Endothelial Nitric Oxide Synthase–Independent Protective Action of Statin Against Angiotensin II–Induced Atrial Remodeling via Reduced Oxidant Injury
Activation of the renin-angiotensin system exacerbates atrial remodeling, leading to atrial fibrillation and thrombosis, especially in a condition with decreased NO bioavailability. Recently, it has been reported that statins reduce the incidence of atrial fibrillation through attenuation of atrial remodeling; however, the mechanisms have not been completely elucidated. Therefore, we aimed to clarify the beneficial effect of statin on atrial remodeling in condition with reduced NO bioavailability. Endothelial NO synthase−/− mice were sham operated or infused with angiotensin II (Ang II) via an osmotic minipump for 2 weeks, and Ang II–infused mice were divided into 3 treatment groups: pitavastatin, Tempol (a free radical scavenger), or vehicle. Echocardiography and electrocardiography showed that Ang II infusion caused left atrial enlargement and a high incidence of atrial fibrillation, whereas pitavastatin and Tempol prevented these abnormalities. In histological analysis, Ang II–induced atrial interstitial fibrosis, perivascular fibrosis, and cardiomyocyte hypertrophy were all attenuated by pitavastatin and Tempol. Immunohistochemical staining showed that Ang II downregulated thrombomodulin and tissue factor pathway inhibitor and upregulated tissue factor and plasminogen activator inhibitor 1 in the left atrium and that pitavastatin and Tempol corrected the thrombogenic condition. Moreover, pitavastatin and Tempol reduced Ang II–induced atrial superoxide production and atrial transforming growth factor-β1 expression and Smad 2/3 phosphorylation. Atrial rac1-GTPase activity, known to activate NADPH oxidase, was attenuated by pitavastatin but not by Tempol. In conclusion, pitavastatin exerts endothelial NO synthase–independent protective actions against Ang II–induced atrial remodeling and atrial fibrillation with enhanced thrombogenicity through suppression of oxidant injury.
Activation of the renin-angiotensin system plays a central role in cardiac remodeling, including atrial remodeling, leading to atrial fibrillation (AF) and cardiogenic embolism.1–4 It has been reported recently that statins reduce the incidence of AF through attenuation of atrial remodeling.5 It is well known that statins have not only cholesterol-lowering effects but also pleiotropic effects on the cardiovascular system, including improvement of endothelial function and anti-inflammatory and antioxidant effects.6–8 Although it is known that these cholesterol-lowing independent effects of statins are attributable to enhancement of endothelial NO synthase (eNOS) expression and activation,9,10 we reported previously that pitavastatin exerts eNOS-independent protective actions against angiotensin II (Ang II)–induced left ventricular remodeling through suppression of oxidative stress and the transforming growth factor (TGF)-β1–Smad 2/3 signaling pathway.11 However, it has not been determined whether pitavastatin has eNOS-independent protective actions against atrial remodeling and incidence of AF. Superoxide is mainly produced by NADPH oxidase in the cardiovascular system, and, recently, rac1, a small G protein, was shown to be a key molecule for the assembly and function of NADPH oxidase components.12 We, therefore, hypothesized that pitavastatin has an eNOS-independent protective action against Ang II–induced atrial remodeling and AF through attenuation of rac1-mediated oxidative stress.
Animals and Experimental Protocol
Ten-week–old male eNOS−/− mice (The Jackson Laboratory, Bar Harbor, ME) were subjected to sham operation or subcutaneous infusion of Ang II (WAKO) at a rate of 2.0 mg/kg per day for 14 days via an osmotic minipump (Alzet model 1002, Alza Corp). The Ang II-infused eNOS−/− mice were divided into 3 groups: ad libitum administration of pitavastatin (NK 104, Kowa Pharmaceutical Co, Ltd), Tempol (Sigma-Aldrich Japan; a free radical scavenger), or vehicle. Pitavastatin and Tempol were dissolved in drinking water at concentrations of 2.5 and 12.5 mg/L, respectively. The average daily amount of drinking water of eNOS−/− mice was ≈2.5 mL, and average body weight was ≈22 to 24 g (Table). Therefore, the estimated intakes of pitavastatin and Tempol were ≈0.3 and 1.5 mg/kg per day, respectively. We stated previously that plasma pitavastatin concentration in these pitavastatin-treated mice was comparable to and below that observed in clinical use.11 After 14-day infusion of Ang II, the mice were euthanized for analyses. All of the experimental procedures were performed in accordance with the guidelines of the animal research committee at the University of Tokushima Graduate School.
Measurement of Blood Pressure and Heart Rate
Blood pressure and heart rate were measured using a noninvasive computerized tail-cuff system (BP98A, Softron).
Measurement of Plasma Lipids and Glucose Levels
Plasma levels of lipids and glucose were analyzed by enzymatic methods.
Transthoracic echocardiography was performed using a 15-MHz imaging transducer (Aplio 80, Toshiba Medical Systems). The volume of the left atrium (LA) was calculated by biplane modified Simpson method.
Histological Analysis and Immunohistochemical Staining
The whole heart was resected, fixed, and stained with Azan. The cross-sectional areas of atrial cardiomyocytes, interstitial fibrosis, perivascular fibrosis, and medial thickening of atrial coronary arteries were calculated. Samples of atria were stained using antibodies for TGF-β1, tissue factor, plasminogen activator inhibitor type 1, thrombomodulin, and tissue factor pathway inhibitor. All of the antibodies were obtained from Santa Cruz Biotechnology.
Electrocardiography was performed using a PowerLab system (AD Instruments).
Western Blot Analysis
Atrial TGF-β1 expression and downstream Smad 2/3 activation were evaluated by Western blot analysis. The antibodies were obtained from Santa Cruz Biotechnology.
Superoxide Detection and Measurement of Rac1 Activation
Atrial superoxide production was evaluated by in situ dihydroethidium staining, and atrial activated rac1 was determined by a Rac Activation Assay kit (Upstate Biotechnology).
RNA Extraction and Quantitative Real-Time PCR
The atrial mRNA expression levels of gp91phox, p22phox, p67phox, p47phox, and rac1 were determined by quantitative real-time PCR with SYBR green detection. Primers for gp91phox, p22phox, and p47phox were obtained from Takara Bio, and primers for p67phox and rac1 were obtained from Qiagen.
All of the data are expressed as mean±SE. For comparisons among groups, statistical significance was assessed using 1-way ANOVA, and the significance of each difference was determined by post hoc testing with the Tukey-Kramer method.
An expanded Methods section is available in the online Data Supplement at http://hyper.ahajournals.org.
Although Ang II decreased body weight in eNOS−/− mice, both pitavastatin and Tempol treatment maintained body weight in eNOS−/− mice during Ang II infusion (Table).
Blood Pressure and Heart Rate
Systolic and diastolic blood pressure were elevated by Ang II infusion. Pitavastatin and Tempol treatment did not affect blood pressure. There were no significant differences in heart rate among the groups (Table).
Plasma Lipid Profile and Glucose Level
Ang II infusion caused a significant elevation of plasma total cholesterol, triglyceride, and fasting plasma glucose levels in vehicle-, pitavastatin-, and Tempol-treated mice, with no significant differences among the groups. There were also no significant differences in high-density lipoprotein cholesterol among the groups (Table).
Pitavastatin and Tempol Prevent Ang II–Induced Atrial Enlargement
Ultrasonographic studies revealed that Ang II stimulation caused a significant increase in LA volume in vehicle-treated mice; however, Ang II treatment did not significantly increase LA volume in pitavastatin- and Tempol-treated mice. Although atrial weight was also increased by Ang II stimulation, pitavastatin and Tempol treatment attenuated this change (Figure 1).
Pitavastatin and Tempol Attenuate Ang II–Induced Cardiomyocyte Hypertrophy, Cardiac Interstitial Fibrosis, and Perivascular Fibrosis of Coronary Arteries in LA
Quantitative analyses of microscopic findings of the LA revealed that Ang II caused hypertrophic changes of cardiomyocytes and marked interstitial fibrosis, along with accelerated perivascular fibrosis of atrial coronary arteries; however, pitavastatin and Tempol treatment prevented Ang II–induced LA remodeling. There were no significant differences in the medial area of atrial coronary arteries in any group (Figure 2).
Pitavastatin and Tempol Prevent Ang II–Induced AF
Electrocardiography showed that Ang II infusion caused a high incidence of persistent AF of 33.3% in eNOS−/− mice, whereas both pitavastatin and Tempol treatment decreased the incidence of Ang II–Induced AF to 8.3% and 12.5%, respectively (Figure 3).
Pitavastatin and Tempol Attenuate Ang II–Induced Oxidative Stress
Dihydroethidium-positive spots indicating superoxide production were markedly increased in the atrial myocardium and atrial coronary arteries by Ang II stimulation in vehicle-treated mice. In contrast, pitavastatin and Tempol treatment reduced the increase in dihydroethidium-positive spots in Ang II–infused eNOS−/− mice (Figure 4A).
Pitavastatin and Tempol Attenuate Ang II–Induced Atrial TGF-β1 Expression and Smad 2/3 Activation
Marked enhancement of atrial TGF-β1 expression in Ang II–infused eNOS−/− mice was demonstrated by immunohistochemical staining and Western blot analysis. In addition, Ang II stimulation caused marked enhancement of atrial Smad 2/3 phosphorylation in vehicle-treated mice. In contrast, pitavastatin and Tempol treatment attenuated these changes in Ang II–treated eNOS−/− mice (Figure 4).
Pitavastatin But Not Tempol Attenuates Ang II–Induced Atrial Rac1 Activation
Atrial mRNA expression of p22phox, p67phox, and rac1 was enhanced by Ang II infusion; however, pitavastatin and Tempol treatment did not attenuate these changes. There were no significant changes in mRNA expression of gp91phox and p47phox in any group, whereas a pull-down assay revealed that Ang II infusion markedly increased the active form of rac1 in the atrium; however, accelerated rac1 activation was attenuated by pitavastatin treatment but not by Tempol treatment (see Figure S1 in the online Data Supplement and Figure 5).
Pitavastatin and Tempol Ameliorate Expression of Thrombogenicity Regulating Factors in the Atrial Endocardium
Immunohistochemical staining showed that Ang II downregulated the expression of thrombomodulin and tissue factor pathway inhibitor and upregulated the expression of tissue factor and plasminogen activator inhibitor type 1 in the endothelium of the LA, indicating that Ang II enhanced thrombogenicity in the LA wall; however, pitavastatin and Tempol treatment corrected the Ang II–induced aberrant expression of these thrombogenicity regulating factors (Figure 6).
LA remodeling refers to a time-dependent adaptive response of cardiac myocytes to counteract external stressors and is accompanied by structural and functional changes, which evoke AF through electric remodeling.13–15 AF is associated with not only decreased quality of life but also an increased incidence of congestive heart failure and stroke, leading to increased morbidity and mortality. Therefore, prevention of LA remodeling may have a clinical impact on promoting public health.
Although it has been reported that the atrium is more susceptible to fibrotic changes than the ventricle,16 the precise mechanisms are still unknown. It has been shown that the plasma level of NOx, an NO metabolite, was significantly decreased in AF patients17 and that AF was associated with a marked decrease in eNOS protein expression.18 In addition, long-term inhibition of NO synthesis has been shown to induce fibrotic changes in the LA, as well as in the left ventricle.19
Recently, it has been reported that angiotensin-converting enzyme inhibitors and Ang II receptor blockers were associated with a low incidence of AF.20,21 In addition, statins were recognized to be effective for preventing AF after cardioversion and cardiac surgery in patients with coronary artery disease.22–24 These effects of statins are thought to be independent of their lipid-lowering effect, as shown in our study.
Carnes et al25 reported that antioxidant treatment using ascorbic acid prevented the onset of AF. Because we showed that Tempol attenuated Ang II–induced atrial remodeling even in eNOS−/− mice, oxidative stress may be a therapeutic target for preventing atrial remodeling and AF in patients with endothelial dysfunction. In addition, both pitavastatin and Tempol prevented Ang II–induced body weight loss in eNOS−/− mice. There is a possibility that pitavastatin protects against not only atrial remodeling but also Ang II–induced muscle wasting by its antioxidant effect.26
In the cardiovascular system, NADPH oxidase is thought to be a key factor in reactive oxygen species production. In addition, rac1 activation is necessary for NADPH oxidase activation and has been shown to participate in the signaling pathways of left ventricular hypertrophy and atrial remodeling.12,27 Satoh et al28 showed that Ang II–induced cardiac hypertrophy was not observed in cardiac rac1−/− mice. Furthermore, statins downregulate rac1-GTPase activity by reducing isoprenylation through suppression of geranyl-geranyl pyrophosphate production and prevent the development of cardiac hypertrophy.12,29 In the present study, we found that pitavastatin, but not Tempol, completely suppressed Ang II–induced rac1 activation. This result indicates that rac1 activation is an upstream event in the production of reactive oxygen species by renin-angiotensin system activation.
TGF-β1 is also an essential molecule for cardiac remodeling, because cardiac hypertrophy was not observed in Ang II–treated TGF-β−/− mice.30 We showed that the TGF-β1–Smad 2/3 signaling pathway is also involved in LA remodeling.
In conclusion, pitavastatin exerts eNOS-independent protective actions against Ang II–induced atrial remodeling, with a reduced incidence of AF and thrombogenicity through inhibition of the TGF-β1–Smad 2/3 signaling pathway by suppression of oxidant injury.
This animal study indicates that pitavastatin treatment is a potential strategy for the prevention of atrial remodeling and incidence of AF in renin-angiotensin system–activated patients and even in the patients with endothelial dysfunction. The hypothesis on the basis of our results that pitavastatin prevents the incidence of AF in subjects with impaired endothelial function needs to be confirmed by a larger cohort study.
Sources of Funding
This work was supported in part by Grants-in-Aid for Scientific Research; the Center-of-Excellence Program of the 21st Century from the Ministry of Education, Science, Sports, and Culture of Japan; and by Grant for a Study Group on Aseptic Femoral Neck Necrosis from the Ministry of Health, Labor, and Welfare of Japan.
- Received November 10, 2009.
- Revision received November 21, 2009.
- Accepted January 30, 2010.
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