persistent. The other known mouse models that exhibit AF are characterized by increased mortality, lethal phenotype, or severe left ventricular pathology (19–24). In contrast, the RacET mice studied here show a normal life span and no obvious extracardiac pathology. RacET exhibit only mild LV hypertrophy, and pulmonary or hepatic congestion was not different from WT. In summary, compared with the relatively few other mouse models with atrial arrhythmias, RacET provide a more specific model for the study of AF. Aging is an important factor for the development of AF in the RacET, paralleling the increased incidence of AF in elderly humans. It seems possible that other mouse models of increased cardiac oxidative stress or inflammation might develop AF if studied for longer periods of time (e.g., >1.5 years).
In addition to superoxide produced by the NADPH oxidase, further downstream effects and protein interactions of Rac1 signalling such as PAK and Src or Rho family members are likely to contribute to cellular transformation. Interestingly, a recent study demonstrated that localization of the gap junction protein connexin 43 was determined through the Rac1 pathway downstream of N-cadherin in cardiac myocytes (25). Information from transgenic mice with atrial arrhythmias will be helpful to further elucidate downstream effects of Rac1 and NADPH oxidase. Candidates are inflammatory cytokines and growth factors (15,17,21), extracellular matrix, cell–cell interactions, and membrane proteins (19) as well as regulators of potassium and calcium currents (16,19,20,24).
Signal transduction during AF substantially differs between atria and ventricles (e.g., several current pharmacological agents for the treatment of AF are limited by ventricular pro-arrhythmia) (26). In addition, a recent micro-array analysis of a pig model of AF suggested that genomic changes differ between the right and the left atrium (27). Atrial fibrillation is usually initiated and maintained in the LA, because the cycle length is shorter in the left compared with the right atrium during AF. However, the available information on signal transduction during AF in human LA is limited. We therefore compared samples of the LA from patients in SR and permanent AF undergoing mitral valve surgery. The atria of both groups were only moderately enlarged and did not differ in size. Left atrium of AF patients exhibited increased interstitial fibrosis as an indicator of structural remodeling, in agreement with previous studies (14,28). The study demonstrates that the left atria of patients with AF are characterized by a marked up-regulation of the membrane expression and activity of Rac1 that correlates directly with increased sensitivity of NADPH oxidase activity.
The HMG-CoA reductase inhibitors (statins), in addition to inhibiting cholesterol synthesis, down-regulate Rac1-GTPase activity by reducing isoprenylation and translocation of Rac1 to the cell membrane (29). Animal models have demonstrated that inhibition of Rac1 by statin treatment decreases NADPH oxidase–induced superoxide production in cardiac myocytes and reduces left ventricular hypertrophy (6,7,9,11,12,29). On the basis of these data, we tested the effect of long-tern statin treatment on atrial pathology in RacET mice. Statin treatment was able to reduce Rac1 activity despite overexpression of constitutively active V12Rac the transgenic hearts. Statin treatment of RacET for 10 months did not alter the size of atria or ventricles. Echocardiography showed a small improvement of fractional shortening and a reduction of wall thickness. Atrial collagen content was not reduced. However, inhibition of Rac1 activity by statin treatment was associated with a 50% reduction of the incidence of AF. We believe that in addition to inhibition of Rac1, the effect of statins on prenylation of other proteins as well as anti-inflammatory effects of statins are likely to contribute to the observed effects. The data are in agreement with recent findings in dogs, where simvastatin suppressed tachypacing-induced shortening of atrial refractoriness (30) and atorvastatin prevented maintenance of AF in a model of sterile pericarditis (31). Furthermore, several lines of clinical evidence support an antiarrhythmic effect of statins in AF, demonstrating reduction of AF in patients with coronary disease, after surgery and after electrical cardioversion, and in patients with paroxysmal AF. The recent ARMYDA-3 (Atorvastatin for Reduction of MYocardial Dysrhythmia After cardiac surgery) study prospectively showed that statin treatment prevents postoperative AF (32–34). Our data demonstrate that inhibition of Rac1 GTPase represents an important molecular mechanism underlying these effects. We speculate that inhibition of Rac1 signalling might significantly contribute to other anti-AF treatment strategies (e.g., by inhibitors of the aldosterone-renin-angiotensin system) (35).
In summary, left atria of patients with AF exhibit up-regulation of Rac1 correlating with increased NADPH oxidase activity. In mice, chronic cardiac overexpression of Rac1 represents a novel model for AF. Rac1 GTPase contributes to the pathogenesis of AF and might represent a target for the prevention and treatment of AF.
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Acknowledgments
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The authors thank Simone Jäger and Ellen Becker for their excellent technical assistance.
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Footnotes
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This study was supported by the Deutsche Forschungsgemeinschaft (to Dr. Laufs) and the Universität des Saarlandes (HOMFOR).
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References
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