Ding, Xue

Yang, Wei

Elsevier - Publisher Connector 

J O U R N A L O F T H E A M E R I C A N C O L L E G E O F C A R D I O L O G Y , V O L . 6 6 , N O . 1 6 , S U P P L S , 2 0 1 5 C19GW26-e1082Activation of Cloned BKCa Channels by Hydrogen Peroxide ExhibitedIncreased Anti-oxidative Stress of HEK293 cellsWenjun Huang, Xiaorong ZengKey laboratory of medical electrophysiology of Ministry of Education,Luzhou Medical CollegeOBJECTIVES The present study was designed to clarify whetherHydrogen peroxide (H2O2) is an activator of BKCa channels in humanembryonic kidney 293 (HEK293) cell and the function for anti-oxidativestress will change after stable expressing BKCa channel in HEK293 cell.METHODS The cDNA encoding the a-subunit of BKCa channel, hSloa,was transfected into HEK293 cells. Stable cell line expressing BKCachannel was established by screening with G418. The cell viability inHEK-hSloa cells was detected using cell viability test (Cell CountingKit-8,CCK8). The time-dependent (0, 6h, 12h, 24h, 48h) and concen-tration-dependent (0, 50mM, 100mM, 200mM, 400mM) curves of H2O2effect on cell viability were also detected by CCK8. Changes in intra-cellular ROS levels were determined by measuring the oxidativeconversion of cell permeable 2’,7’-dichloroﬂuorescein diacetate(DCFH-DA) to ﬂuorescent dichloroﬂuorescein (DCF) in ﬂuorospectro-photometer. Single-channel patch clamp experiments were performedin HEK-hSloa cells to test the effect of H2O2 on BKCa channels.RESULTS Optimized time and concentration of H2O2 effect weredetermined by time-dependent and concentration-dependent curvesof H2O2 effect on cell viability. When the cells were cultured indifferent concentration of H2O2 for different time, treatment of HEK-hSloa cells with 100mM H2O2 for 24 h exhibited greater cell viability oncell survival compared to HEK293 cells. H2O2 (100mM) signiﬁcantlyincreased the intracellular level of ROS. HEK-hSloa cells with 100mMH2O2 exhibited lower ROS level compared to HEK293 cells. Pretreat-ment with TEA (2mM) signiﬁcantly elevated intracellular concentra-tion of ROS by H2O2 in HEK-hSloa cells. Single-channel patch clampexperiments indicated that 100mM H2O2 would activate BKCa channelsobviously with increased open probability.CONCLUSIONS These results indicated that H2O2 could activate BKCachannel; HEK-hSloa cells under treatment of 100mM H2O2 for 24 hexhibited increased anti-oxidative stress ability; activation of clonedBKCa channels by Hydrogen peroxide was implicated in increasedanti-oxidative stress of HEK293 cells.GW26-e2238The Contribution of Platelet MicroRNA Expression and its Interaction withCYP2C19 Allele Variants to Clopidogrel Antiplatelet Responsiveness inAcute Coronary Syndrome PatientsLi Peng, Tong Yin, Caiyi LuDepartment of Cardiology, Chinese PLA General HospitalOBJECTIVES To investigate relationship between platelet miRNA(miR-223, miR-221, miR-21) expression and clopidogrel antiplatelet responsive-ness, as well as the contribution of platelet microRNA expression and itsinteraction with CYP2C19 allele variants on clopidogrel antiplateletresponsiveness in acute coronary syndrome (ACS) patients.METHODS We enrolled consecutive 272 patients with ACS fromDecember 2013 to December 2014 in our department of Cardiology. 39cases were screened by the detection of the platelet aggregation. Then39 cases were divided into two groups by the four quantiles: highplatelet reactivity group of extreme values (high response group), 21cases; and low platelet extreme value group (low response group), 18cases. Bioinformatic analysis was used to investigate the candidatemicroRNA. Real time polymerase chain reaction (real time PCR)wasperformed to analyze the expression levels of all miRNAs which is bandto platelet P2Y12 receptor, as well as to genotype candidate singlenucleotide polymorphisms (SNP) including CYP2C19*2 and CYP2C19*3.Univariate and multivariate analysis and receiver operating charac-teristic curve (ROC curve) were utilized to assess the relationship be-tween miRNA expression levels and clopidogrel platelet reactivity, thenon-conditional Logistic regression was used to evaluate the interac-tion between expression of miRNA and CYP2C19 * 2 allelic variant.RESULTS ① According to bioinformatic analysis and forecast, weﬁnally conﬁrmed three candidates microRNA, including miR-223,miR-221 and miR-21. ② Compared with the low response group, thelevels of three miRNAs expression were up regulated with statisticaldifferences (miR-223: p ¼ 0.002; miR- 221: p ¼ 0.004; miR-21: p ¼0.007) in the high response group.③ ROC curve analysis showed thatthe relative expression levels of the three miRNAs can effectivelyidentify the high platelet response (miR-223: AUC ¼ 0.704, miR-221:AUC¼0.762, miR-21: AUC ¼0.796). ④ After multivariate adjustment,we found that the three microRNAs were independent predictors ofhigh platelet reactivity. ⑤ Interaction analysis revealed signiﬁcantlyhigher expression levels of miRNAs in CYP2C19 * 2 allele carriers whowere high platelet responders (miR-223: p¼ 0.037; miR-221: p ¼ 0.001;miR-21: p < 0.001),but not in CYP2C19 * 2 non-carriers (p ＞ 0.05).CONCLUSIONS Our study ﬁnds that up-regulation of three kinds ofmicroRNAs (miR-223, miR-221 and miR-21) expression is associatedwith clopidogrel high platelet reactivity. What’s more, we ﬁnd that theinteraction between the expression levels of miRNAs and the CYP2C19* 2 variant carriers may have an impact on clopidogrel platelet reac-tivity. Our results suggest that miR-223, miR-221 and miR-21 mayserve as the novel biomarkers for assessment clopidogrel antiplateletresponsiveness in ACS patients.GW26-e0716Forestalling SIRT1 Carbonyl Stress by ALDH2 Restores Autophagy in AgedHeartYishi Wang,1 Chunhu Gu,2 Lu Yu,3 Heng Ma11Department of Physiology, Fourth Military Medical University;2Department of Cardiovascular Surgery, Xijing Hospital, FourthMilitary Medical University; 3Department of Pathology, Xijing Hospital,Fourth Military Medical UniversityOBJECTIVES Cardiac aging is an intrinsic process that results inimpaired cardiomyocyte proteostasis. We previously demonstratedthat ALDH2 activation is sufﬁcient to protect the heart againstextravagant aging-related myocardial ‘aldehydic load’ induced pro-teoxicity. However, it is not known whether ALDH2 can regulate theclearance of damaged proteins in aged heart. Here, we determined thebeneﬁts of activation of ALDH2 on the autophagy by forestallingSIRT1carbonyl stress in aged heart.METHODS Young adult, aged (21–22 mo.) male C57BL/6 mice andALDH2 KO mice were perfused in the presence or absence of selectiveALDH2 activator (Alda-1,6 mg/kg/d).RESULTS Aging impairs cardiac ALDH2 activity. However, accumu-lation of 4-HNE-protein adducts and protein carbonyls concomitantwith impairment in the autophagy ﬂux seen in aged heart wasameliorated in Alda-1 treated aged mice, suggesting that increasingthe ALDH2 activation contributes to the reduction of proteotoxicityand restoration of autophagy. Furthermore, ALDH2 KO mice pro-gressively developed age-related heart dysfunction and increasedcardiomyocyte ER stress. ALDH2 KO mice showed reduction in the lifespan, as compared with that of WT controls, strongly supporting thatALDH2 ablation leads cardiac aging. In addition, ALDH2 activation byAlda-1 led to a signiﬁcant reduction of SIRT1 carbonylation and acet-ylation of FoxO1 as well as increased Rab7 expression in aged hearts,whereas SIRT1 was required for Alda-1-induced increases in auto-phagic ﬂux. Although cardiac function was improved in sustainedAlda-1treated aged mice after 48 hours of food starvation, it wassigniﬁcantly deteriorated in Sirt1þ/ hearts, in which ALDH2 activationinduced autophagy is inhibited.CONCLUSIONS These results highlight that both accumulation ofproteotoxic carbonyl stress along with autophagy decline contributeto heart senescence. ALDH2 activation is sufﬁcient to improve theautophagy by reducing the carbonyl modiﬁcation on SIRT1, which inturn plays an important role in maintaining cardiac health duringaging.GW26-e1051MicroRNA-185 Aggravates Congestive Heart Failure by Targeting AKT1Xue Ding, Wei YangCardiovascular Department, the First Afﬁliated Hospital of HarbinMedical University, Harbin, Heilongjiang Province, P.R. ChinaOBJECTIVES Mounting evidence has revealed the master regulatoryrole of microRNAs (miRNAs) in a variety of cardiac processes,including growth, development, cardiac function and response todiverse stress. However, the underlying mechanism of miRNA incongestive heart failure (CHF) is still less well studied. In the pre-sent study, we sought to investigate the biological role of miR-185in CHF.METHODS Quantitative real-time PCR (Q-PCR) was carried out tomeasure the circulating miR-185 in the serum of congestive heartfailure patients (n¼80). Meanwhile, a CHF rat model was establishedby intraperitoneal injection of doxorubicin. Echocardiography andpressure analyses were performed on all animals. Furthermore, Pri-mary cardiomyocytes were cultured and transfected with miR-185C20 J O U R N A L O F T H E A M E R I C A N C O L L E G E O F C A R D I O L O G Y , V O L . 6 6 , N O . 1 6 , S U P P L S , 2 0 1 5mimics or inhibitors to assess the in vitro effect of miR-211 on CHF.The target gene of miR-185 was predicted by using bioinformatic al-gorithm. A further luciferase reporter assay was performed to conﬁrmthe speciﬁc binding between miR-185 and the 3’-UTR of its target.Western-blots assays were used to check relevant gene expression.RESULTS We found that circulating miR-185 was signiﬁcantly upre-gulated in CHF patients as well as in the heart tissues from CHF ratmodel compared with respective controls. Silencing of miR-185exhibited a cardiac protective role in doxorubicin induced micemodel. Using Targetscan and Miranda, we identiﬁed AKT1 as thetarget of miR-185, which was conﬁrmed by dual luciferase reporterassays. Ectopic expression of miR-185 enhanced the apoptosis of pri-mary cardiomyocytes induced by doxorubicin by directly targetingAKT1. Downregulation of AKT1 phenocopied the pro-apoptotic effectof miR-185. Furthermore, administration of constitutively active AKT1promoted primary cardiomyocytes proliferation, and reversed pro-apoptotic effect of miR-185 overexpression.CONCLUSIONS MiR-185 exacerbates CHF and reduces cardiomyocyteproliferation through downregulation of AKT1, suggesting that miR-185 might be a target to manage cardiac dysfunction.GW26-e4759The Anti-atherosclerotic Effect of Pseudolaric acid B in High-Fat Fed ApoEKnockout Mice: Alterations in Circulating and Atherosclerotic PlaqueMonocytes/Macrophages and The Underlying MechanismsXiao Li,1,2 Xin Zhou,1,2 Tan Li,1 Yu-Xiu Li,1,2 Tie-Min Jiang,1,2Yu-Ming Li,1,2 Ji-Hong Zhao1,21Tianjin Key Laboratory of Cardiovascular Remodeling and TargetOrgan Injury, Tianjin, China; 2Institute of Cardiovascular Disease andHeart Center, Pingjin Hospital, Logistics University of the ChinesePeople’s Armed Police Forces, Tianjin, ChinOBJECTIVES Atherosclerosis (AS) is the pathological basis of cardio-vascular and cerebrovascular diseases. The chronic inﬂammation andlipid inﬁltration theory are thought to be the pathogenesis of AS.Mononuclear phagocyte (MNP) were key players involved in the lipidmetabolism of plaque and chronic inﬂammation. Pseudolaric acid B(PB) is an extract of pseudolaricis. Recently, we show that PB couldactivate peroxisome proliferator activated receptor g, while is animportant molecular target involved in the process of inﬂammationreaction and lipid metabolism. Accordingly we hypothesis that PB hastherapeutic potential for AS.METHODS In vivo study, ApoE-/-mice (8 weeks) were consumed highfat diet for 8 weeks, then randomly grouped into the baseline group,high fat diet group, PB treated with high fat diet group, normal dietgroup and PB treated with normal diet group (n¼7 each group). We didexperiments as followings, the proportions of monocyte subsets wereanalyzed. Confocal microscopy was used to investigate the density ofthe proliferation macrophages in plaque of aortic root. Oil Red Ostaining was used to explore the plaque size of aortic, common carotidartery and the aortic root. plasma triglycerides and total cholesterolconcentration were measured by ELISA. In vitro study. Flow cytom-etry was used to explore the efﬂuence and uptake of lipid inRAW264.7 cells by Dil-ox-LDL. RAW264.7 cells were intervened withoxidized low density lipoprotein for 24 hours, which would form foamcells, then divided into the model group, PB intervention group andPB with PPARg antagonist block group. The Foam cells were subject tothe following experiments, Oil Red O staining was used to investigatefoam cell. The gene expression of PPARg, LXRa, ABC1 and CD36 wasmeasured by real-time PCR.RESULTS In vivo study showed that, the proportion of Ly6Chimonocyte subsets are lower in PB treated with normal diet（33.01%3.66% vs. 47.20%3.43%, P<0.05). The density of the proliferationmacrophages are lower in PB treated with normal diet (P<0.05). Thelevels of plasma triglycerides and total cholesterol are lower in thegroups of PB treated with normal diet (all P<0.05). The load of plaquein the root of aorta, common carotid arteries and aorta tended to belower in the groups of PB (12.02%1.69% vs. 5.70%1.38%, 7.14%1.57% vs. 3.71%0.95% and 24.72%3.80% vs. 15.3%1.88%, respec-tively all P<0.05). In vitro study showed that PB can decrease the levelof lipid in macrophage (P<0.05). PB can increase the gene expressionof PPARg (1.070.36 vs. 3.150.42, P<0.05), LXRa (0.990.06 vs.2.050.22, P<0.05) and ABC1 (1.880.16 vs.0.960.06, P<0.05).CONCLUSIONS PB with normal diet not only delay the progression ofatherosclerosis but also reduce plaque burden. PB could activate thesignal pathway of PPARg-LXRa-ABC1 and PPARg-CD36 to regulate thelipid droplet and efﬂux of foam cells, and inhibit the formation offoam cells.GW26-e0194Apelin Ameliorates Myocardial Insulin Resistance and Improves MyocardialInjury in DiabetesHangxiang Zhang,1 Jiaojiao Chu,2 Tao Shen,2 Chongqing Yang,3Xiuqing Huang,2 Shu Wang,2 Xiaoming Wang,1 Jian Li2,41Department of Gerontology, Xi-Jing Hospital, Fourth Military MedicalUniversity, Xian 710032, China; 2Key Laboratory of Geriatrics, BeijingInstitute of Geriatrics & Beijing Hospital, Ministry of Health, Beijing100730, China; 3Peking Union Medical College and Chinese Academy ofMedical Sciences, Beijing, China; 4Department of Pathology, BeijingHospital, Ministry of Health, Beijing, ChinaOBJECTIVES Apelin as a beneﬁcial adipokine has been linked to in-sulin resistance and cardiovascular protection in diabetes, however,the underlying molecular mechanisms of how apelin affects myocar-dial insulin resistance and myocardial injury remain poorlyunderstood.METHODS We collected 131 T2DM patients serum, myocardial tissuesof 8 patients with T2DM and 6 accidental deaths without T2DM atBeijing Hospital. After the TNF-a administration, C57BL/6J male micewere randomized to receive either vehicle, apelin-13(10mg/kg), or F13A(a competitive antagonist for apelin receptor 20mg/kg) by i.p.injection.RESULTS Plasma samples of 131 patients with type 2 diabetes mellitus(T2DM) and 54 healthy controls, and myocardial tissues of 8 T2DMpatients and 6 accidental deaths without T2DM were collected.Increased levels of plasma apelin, ROS and TNF-a were accompaniedby insulin resistance and myocardial injury in patients with T2DM,similar results were conﬁrmed in diabetic ob/ob mice, suggesting thatapelin, TNF-a and ROS might be correlated with the states of insulinresistance. In vitro, TNF-a could stimulate myocardial insulin resis-tance, ROS generation, and apelin expression in H9c2 cells. However,exposure apelin-13 to TNF-a-treated H9c2 cells and intraperitonealinjection of apelin-13 to TNF-a-induced insulin-resistant C57BL/6Jmice rescued TNF-a-induced higher intracellular glucose content andimpaired insulin signaling pathway. Moreover, the results demon-strated that an intraperitoneal injection of apelin-13 improved MI-induced increased glucose content and cardiac injury as evidenced byattenuated myocardial infarct size and decreased myocardialapoptosis in both ob/ob mice and C57BL/6J mice. Further studiesindicated that apelin-13 suppressed ROS generation through down-regulation of iNOS expression. Finally, F13A, a competitive antagonistfor apelin receptor APJ, suppressed the effects of apelin-13 on insulinsignaling pathway in both insulin-resistant H9c2 cells and insu-lin-resistant mice. Interestingly, F13A also impaired the effects ofapelin-13 on iNOS expression, demonstrating that apelin amelioratedTNF-a-induced myocardial insulin resistance by its receptor APJ.CONCLUSIONS We provide novel experimental evidence that apelincould ameliorate myocardial insulin resistance and improve myocar-dial injury though suppressing ROS generation via down-regulation ofiNOS expression, in term, elevating insulin signaling transduction.GW26-e0456Exosomes Derived From Dendritic Cells Activate CD4þ T LymphocytesAfter Myocardial InfarctionHaibo Liu,1,2 Wangjun Yu,3 Chunming Wang,1 Xiaofang Guo,3Kang Yao,2 Jie Yuan,2 Yunzeng Zou,2 Junbo Ge21Department of Cardiology, Yinzhou People’s Hospital Afﬁliated toMedical school of Ningbo University; 2Shanghai Institute ofCardiovascular Diseases, Zhongshan Hospital, Fudan University;3Clinical Laboratory Center, Yinzhou People’s Hospital, NingboUniversityOBJECTIVES Activation of CD4þ T cells plays key roles in facilitatingwound healing after myocardial infarction (MI). However, the mech-anism of how CD4þ T cells are activated post-MI remains largely un-known. This study was aimed to clarify whether exosomes derivedfrom dendritic cells (DCs) mediate activation of CD4þ T cells post-MI.METHODS Bone marrow-derived DCs (BMDCs) of mouse were incu-bated with the supernatant of necrotic (Necrosis group) or normal HL-1 myocardial cells (Control group), or without stimulation (Negativegroup). DEXs were then isolated from the BMDCs and identiﬁed byelectron micrograph and Western Blotting using the exosomal markerproteins. For DEXs tracker and dynamics experiments, the labeledDEXs (DiR) were injected into mice through tail vein and monitored byﬂuorescence imaging. For DEXs uptake experiments, the labeled DEXs(PKH67) were added to CD4þ T cells suspensions or injected into micetail vein, the CD4þ T cells and frozen splenic sections were ﬁxed for

GW26-e1051 MicroRNA-185 Aggravates Congestive Heart Failure by Targeting AKT1 

Xue Ding

Wei Yang

Crossref

Journal of the American College of Cardiology