Role of Nrf2 in dh404-induced suppression of cardiomyocyte hypertrophy and death, and cardiac fibroblast proliferation in vitro.

<p>(A) Effect of dh404 (200 nmol/L) on NE (20 µmol/L)-induced [³H]leucine uptake (left pannel) and H₂O₂ (100 µmol/L)-induced cell death of the cardiomyocytes infected with adenovirus of control (cont) shRNA and Nrf2 shRNA. *p<0.05 vs cont shRNA (-), n = 4. (B) Left pannel - Effect of dh404 (200 nmol/L) on Ang II (0.1 µmol/L)-, NE (20 µmol/L), and PE (20 µmol/L)-induced [³H]thymidine uptake in the cardiac fibroblasts. *p<0.05 vs control dh404 (-), n = 6. Right pannel – Effect of dh404 (200 nmol/L) on NE (20 µmol/L)-induced [³H]thymidine uptake in the cardiac fibroblasts infected with adenovirus of control (cont) shRNA and Nrf2 shRNA. *p<0.05 vs control dh404 (-), n = 4.</p

Effect of dh404 on myocardial fetal gene expression of mice after TAC.

<p>Hearts of mice were harvested, and left ventricles were dissected for RNA purification 4 weeks after the operation. Expression of ANF, BNP, αMHC, and βMHC was quantified by Q-PCR. *p<0.05 vs sham.</p

Echocardiography and pathology of mice treated with and without dh404 for 4 wks after TAC.

<p>Two dimension guided M-mode echocardiography and pathology were performed 4 weeks after the initiation of TAC or sham surgery in C57BL/6J mice treated with vehicle or dh404 as indicated.</p><p>Abbreviations: IVSd, interventricular septum diastolic; LVIDd, left ventricular internal dimension diastolic; LVIDs, left ventricular internal septum diastolic; LVPWd, left ventricular posterior wall diastolic; FS, fractional shortening; BW, body weight; HW/Tibia L, heart weight/tibia length ratio; LW/Tibia-L, lung weight/tibia length ratio.</p>a<p>p<0.05 vs sham (0);</p>b<p>p<0.05 vs TAC (0);</p>c<p>p<0.05 vs TAC + dh404 (5 mg/kg/d);</p>d<p>p<0.05 vs TAC + dh404 (10 mg/kg/d).</p

Effect of dh404 on myocardial fibrosis in mice after TAC.

<p>Myocardial fibrosis was assessed by staining of collagen with a Masson’s Trichrome Kit. In the left panels representative photomicrographs of a left ventricular (LV) and interventricular septum (IVS) sections from TAC hearts with and without dh404 treatment are presented. In the right panel the fibrotic areas of LV and IVS interstitial fibrosis as a percentage of total microscopic area per heart are presented. The numbers represent the number of sham and TAC hearts analyzed.</p

Effect of dh404 on myocardial apoptosis in mice after TAC.

<p>(A) In the left panel, the representative TUNEL staining of apoptotic cells in left ventricle are shown. TUNEL-positive, i.e., apoptotic nuclei in TAC hearts are stained red, nuclei blue and the myocardium is green (Alexa Fluor 488 Phalloidin to mark F-actin). Magnification, X 630. In the right panel, the apoptotic index results are given. TUNEL-positive cells were quantified as a percent of all nuclei in the section of LV. The number of hearts analyzed for each group is indicated. (B) In the left panel, the representative staining of cleaved caspase-3 positive cells in the left ventricle are shown. Cleaved caspase-3-positive, (i.e., cells with activated caspase-3) in TAC hearts are stained red and nuclei are blue. Cardiomyocytes were green utilizing an antibody of anti-cardiac myosin heavy chain. Magnification, X 630. In the right panel, the relative caspase-3 activity is summarized. Cleaved caspase-3-positive cells were quantified as a percent of all in a section of LV. The number of hearts analyzed for each group is indicated.</p

Effect of dh404 on myocardial Nrf2 expression of mice after TAC.

<p>C57BL/6J mice at age of 8 weeks were treated with vehicle or dh404 (10 mg/kg/d) and subjected to sham or TAC operations. Their hearts were harvested 4 weeks after the operation and analyzed for Nrf2 protein expression. Left panel - representative immunohistochemical staining of Nrf2 protein expression in left ventricles from TAC mice with or without dh404 treatment. Nrf2 nuclear translocation is indicated by arrows. Nrf2 staining was performed in 4 or more sections per heart. Nrf2 is stained red, nuclei blue and cardiomyocytes green utilizing an antibody of anti-cardiac myosin heavy chain. Right panel - a semi-quantitative analysis of Nrf2 protein levels in the heart. The number of hearts analyzed is indicated.</p

Effect of dh404 on myocardial oxidative stress of mice after TAC.

<p>Eight wk old C57BL/6J mice were grouped and treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044899#pone-0044899-g005" target="_blank">Figure 5</a>. Left panel - representative confocal microscopic images of left ventricular 4-HNE staining and 8-OHdG staining. Areas positive for 4-HNE and 8-OHdG are shown in red, nuclei in blue were labelled with DAPI, and. cardiomyocytes in green were marked using anti-Tropomyosin I antibody against cardiac myocyte tropomyosin. Right panel - levels of 4-HNE and 8-OHdG were semi-quantified by measuring IOD of eight randomly chosen fields in each myocardial tissue section. The number of hearts (<i>n</i>) analyzed is indicated.</p