47 Induced Cardiomyocyte Apoptosis

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JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, VOL. 70, NO. 16, SUPPL C, 2017

CONCLUSIONS Chronic exercise training modulated the ANS imbalance without increasing the incidence of spontaneous arrhythmia in MI mice mainly through the normalization of Ca2＋dynamics. GW28-e0265 Myocardium-targeted transplantation of PHD2 shRNA-modified mesenchymal stem cells by ultrasound-targeted cationic microbubble destruction improves cardiac function in myocardial infarction of rats Zhang Li,1 Sun Zhenxing,1 Xie Mingxing1 1 Department of Ultrasound, Uinon Hospital, Tongji Medical College of Huazhong University of Science and Technology OBJECTIVES to prepare a cationic lipid microbubble and investigate its characterization and ultrasonic imaging in vivo; to explore the silencing effect of shPHD2-EGFP after its transfection into MSCs mediated by UTMD; to study if the MSCs transfected with shPHD2EGFP can be easier to transplant into myocardial tissue by UTMD and enhances their cardioprotective effects after transplantation into infarcted hearts. METHODS Cationic lipid microbubble was prepared by filming-rehydration and machine vibration. Its concentration, average diameter and zeta potential were measured. The binding capacity with plasmid and contrast-enhanced ultrasound effect was also assessed. The plasmid that silence PHD2 gene and carry GFP gene was constructed. Firstly, the cultured MSCs were divided into three groups, including Plasmid, Plasmid + Ultrasound and Plasmid + UTMD to prove that UTMD enhances gene transfection and find optimal transfection parameters by UTMD. Then, to demonstrate that shPHD2-EGFP silence PHD2 gene and induce HIF-1a and angiogenic factor gene, the experimental regimens included Control, EGFP and shPHD2-EGFP; to prove that PHD2 silencing enhances MSCs survival, the MSCs were divided into four groups, including Control, BMSC, BMSC-EGFP and BMSC-shPHD2-EGFP. Rat myocardial infarction models were conducted. Firstly, 30 rats were divided into three groups, including MI, MI-MSCEGFP and MI-MSCshPHD2-EGFP. The MSCs transplantation effect was tested. Then, other 120 rats were divided into four groups, including Control, MI-PBS, MI-BMSCsEGFP and MI-BMSCsshPHD2EGFP. The echocardiography, histological character and myocardial apoptosis were evaluated. RESULTS The concentration of CMB was (4.12
0.29)  109/ml. its average diameter and zeta potential was (1.67
0.32) mm and (29.18
1.36) mV respectively. The saturated DNA loading capacity for 1108CMB was (3.56
0.31) mg. And enhancement effect in rat heart was good. Compared with Plasmid and Plasmid + Ultrasound groups, the gene transfection efficiency was highest. US intensity (1.0W/cm2) and exposure time (30s) were relative optimal transfection parameters. The shPHD2-EGFP down-regulated PHD2 and up-regulated HIF-1a after it was delivered into MSCs by UTMD. Meanwhile, the shPHD2-EGFP decreased MSC apoptosis. The number of MSCs for transplantation in MI-Plasmid + UTMD group was higher than MIPlasmid and MI-Plasmid + ultrasound groups. Four weeks after transfection, compared with the other groups, myocardial apoptosis and scar size at the infarct area reduced in the MI-BMSCsshPHD2EGFP group (P<0.05). Capillary densities in MI-BMSCsshPHD2-EGFP group were increased and left ventricular function had improved (P<0.05) over other groups. CONCLUSIONS UTMD successfully promoted transfection efficiency of plasmid DNA in vitro and increased marrow stem cell homing in vivo, thus improving the efficacy of therapeutic angiogenesis and myocardial function. GW28-e0312 High uric acid induces inhibition of viability through ERK/P38 pathway via oxidative stress in cardiomyocyte Zhi Li1,2 1 Department of Internal Medicine, The Second Affiliated Hospital of Shantou University Medical College; 2Department of Internal Medicine, The First Affiliated Hospital of Shantou University Medical College, Shantou OBJECTIVES Clinical studies have shown hyperuricemia strongly associated with cardiovascular disease. However, the molecular mechanisms of high uric acid （HUA） associated with cardiovascular disease remain poorly understood. In this study, we investigated the effect of HUA on primary cardiomyocytes and H9c2 cardiomyocytes, a rat cardiomyocyte cell line.

METHODS We exposed cardiomyocytes to HUA, then cell viability was determined by MTT assay, and reactive oxygen species (ROS) production was detected by fluorescent. Western blot analysis was used to examine the levels of phosphorylated extracellular signal-regulated kinase (ERK), phospho-P38, phosphorylated phosphatidylinositol 3kinase (PI3K) and phospho-Akt. We monitored the impact of HUA on phospho-ERK and phospho-P38 levels in myocardial tissue of an acute hyperuricemia mouse model established by potassium oxonate treatment. RESULTS Our data showed that HUA decreased cardiomyocytes viability and increased ROS production in cardiomyocytes; pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, and PD98059, an ERK inhibitor, reversed HUA-inhibited viability in H9c2 cardiomyocytes. Further examination on signal transduction pathways revealed that HUA-induced ROS is involved in the activation of ERK/ P38 and inhibition of PI3K/Akt in cardiomyocytes. Furthermore, the acute hyperuricemic mice model showed increased phospho-ERK/P38 in myocardial tissues. CONCLUSIONS These results indicate that HUA induce oxidative damage and inhibit viability of cardiomyocytes by activating the ERK/ P38 signal pathways, which is a novel potential mechanism of hyperuricemic-related cardiovascular disease. GW28-e0320 sGC signaling and MtFt attenuate doxorubicin-induced cardiomyopathy Xiaoxiao Zhao,1,2 Like Guan2 1 Kyung Hee University Medical Center Hospital Center cardiological lab, Souel, Korea; 2Department of Cardiology, Yanbian University Hospital, Yanji, China OBJECTIVES The cardiotoxicity induced by the highly effective anticancer agent doxorubicin (DOX) involves increased oxidative stress, mitochondrial iron overload, DNA damage, autophagy, necrosis and apoptosis, underlining which also associated with its secondary tumorigenicity. soluble guanylate cyclase (sGC) signaling has protective effect on cardiovascular disease and chemosensitization effect on cancer. The present study investigates the role of Bay60-2770, a more effective activator of oxidized and heme-free sGC, and the role in alleviating DOX induced dilated cardiomyopathy. METHODS H9c2 cardiomyocytes were treated with 10 mM Bay 602770 24hr prior to Dox (0.5-10 mM), and cell viability, 3-Nitrotyrosine and p-P53(ser15) were subsequently measured. In order to determine the role BAY60-2770 in ROS generation, we examined DCFHDA and MitoSOX RED under DOX exposure. Also we did Cyto-ID autophagy detection. Additionally, 9-week-old male Sprague Dawley rats were orally administered with BAY60-2770 1 hour prior to every DOX treatment. Echocardiography and hemodynamic values were then analyzed. Autophagy proteins and mitochondrial iron regulating proteins expression levels were examined by western blot analysis. RESULTS BAY60-2770 ameliorated cell viability and 3-Nitrotyrosine induced by DOX in H9c2 cardiomyocytes. BAY60-2770 increased autophagosomes in DOX cells and up-regulated P62 expression level in heart tissue. p-P53(ser15), intracellular ROS and mitochondrial ROS attenuated by BAY60-2270 in DOX-treated H9c2 cells. BAY60-2270 enhanced the protein expression of Mitochondrial ferritin (MtFt) in DOX administered heart. Echocardiography showed that pre-treatment with BAY60-2770 significantly improved reduced LV function that is induced by DOX treatment. CONCLUSIONS BAY60-2770 reduces DOX-induced mitochondrial ROS generation and subsequent DNA damage by up-regulating MtFt. BAY60-2770 improved autophagy and cell viability, decreased cell death. BAY60-2770 ameliorated cardiac function. These novel results highlight the therapeutic potential of sGC signaling and MtFt to prevent doxorubicin induced cardiomyopathy. GW28-e0322 Increasing Calpain Expression Regulates hERG Mutation L539fs/47 Induced Cardiomyocyte Apoptosis Shuting Ma,1 Yun Zhao,1 Yuejiao Wei,1 Chaofeng Sun1 1 Department of Cardiovascular Medicine, the First Affiliated Hospital of Xi’an Jiaotong University College of Medicine OBJECTIVES Among the 15 types of congenital long QT syndrome, the LQTS 2 caused by hERG mutate is the most common type in China. Nowadays, more and more researches have proved LQTS has not only

JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, VOL. 70, NO. 16, SUPPL C, 2017

abnormal cardioelectrical activities, but also cardiovascular structural deficiency and remodeling: LQTS is associated with contraction inability, left atrial and ventricular dilated and heart deformity; ARVC, DCM, LVNC are also accompanied with LQTS; fetuses and infants heart deformity or sudden death are associated with LQTS as well. The mechanisms of LQTS structural deficiency are still under exploring. By biopsy, LQTS patients cardiomyocytes characterized by increased apoptosis; hERG mutation mice’s hearts are developmental defected and increased apoptosis. These proofs above pointed apoptosis of cardiomyocyte might be the potential mechanism of LQTS induced heart deformity. L539fs/47-hERG is a complicated mutation in a Chinese family consists of 19 bps deletion at 1619-1637 accompanied with a 1692 A/G nonsense mutation, which causes hERG protein shorten and channel function defected. It can also prolong the phase 2 repolarization which slow down the L-type Ca2+ channel deactivation, make Ca2+ accumulate in cytoplasmic. High Ca 2+ concentration can cause cell apoptosis via activation of Calpain. Calpain is a proteinase widely distributed in mammal cells. It is activated by Ca2+ and then induce CASPASE and None-CASPASE cell apoptosis pathways. According to these above evidences, we make the hypothesis that hERG mutation could induce cell apoptosis via activating Calpain pathway. In order to prove this theory and explore the potential mechanism of hERG mutation induced apoptosis, we decide to use L539fs/47-hERG cell model to detect the level of apoptosis and the expression of Calpain simultaneously. METHODS We have constructed L539fs/47-hERG-pcDNA3.0 and WThERG-pcDNA3.0 plasmids, each of them was transiently transfected to HEK293 cell respectively to mimic L539fs/47-hERG mutation or WThERG cell models. After 24 hours incubation, the laser confocal scan microscope was used to detect hERG protein distribution. Hoechst33342 cell apoptosis stain was conducted and observed under fluorescence microscope. The whole cell protein extract Calpain1 expression level was detected through Western Blot technic. RESULTS At 24 hours after transfection, it can be seen that abnormal hERG protein accumulated in endoplasmic reticulum and failed to rivet into membrane. Using inverted phase contrast microscope, L539fs/47-hERG homozygous cell typing has more floated and bright cell in the medium compared with the heterozygosis typing or WThERG cell. After Hoechst33342 stain it can be seen that the number of apoptosis cells are significantly higher in L539fs/47-hERG cell than WT-hERG (P<0.05). Comparing with the WT-hERG, the Calpain1 is elevated in L539fs/47-hERG mutation cell (P¼0.010). CONCLUSIONS The results indicated that L539fs/47-hERG mutation can induce cell apoptosis, and the elevation pf Calpain1 expression may play a regulatory role in this pathophysiology process. It’s the first time to prove that LQTS type 2 mutation L539fs/47-hERG can cause cell apoptosis, which gives researchers a new sight to comprehension the potential mechanism of LQTS patients’ structural deficiency and systole abnormal. Calpain play a considerable role in regulating L539fs/47hERG induced apoptosis, which provide clinicians a new potential medication target in LQTS type 2 patients management.

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the SCr levels between the pre- and post- CM or NS exposure. In addition, the cys-C levels were also higher after CM exposure than that before in the FM+CM15 group (0.08
0.03 vs. 0.18
0.05 mg/L, p < 0.001). There were minor changes in the FM + NS group before and after NS administration. Furthermore, only rats in the FM + CM15 group developed CI-AKI based on the definitions for SCr or cys-C. The histopathological scores were significantly higher in the FM + CM15 group than in the FM + NS group. CONCLUSIONS We developed a simple and reliable animal model for LOCM-induced AKI that is similar to clinical CI-AKI based on different definitions for AKI. GW28-e0341 Apelin-13 infusion salvages the peri-infarct region to preserve cardiac function after severe myocardial injury Jie Qin,1 Lingrong Peng,1 Chuangfeng Li,1 Wenjie Tang1 Department of Radiology, the Third Affiliated Hospital of Sun Yat-sen University

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OBJECTIVES To assess the restorative effects and mechanism of Apelin-13 (A13) on the peri-infarct region in murine acute myocardial injury (AMI) model. METHODS 51 FVB/N mice (12weeks, 30g) underwent AMI. A week following injury, continuous micro-pump infusion of A13 (0.5mg/g/ day) and saline was initiated for 4-week duration. Dual contrast MRI was conducted on weeks 1, 2, 3, and 5, consisting of delayed-enhanced and manganese-enhanced MRI. Four mice in each group were followed for an extended period of 4weeks without further infusion and underwent MRI scans on weeks 7 and 9. RESULTS A13 infusion demonstrated preserved LVEF compared to saline from weeks 1 to 4 (21.9
3.2% to 23.1
1.7%* vs. 23.5
1.7% to 16.9
2.8%, *p¼0.02), which persisted up to 9weeks post-MI (+1.4%* vs. -9.4%, *p¼0.03). Mechanistically, dual contrast MRI demonstrated significant decrease in the peri-infarct and scar % volume in A13 group from weeks 1 to 4 (15.1 to 7.4% and 34.3 to 25.1%, p¼0.02, respectively). This was corroborated by significant increase in 5ethynyl-2’-deoxyuridine (EdU(+)) cells by A13 vs. saline groups in the peri-infarct region (16.5
3.1% vs. 8.1
1.6%; p¼0.04), suggesting active cell mitosis. Finally, significantly enhanced mobilization of CD34(+) cells in the peripheral blood and up-regulation of APJ, fibrotic, and apoptotic genes in the peri-infarct region were found. CONCLUSIONS A13 preserves cardiac performance by salvaging the peri-infarct region and may contribute to permanent restoration of the severely injured myocardium. GW28-e0342 Complement factor 5 blockade reduces porcine myocardial infarction size and improves immediate cardiac function Jie Qin,1 Lingrong Peng,1 Chuangfeng Li,1 Wenjie Tang1 Department of Radiology, the Third Affiliated Hospital of Sun Yat-sen University

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GW28-e0340 A novel simple experimental model for low-osmolar contrastinduced acute kidney injury using different definitions based on the levels of serum creatinine and cystatin C Yuanhui Liu,1 Ning Tan,1 Yong Liu,1 Jiyan Chen1 1 Guangdong General Hospital, Guangdong Cardiovascular OBJECTIVES We aimed to develop a novel simple experimental model in mice for low-osmolar contrast-induced acute kidney injury (CI-AKI) with different definitions for AKI that would be comparable to CI-AKI in humans.

OBJECTIVES The purpose was to investigate the effects of inhibition of complement factor 5 (C5) on infarct size and ventricular function in a porcine model of myocardial infarction. METHODS In pigs (Sus scrofa), the left anterior descending coronary artery was occluded (40 min) and reperfused (240 min). Coversin or placebo was infused 20 min after occlusion and throughout reperfusion in 16 blindly randomized pigs.

METHODS Fifty Sprague-Dawley rats were divided into five groups of 10 rats each: (1) sham group (normal saline [NS] + NS); (2) NS plus contrast medium (CM15) (NS+CM15); (3) furosemide (FM) plus NS (FM + NS); (4) FM+CM10; and (5) FM + CM15. After they were acclimatized for 7 days, and 6 h before CM administration, FM (10 mL/kg) was administered to groups 3, 4 and 5. Then, after restricted access to water for 6 h in all the groups, groups 2 and 5 received low-osmolality CM (LOCM) (iopromide, 15 mL/kg) in the tail vein, and group 4 received LOCM (10 mL/kg) under ether anesthesia. Serum creatinine (SCr) and cystatin C (cys-C) levels were measured and histopathological scores were determined in kidney tissues.

RESULTS Coversin significantly reduced myocardial infarction in the area at risk by 39% (p ¼ 0.03, triphenyl tetrazolium chloride staining) and by 19% (p ¼ 0.02) using magnetic resonance imaging. The methods correlated significantly (R ¼ 0.92, p < 0.01). Tissue Doppler echocardiography showed increased systolic displacement (31%, p < 0.01) and increased systolic velocity (29%, p ¼ 0.01) in coversin treated pigs. Interleukin-1b in myocardial microdialysis fluid was significantly reduced (31%, p < 0.05) and tissue E-selectin expression was significantly reduced (p ¼ 0.01) in the non-infarcted area at risk by coversin treatment. Coversin ablated plasma C5 activation throughout the reperfusion period and decreased myocardial C5b-9 deposition, while neither plasma nor myocardial LTB4 were significantly reduced.

RESULTS In the FM + CM15 group, SCr concentration was significantly higher after CM exposure than before (32.9
4.57 vs. 158.7
14.48 mmol/L, p < 0.001). In the other groups, there were minor changes in

CONCLUSIONS Coversin substantially reduced the size of infarction, improved ventricular function, and attenuated interleukin-1b and Eselectin in this porcine model by inhibiting C5.