GW26-e4609 Trimetazidine Attenuates Cardiac Remodeling Via Suppression AKT Signaling Pathway

JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, VOL. 66, NO. 16, SUPPL S, 2015

visualized by enhanced chemiluminescence detection kit. b-actin was used as an internal control. RESULTS Results showed that tanshinone IIA could protect H9c2 cells from H2O2-induced cell death concentration-dependently. MiR-133 was involved in tanshinone IIA action. Decreases of miR-133 expression induced by increasing concentration of H2O2 could be completely reversed by tanshinone IIA treatment. Inhibition of miR-133 function by transfection of specific inhibitor abolished the cardioprotective effects of tanshinone IIA against H2O2-induced cell death. Furthermore, our results also showed that tanshinone IIA could activate Akt kinase by phosphorylation at serine 473. We furtherly determined the Bcl-2 protein levels by western blot and found that tanshinone IIA could completely reverse the decreases of Bcl-2 protein induced by H2O2; but the effect of tanshinone IIA on Bcl-2 protein in oxidative environment was suppressed by PI3K inhibitor, wortmannin, which indicated that tanshinone IIA exerted cardioprotective effects against H2O2-induced cell death through the activation of PI3K/Akt signal transduction pathway and consequent upregulation of Bcl-2 expression. CONCLUSIONS The present study indicates that tanshinone IIA is a promising natural cardio-protective agent. GW26-e4380 Screening and Differences for Glycolytic Metabolism Related Genes Between Two Ventricles in Monocmtaline Induced Pulmonary Arterial Hypertension Rats Meihong Qiu,1 Weihua Zhang,2 Rui Zhang,3 Hongda Zhang,3 Zhicheng Jing,3,4 Shouyan Zhang1 1 Luoyang Central Hospital affiliated to Zhengzhou University; 2First Hospital of Jilin University; 3Shanghai Pulmonary Hospital, Tongji university; 4Fuwai Hospital, Chinese Academy of Medical Science and Peking Union Medical College OBJECTIVES To investigate the differences of cardiac energy metabolism between left and right ventricle in the monocmtaline (MCT) induced pulmonary arterial hypertension(PAH) rats. METHODS PAH was induced by a single subcutaneous injection of MCT (50 mg/kg) in rats, who developed right heart failure eventually. At the same time, control group was injected with normal saline. MCTPAH rats were randomly divided into three groups according to the treatment of MCT: MCT-2week, 3week, 4week group (MCT-2w, 3w, 4w). At the end of study, the hemodynamics and right ventricular hypertrophy were compared in each group. The expression levels of proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labeling (TUNEL) were detected between left and right ventricular myocardial cells. The differences of genes expression from the glycolytic key candidate genes were screened between two ventricles. RESULTS After MCT injection for three weeks, mean pulmonary arterial pressure, right ventricular systolic pressure and right ventricular hypertrophy index were significantly increased. The morphology and structure were changed obviously in left and right ventricular myocardial cells . Proliferation and apoptotic resistance of myocardial cells were markedly increased compared with control group. The expression of HK1 mRNA began to rise in right ventricle of MCT-3w group, which was obviously earlier than left ventricle (MCT-4w group). In the MCT-4w group, the expression of HK2 (hexokinase 2), PDHa1 (pyruvate dehydrogenase complex a1), LDHA (lactate dehydrogenase A) mRNA was significantly increased in left ventricle, while the expression of LDHA only were up-regulated in right ventricle. The HK1 mRNA expression was further confirmed by the expression of HK1 protein and immunohistochemistry analysis. CONCLUSIONS Energy metabolic shift occurred in the left and right ventricles in PAH. Up-regulated expression of HK1 appeared earlier in right ventricle than left ventricle. Interference of glycolysis of right ventricle may be a novel target of PAH in the future. GW26-e4397 Effect of Rosuvastatin on Vascular Remodeling and Vascular Reaction of Spontaneously Hypertension Rats Zhang Lizhen, Zhulin Zhang, Xiurui Ma, lizhen Zhang Shanxi cardiovascular hospital OBJECTIVES To observe the effect of rosuvastatin on remodel and vascular reaction of aorta from spontaneously hypertension rats (SHR).

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METHODS SHR and Wister-Kyoto (WKY) rats were randomly divided into：WKY control group; SHR treated with rosuvastatin 6.25 mg/ (kg/day); SHR treated with rosuvastatin 12.5 mg/(kg/day); rosuvastatin 25 mg/(kg/day); SHR control group. Therapy continued for 8 weeks. Tail systolic blood pressure was weekly measured . At the end of this study, aorta was moved rapidly. A part of aorta was fixed with 4% paraformaldehyde to observe the vascular remodeling by HE stain. The vascular reaction of aorta to different drugs was measured in vitro. RESULTS Compared with WKY, the systolic blood pressure was higher, and vessel lumen diameter (L) decreased, while media thickness (M) and M/L increased in SHR. The contractions to Phe, KCl in aorta were increased in SHR than in WKY. The relaxation to Ach decreased in SHR, but there was no difference of relaxation to SNP. Treatment with rosuvastatin induced a reduction in systolic blood pressure; increasing in L and decrease in M and M/L. Rosuvastatin reduced the contraction of aorta to Phe and KCl; increased the relaxation of aorta to Ach, but had no effect on the relaxation to SNP. CONCLUSIONS The antihypertensive effect of rosuvastatin in SHR was accompanued by protect on endothelial function: retarding the remodeling of aorta; improving the vascular reaction to different vasomotors, resuming the balance of relaxation and contraction. GW26-e4479 Nrf2 Is Crucially Required for Sulforaphane to Prevent High Fat Diet/Low Dose STZ Induced Diabetic Cardiomyopathy Yanli Cheng,1 Shudong Wang,1 Yang Zheng1,2 The First Hospital of Jilin University, Changchun, China; 2University of Louisville, Louisville, KY

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OBJECTIVES The present study aimed to define whether sulforaphane (SFN) protects from type 2 diabetic cardiomyopathy (DCM) entirely through Nrf2. METHODS 8 weeks of Nrf2-knockout (KO) and wild-types (WT, C57BL/ 6J) mice were used to induce a type 2 diabetes (T2DM), by feeding high-fat diet (HFD) for 3 months to induce insulin resistance and then injecting one dose of streptozotocin (STZ, 100 mg/kg body-weight) to induce hyperglycemia. Age-matched control mice were fed a normal diet (ND) for the same period. Both T2DM and control mice were treated with or without SFN at 0.5 mg/kg five days a week for 4 months along with continual feeding with either HFD or ND diet, respectively. Cardiac oxidative damage, hypertrophy and fibrosis were assessed by western blot, real-time qPCR and histopathological examination. RESULTS SFN prevented diabetes-increased cardiac oxidative damage (increased accumulation of 3-nitrotyrosine and 4-hydroxynonenal), hypertrophy (increased the ratio of heart weight to tibia length, b-MHC and atrial natriuretic peptide mRNA expression and wheat germ agglutinin staining) and fibrosis (increased the accumulation of collagen) along with increase in Nrf2 expressions in the WT mice. However, Nrf2-KO diabetic mice suffered from greater cardiac damage and had more severe pathological alterations in the heart. SFN cardiac protection was completely lost in Nrf2-KO diabetic mice. CONCLUSIONS The present study demonstrated for the first time that Nrf2 entirely mediates SFN’s prevention of HFD / STZ-induced type 2 diabetic cardiomyopathy.

GW26-e4609 Trimetazidine Attenuates Cardiac Remodeling Via Suppression AKT Signaling Pathway Zheng Yang, Qizhu Tang Renmin Hospital of Wuhan University OBJECTIVES Trimetazidine (TMZ) is widely used in the therapy of myocardial ischemia. Recent studies suggest that additional TMZ treatment may be able to improve the systolic-diastolic function in heart failure patients. But the effects and underlying mechanisms of TMZ in cardiac remodeling are still unclear. The purpose of this study is to estimate the effects of TMZ in cardiac remodeling and try to explore the underlying mechanisms. METHODS In our study, C57BL/6 mice (male, 8 to 10 weeks, 23.5 27.5 g weight) were subjected to aortic banding (AB) operation or Sham as control. After operation the mice were gavaged with TMZ (20mg/kg) or Placebo for 8 weeks. Then, echocardiography and Pressure-Volume detection were used in the assessment of cardiac

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JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, VOL. 66, NO. 16, SUPPL S, 2015

function. Heart weight/ body weight (HW/BW), lung weight/ body weight (LW/BW)and heart weight/ tibial length (HW/TL) were used to assess the cardiopulmonary damage caused by pressure overload. In the histological analysis, HE stain was used to evaluate the crosssectional area (CSA) of cardiomyocytes, and PSR stain was used to evaluate the collagen volume of left ventricle. Real Time PCR was used in the detection of mRNA expression levels of hypertrophic and fibrotic markers. To find a possible mechanism, the phosphoprotein levels of possible signaling pathways were detected by Western Blot. RESULTS As results, HW/BW and HW/TL were elevated after AB, but less obviously in TMZ group. LW/BW has no significant change after AB. In echocardiography, left ventricle end-diastolic diameter (LVEDD) and left ventricle end-systolic diameter (LVESD) were raised after AB, and more remarkably in Placebo group. Ejection fraction (EF) and fractional shorten (FS) were declined after AB, but descended less obviously in TMZ group. In the histological analysis, HE stain indicated the CSA of cardiomyocytes in TMZ group was ascended less remarkably after AB. Besides, PSR stain show that the collagen volume in TMZ group has a same tendency. In the detection of RT-PCR, the mRNA levels of ANP, BNP and b-MHC in TMZ group were raised much slightly after AB, and fibrotic markers, like CTGF, TGF-b2, Collagen Ia and Collagen III, have same tendencies. In the mechanism research, the phospho-protein levels in AKT signaling pathway, such as p-AKT, p-mTOR, p-4EBP1, p-eIF4e and, have been changed after AB, and TMZ inhibits the activation of AKT signaling pathway. CONCLUSIONS Through the inhibition of cardiac hypertrophy and fibrosis, TMZ presents a protective effect on cardiac remodeling via the suppression of AKT signaling pathway. TMZ is probably a potential therapeutic drug for cardiac remodeling. GW26-e4611 Male-Specific Association of APC rs383830-T Allele With the Risk of Coronary Heart Disease 1

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Jinyan Zhong, Xiaowei Zheng, Huadan Ye, Hanbin Cui, Weiping Du, Zhaoxia Zhang,1 Xiaohong Fei,1 Shaoyi Lin,1 Jian Wang,1 Jia Su,1 Shiwei Duan,2 Xiaomin Chen1 1 Ningbo First Hospital, School of Medicine, Ningbo University, Zhejiang 315200, China; 2Zhejiang Provincial Key Laboratory of Pathophysiology, School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China OBJECTIVES APC is a tumor suppressor gene that is involved in the processes of migration and adhesion, transcriptional activation, and apoptosis. The goal of this study is to evaluate the contribution of APC rs383830 polymorphism to coronary heart disease (CHD) in Han Chinese. METHODS A total of 783 CHD patients and 737 controls were tested in the current association study. RESULTS No significant difference of rs383830-T allele between cases and controls are observed (20.2% versus 18.2%, c2 ¼ 1.950, df ¼ 1, p ¼ 0.163, OR ¼ 1.138, 95% CI ¼ 0.949-1.363). APC rs383830 polymorphism and CHD, a breakdown analysis by gender indicated there was a significant contribution of rs383830-T allele to the risk of CHD in males (p ¼ 0.046, odds ratio ¼ 1.267, 95% confidential interval ¼ 1.004 1.598). CONCLUSIONS In conclusion, our study suggested a male-specific association of APC rs383830 polymorphism with CHD. GW26-e4684 Sphingosine-1-Phosphate Lyase Overexpression Attenuates HypoxiaInduced Pulmonary Arterial Hypertension Shihao Zhao, Wenjun Yan, Fuyang Zhang, Yunlong Xia, Wei Wang, Ling Tao Department of Cardiology, Xijing Hospital, Fourth Military Medical University OBJECTIVES Pulmonary arterial hypertension (PAH), which mostly happens among young people, is associated with high mortality due to right ventricular failure. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite. Previous studies indicate that S1P is increased in PAH patients and animals and may promote PAH. S1P lyase (SPL) is the only enzyme that irreversibly degrades S1P. The aims of our study are to: (1) determine whether SPL is decreased in PAH; (2) identify the role of SPL in PAH. METHODS Adult male SPL transgenic (SPL TG) mice and their wildtype (WT) counterparts were randomly subjected to the following

groups: WT - normoxia, WT-hypoxia, WT – normoxia þ THI (a specific inhibitor of SPL, fed in drinking water, 25mg/L), WT - hypoxiaþTHI, SPL TG - normoxia, SPL TG-hypoxia. The mice of hypoxia groups were kept in a chamber which mimics the condition of an altitude of 5000 meters for 16 hours per day. After 8 weeks, the right ventricular free wall thickness (RVWT) by echocardiography and the weight ratio of right ventricle to left ventricle plus septum (RV / (LVþS)) were used to assess right ventricular hypertrophy. We used HE staining of left lung sagittal sections to calculate (2  wall thickness/external diameter) 100% (%MWT) of pulmonary arteries to evaluate distal pulmonary arterial remodeling. Western blot and real-time PCR were used to examine SPL expression and phosphorylated ERK1/2. S1P levels in plasma and lung were examined by ELISA. RESULTS Compared with WT - normoxia group, mice of WT - hypoxia group showed evident PAH, as evidenced by increased RVWT (0.8570.039 mm vs. 0.5550.075 mm, P <0.01), RV / (LVþS) (0.2820.017 vs. 0.1980.013, P <0.05), and %MWT (0.3520.021 vs. 0.2140.014, P <0.05, WT - hypoxia group vs. WT - normoxia group). Moreover, SPL protein and mRNA levels were significantly decreased, while S1P level was significantly increased in lung tissue of mice in WT - hypoxia group (all P <0.05). Compared with WT hypoxia group, THI treatment significantly increased plasma S1P level and exacerbated pulmonary arterial remodeling and right ventricular hypertrophy (all P <0.05, WT – hypoxia þ THI group vs. WT - hypoxia group). Western blot showed that THI treatment increased ERK1/2 phosphorylation in lung tissue after hypoxia exposure (P <0.05). In contrast, SPL TG mice showed remarkably lower levels of plasma and lung S1P (both P <0.05) and alleviated pulmonary arterial remodeling and right ventricular hypertrophy (RVWT: 0.6260.063 mm vs. 0.8570.039mm; RV/(LVþS): 0.2140.015 vs. 0.2820.017; %MWT: 0.2450.017 vs. 0.3520.021; all P <0.05, SPL TG-hypoxia group vs. WT - hypoxia group). Moreover, SPL overexpression significantly reduced phosphorylated ERK1/2 in lung tissue after hypoxia exposure (P <0.05). CONCLUSIONS These data indicate that SPL downregulation contributes to increased S1P level and ERK1/2 activation in the progression of hypoxia-induced PAH. SPL might be a potential therapeutic target in PAH treatment.

GW26-e4709 Epigallocatechin-3-Gallate Protects HUVECs from PM2.5-Induced Oxidative Stress Injury by Activating Nrf2/HO-1 Pathways Guangzhao Yang,1 Zhao-Jun Wang,2,3 Feng Bai,1 Xiao-Jiang Qin,2 Jing Cao,1 Ming-Sheng Zhang,2,3 Ji-Yuan Lv1 1 The Fist Clinical Hospital, Shanxi Medical University, Taiyuan, Shanxi, China; 2Department of Pharmacology, Shanxi Medical University, Taiyuan, Shanxi, China; 3Department of Physiology, Shanxi Province Key Laboratory of Cellular Physiology, Shanxi Medical University, Taiyuan, Shanxi, C OBJECTIVES The aim of the present study was to investigate whether EGCG could also inhibit PM2.5-induced oxidative stress by activating the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in human umbilical vein endothelial cells (HUVECs). METHODS The effects of EGCG on PM2.5-induced reduction of HUVECs viability were evaluated using CCK-8 assays, and the viability of untreated HUVECs was taken as 100%. The effect of EGCG reduced PM2.5-induced ROS production in HUVECs was determined using flow cytometry and confocal fluorescence microscopy after DCFH2-DA staining. The inhibiting effect of ERK1/2 and p38 MAPK abrogated EGCG-induced Nrf2 and HO-1 expression in PM2.5-treated HUVECs were determined using Western Blotting and Real-Time PCR. Nrf2 silencing was designed to demonstrate the key role of Nrf2 in EGCG-induced Nrf2 and HO-1 upregulation in PM2.5-treated HUVECs. RESULTS CCK-8 assays demonstrated that 200, 300, and 400 mg/mL PM2.5 reduced HUVECs viability to 51.24%  6.90%, 45.97%  6.64%, and 38.32%  4.89%, respectively; the IC50 value was w200 mg/mL. DCFH2-DA staining showed that 200 mg/mL PM2.5 tripled ROS production (3.20  0.08 vs 1.00  0.12, p < 0.05). At 100 m M, EGCG alone did not affect ROS production significantly, but abolished the PM2.5induced increased of ROS production completely. Western blotting showed that 50 and 100 mg/mL PM2.5 increased Nrf2 expression to 162.6% and 267.3% and increased HO-1 expression to 230.0% and 394.9%, respectively. In contrast, at 200, 300, and 400 mg/mL, PM2.5 decreased Nrf2 expression to 57.6%, 6.4%, and 2.7%, and decreased HO-1 expression to 41.6%, 19.7%, and 11.7%, respectively. Real time