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
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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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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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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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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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