Li Juxiang

GW24-e2990 The expression of rock in cardiomyocte exposed to hypoxia and its role in cardiomyocte injury

Objectives Using cultured myocardial cells in vitro and establishing virtual hypoxia environment of cardiomyocyte apoptosis model, to observe the expression of Rock in myocyte exposed to different hypoxia phase and its role in the mechanism of apoptosis. Methods 1. Cardiomyocytes from the hearts of 1-3-day-old neonatal rats were prepared. 2. Established myocardial cell apoptosis model, prepared the simulated hypoxia liquid which have been deoxygenated by bubbling with the mixture of 95% nitrogen and 5% carbon dioxcide for 1 hours. Then exposed to it for 1 h,3 h,6 h,9 h, respectively. Select the time when Rock-1and Rock-2 expression the highest, put the PI3K/AKT blocking agent and Erk5 blocking agent into hypoxia liquid respectively, and then detect related indicators. 3. The cell apoptosis and viability were detected by flow cytometry and MTS respectively. The expression of Rock-1, Rock-2, p-Erk5, Eek5, Caspase-3, PI3k, p-Pi3k, GSK3β at different hypoxia phase were detected by western blotting. Results 1. After exposed to hypoxia liquid for 1 h, 3 h, 6 h, 9 h respectively, the rate of myocyte apoptosis increased and survival rates decreased. After 1 h, Rock-1, Rock-2 and Erk5 in hypoxia-ischaemia liquid began to rise, reached its peak in 3-6 hours, in 9 hours began to decrease, which were significantly higher than the control group (P < 0.01); Caspase-3, Gsk3β in hypoxia-ischaemia liquid, began to rise, which was sustained expression in a high state at following observed time. There was no significace of the expression of Pi3k in the course of hypoxia-ischaemia which was no statistical significance. But After 1 h, p-Pi3k in hypoxia-ischaemia liquid began to rise, reached its peak in 6 hours, in 9 hours began to decrease, which were significantly statistical significance between the two groups (P < 0.05); 3. Put PI3K/AKT blocking agent LY294002 (final concentration 20 umol / L) into liquid, pretreatment for 2h, and then, cardiomyocytes were treated in hypoxia liquid for 6h, myocardial cell apoptosis compared with the group of hypoxia 6 hours was significantly higher (P < 0.01), the survival rate compared with hypoxia group decreased significantly (P < 0.01). The expression of Caspase-3, Rock-1 and Rock-2 were also significantly higher compared with hypoxia 6 h group (P < 0.05) 4. Put Erk5 blocking agent LY294002 (final concentration 20 umol/L) into liquid, pretreatment for 2 h, and then, cardiomyocytes were treated in hypoxia liquid for 6 h, myocardial cell apoptosis compared with the group of hypoxia 6 hours was significantly higher (P < 0.01), the survival rate compared with hypoxia group decreased significantly (P < 0.01). The expression of Rock-1 and Rock-2 were also significantly higher compared with hypoxia 6h group. Conclusions 1. Rock-1 and Rock-2 are involved in the process of hypoxia-induced cardiomyocyte apoptosis, which may play an important role by inhibiting the PI3K/AKT pathways. 2. Erk5 perform anti-apoptosis effection through reducing the activation of Rocks. 3. Hypoxia can activate Gsk3β, Caspase-3, thereby increasing cardiac myocyte apoptosis. Hypoxia can promote cardiac myocyte apoptosis through activating Gsk3β and Caspase-3 expression.

GW24-e2379 Expression of prohibitin and proliferation in vascular smooth muscle cells after balloon injure in rat carotid artery

Objectives To study the expression Prohibitin in proliferation vascular smooth muscle cells (VSMCs) after balloon injure in rat carotid artery. Methods Twenty-four male SD rats of SPF grade were randomly divided into control groups with 6 animals and injured group with 18 animals. Based on the model of balloon injure in rat carotid artery, the artery tissues were harvested on 7, 14, 28 days after operation. The following investigation was carried out: Immunohistostaining and Western-blotting analysis the expression of Prohibitin in proliferation VSMCs. Results The arterial intimal thickening and neointima formation, at 7 days after vascular balloon catheter injury, there were 4-6 layers of cells. 14 days after balloon injury, there were more than 8 ∼ 10 layers of cells, and injury induces arterial intimal thickening after 28 days. The intimal proliferation area was more thickener in 28 days than 14 days, and 14 days more thickener than 7 d after injur, the 7 days more thickener than the control group, P < 0.05. There was PHB expressed in VSMCs of normal vascular, but PHB expression was significantly increased in injured rat carotid arteries on 7 day compared with normal and further to increase on 14 day and 28 day after injury (P < 0.05) Conclusions The results indicated there was a small amount PHB expressed in VSMCs of normal vascular, the expression of PHB may be associated with VSMCs proliferation and migration after balloon injury.

GW24-e1392 The Role of ER stress in the BH3-only protein Bim-mediated cardiomyocytes apoptpsis induced by hypoxia

Objectives To investigate the role of endoplasmic reticulum stress in the injury induced by Bim of hypoxic cardiomyocytes. Methods The Rat cardiomyocytes, 1-3 days after birth, were cultured primarily and identified by using antibody targeting α-actin of striated muscle. Bim-siRNA were transfected transiently into cardiomyocytes by lipofectamine 2000. Establish a model of hypoxia (The cells after 48 h transfection, were given a deal with hypoxia for 12 h). The experiments were divided into five groups: Blank Control Group, Hypoxia Group, Hypoxia + Negative Control siRNA Group, Hypoxia + Mock control Group, Hypoxia + Bim-siRNA Group. The cell survival rate was determined by MTT method; the cell apoptosis rate and the change of the calcium concentration in cells were detdemined by flow cytometry; the expression of Bim and the markers of endoplasmic reticulum stress Caspase-12 and InSP3 were assessed by Western Blotting. Results Immunohistochemical identification confirmed that rat cardiomyocytes were successful cultured. Green fluorescence was observed in the cells transfected of negative control siRNA group though the fluorescence microscope. The results of Western blot showed that the transfection of Bim-siRNA decreased the expression of Bim effectively (p < 0.01). Compared with the blank control group, The MTT assay determined that the survival rate of cardiomyocytes was decreased (p < 0.05) after the injured by hypoxia. And the results of flow cytometry showed that hypoxia increased cell apoptosis rate (P <0.01) and the concentration of calcium (p < 0.01), while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P <0.05 or P <0.01). Compared with the hypoxia + Negative Control siRNA group, the transfection of Bim-siRNA increased the cell survival rate, decreased cell apoptosis rate and the concentration of calcium (p < 0.01). The results of Western blotting showed that the transfection of Bim-siRNA reduced the expression of endoplasmic reticulum stress markers Caspase-12 and InSP3 (p < 0.05 or p < 0.01), and reduced the effects that hypoxia increased the expression of Caspase-12 and InSP3 (p < 0.05 or p < 0.01). Conclusions The down-regulation of the expression of Bim can suppress the apoptosis of cardiomyocytes induced by hypoxia. Its mechanism is associated with the endoplasmic reticulum stress. It is likely to be a new direction for treatment of coronary atherosclerotic heart disease.

GW24-e1393 The effect of Bim silencing by siRNA on apoptosis induced by hypoxia in rat cardiomyocyte

Objectives To investigate the effect of BH3 only protein Bim (Bcl-2 interacting the mediator of the cell death) on apoptosis induced by hypoxia in rat cardiomyocyte. Methods The Rat cardiomyocytes, 1-3days after birth, were cultured primarily and identified by using antibody targeting α-actin of striated muscle. Bim-siRNA were transfected transiently into cardiomyocytes by lipofectamine 2000. Establish a model of hypoxia (The cells after 48 h transfection, were given a deal with hypoxia for 12 h). The experiments were divided into five groups: Blank Control Group, Hypoxia Group, Hypoxia + Negative Control siRNA Group, Hypoxia + Mock control Group, Hypoxia + Bim-siRNA Group. With the inverted microscopy, the Cardiomyocyte beat frequency and rhythm can be observed and recorded; the content of lactate dehydrogenase (LDH) in cell culture fluid was detected by automatic biochemical analyser; The cell survival rate was determined by MTT method; the cell apoptosis rate was determined by flow cytometry; the expression of Bim, Bax, Bcl-2 and p-p38MAPK was assessed by Western Blotting. Results Immunohistochemical identification confirmed that rat cardiomyocytes were successful cultured. The beat frequency of cardiomyocyte was slowed down after hypoxia stimulation, the rhythm was disordered (P < 0.01) and the content of lactate dehydrogenase (LDH) in cell culture fluid increased obviously (P < 0.01). The MTT result showed that the survival rate was decreased (p< 0.05). And the results of flow cytometry showed that hypoxia increased cell apoptosis rate (P < 0.01), while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P < 0.05 or P < 0.01).the results of Western blotting showed that the transfection of Bim-siRNA decreased the expression of Bim (p<0.01). It confirmed that Bim was silenced by Bim-siRNA effectively. the hypoxia increased the expression of Bax and p-p38 (p<0.05),and decreased the expression of Bcl-2(p<0.01),while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P <0.05). These were greatly related to the decreasing of apoptosis. Conclusions The down-regulation of the expression of Bim can suppress the apoptosis of rat cardiomyocyte induced by hypoxia. Its mechanism is associated with the down-regulation of p-p38MAPK, Bax expression and up-regulation of Bcl-2. It is likely to be a new direction for treatment of coronary atherosclerotic heart disease.

GW24-e2942 The change of endogenous asymmetric NG, NG-dimethyl-L-arginine in patients with unstable angina pectoris

Objectives To investigate the change of asymmetric NG, NG-dimethyl-L-arginine (ADMA) and elucidate the possible mechanism of endothelial dysfunction in patients with unstable angina. Methods Screening 103 unstable angina patients hospitalised in our hospital from June 2010 to Jury 2012, the diagnosed standards of unstable angina are typical angina pectoris, positive ECG or exercise stress test and with more than 70% stenosis of single or multiple coronary arteries by angiography, excluded other diseases, such as hypertension, diabetes, hypertrophic cardiomyopathy et al. We selected 20 normal persons with matching age and sex as control. The levels of ADMA, NO, ET-1, SOD, MDA, blood lipid, blood glucose, blood viscosity, fibrinogen were measured respectively. Results 1. Compared with control group, the levels of ADMA, ET-1 were significantly increased and the level of NO was significantly decreased in unstable angina group (5.60 ± 1.79 vs 1.01 ± 0.05, 70.96 ± 16.05 vs 50.58 ± 7.52, 52.8 ± 7.59 vs 75.6 ± 18.3, P < 0.05 respectively). ADMA was positive correlated with the ratio of ET-1/NO. The levels of ADMA and ET-1 were also increased significantly in unstable angina group.. There were statistical significance between control and unstable angina group for the levels of SOD and MDA (70.3 ± 17.8 vs 104 ± 18.8 and 6.25 ± 0.79 vs 4.09 ± 0.58, P < 0.05 respectively). The levels of blood lipid (TC, TG, LDL, HDL-C, Lpa) in most patients of CHD were abnormal, and were higher than those in control group (P < 0.05). There were no differences for the levels of blood glucose, blood viscosity, fibrinogen between two groups. Conclusions This study shows endothelial dysfunction in the patients with unstable angina is associated with the increase of ADMA. Oxide stress may play an important role in unstable angina.

GW24-e2989 Linear ablation of left atrium for treatment atrial fibrillation guided by three dimensional electroanatomical mapping but without lasso catheter

Objectives There were few data about linear ablation of left atrium (LA) without lasso catheter guiding, We report the results of linear ablation of LA guided by three dimensional (3-D) electroanatomical mapping (Carto) and mapping catheter but without lasso catheter in 106 patients. Methods There were 106 patients (mean age, 51.4 ± 9.9 years). Seventy-eight patients had paroxysmal AF, 12 persistent AF and 16 permanent AF. After the anatomical model of LA and all pulmonary veins (PVs) had been established, circumferential ablations of the left pulmonary vein antrum and the right pulmonary vein antrum were performed. The endpoint of ablation was abolishment or dissociation of the pulmonary vein potentials (PVPs) according to the mapping at the two sides of ablation line with large-tip mapping catheters. Oral amiodarone or Flecainide was taken for at least 3 months by patients with persistent AF, or those whose PVPs had not been isolated completely. The recurrence of atrial tachyarrhythmia was observed 3 months after the procedure. Results Onset of atrial fibrillation occurred in 52 patients during ablation procedure. Thirty-two patients restored to sinus rhythm eventually after the procedure. Abolishment or dissociation of PVPs was accomplished during the procedure in 94 patients (88.7%). The duration of procedure and exposure to X-ray were (213 ± 45) minutes and (32.5 ± 12.8) minutes, respectively. Among the 87 patients followed up for over 3 months, 62 were free of atrial tachyarrhythmias (including 8 patients who were still taking oral amiodarone). The success rate was 71.3% in the first procedure. Two patients had pericardial effusion treated by pericardial puncture and effusion drainage. No pulmonary vein stenosis, atrioesophageal fistula, pericardial effusion, stroke or procedural death occurred. Conclusions Combination of 3-D electroanatomical mapping with large-tip catheter mapping at two sides of ablation line to guide the linear ablation of left atrium procedure can confirm the isolation of PVPs.

ASSA13-18-2 Aging Attenuates the Inter-Arm Diastolic Blood Pressure Difference Induced by One Arm Exercise

Objectives It is known that one arm exercise increases the inter-arm diastolic blood pressure difference (dIAD) in young people, but no research was performed in middle-aged and more senior population. This study aimed to determine whether ageing impacts the exercise-induced dIAD in healthy subjects. Methods Normotensive adults (n = 120) were recruited and divided into the young (22.5 ± 1.5y), middle-aged (42.8 ± 4.6y) and senior (61.0 ± 7.0y) groups. The right arm exercise involved performing cycling movements at 60 times/min for three minutes. Bilateral brachial BPs were simultaneously measured with two automatic BP measurement devices before (baseline), immediately (0), 5, 10 and 15 min after the exercise. The difference of bilateral diastolic BPs was calculated as BP l-r and its absolute value of equal or over 10mmHg was recognised as IAD. Results At baseline, the SBP l-r and DBP l-r were similar in three age groups. One arm exercise induced markedly DBP decline in the exercised arm, and then increased DBP l-r and dIAD prevalence in three age groups in an age-dependent manner. The dIAD prevalence increased from baseline of zero to 85% at 0 min in young, 37% in middle-aged and 30% in senior groups. One arm exercise did not significantly change SBP l-r and systolic IAD prevalence in three groups. A reverse correlation was found between the DBPl-r 0 and ages (r = –0.359, P < 0.05), but no correlation between ageing and SBPl-r 0. Conclusions Aging attenuates the levels and duration of inter-arm DBP difference induced by one arm exercise in healthy adults.

GW24-e0318 Analysis of SCN5A mutation in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia

Objectives Arrhythmogenic right ventricular cardiomyopathy/Dysplasia (ARVC/D) is a genetically determined disorder, characterised by the two component: cardiomyopathy and arrhythmia. To date, the molecular pathogenesis underly this phenomenon is poorly understood. Whether the ion channel defect involved in the ARVC/D is unknown. The aim of this study was systematically evaluate the sodium channel variants in ARVC/D. Methods The patients according to the diagnostic guideline of ARVC/D revised in 2010 were collected. Genomic DNA was extracted from peripheral blood lymphocytes. All the exons and exon-intron boundaries of the SCN5A gene and desmosomal genes known to be associated with ARVC/D, including DSC2, DSG2, DSP, JUP and PKP2 were sequenced through the direct DNA sequencing. Results A total of 13 unrelated index patients were collected. A new missense heterozygote mutation I137M in SCN5A gene was found in one proband 5. The mutation sited at the exon 4 of the SCN5A and the S1 segment in Domain I of Nav1.5, consisted of an C-to-G substitution at nucleotide site 411 (c.411C>G), which predicted a substitution of isoleucine for methionine at codon site 137 (p. Ile137Met, I137M). I137M was not detected in the 400 healthy control chromosomes from individuals of the same ethnic background, which indicated that this mutation was a conservative site in SCN5A gene and the encoding protein- Nav1.5 may have a functional defect. Conclusions Our study for the first time systematically evaluates the sodium channel variants in patients with ARVC/D and find a new SCN5A mutation- I137M. The result increases the insight of genetic pathogenesis in ARVC/D. The mutational sodium channel may destroy the “desmosomal-related complex” and cause the genesis of ARVC/D.

ASSA13-10-16 The Role of Endoplasmic Reticulum Stress in The Injury Induced by Bim of Hypoxic Cardiomyocytes

Objective To investigate the role of endoplasmic reticulum stress in Bim-induced cardiomyocytes injured by hypoxia. Methods Bim-siRNA were transfected transiently into H9C2 cells by lipofectamine 2000. Establish a model of hypoxia (The cells after 48 h transfection, were given a deal with hypoxia for 12 h). The experiments were divided into five groups: Blank Control Group, Hypoxia Group, Hypoxia + Negative Control siRNA Group, Hypoxia + Mock control Group, Hypoxia + Bim-siRNA Group. The cell survival rate was determined by MTT method; the cell apoptosis rate and the change of the calcium concentration in cells were detdemined by flow cytometry; the expression of Bim and the markers of endoplasmic reticulum stress Caspase-12 and In SP3 were assessed by Western Blotting. Results Green fluorescence was observed in the cells transfected of negative control siRNA group though the fluorescence microscope. Compared with the blank control group, The MTT assay determined that the survival rate of H9C2 was decreased (p < 0.05) after the injured by hypoxia. And the results of flow cytometry showed that hypoxia increased cell apoptosis rate (P < 0.01) and the concentration of calcium (p < 0.01), while the transfection of Bim-siRNA reduced the effects caused by hypoxia (P < 0.05 or P < 0.01). Compared with the hypoxia group, the transfection of Bim-siRNA increased the cell survival rate, decreased cell apoptosis rate and the concentration of calcium (p < 0.01 or p < 0.05). While there was no significant difference among Hypoxia Group, Hypoxia + Negative Control siRNA Group and Hypoxia + Mock control Group (p > 0.05). The results of Western blotting showed that the transfection of Bim-siRNA reduced the expression of Bim obviously (p < 0.01); meanwhile, reduced the expression of endoplasmic reticulum stress markers Caspase-12 and InSP3 (p < 0.05 or p < 0.01), and reduced the effects that hypoxia increased the expression of Caspase-12 and InSP3 (p < 0.05 or p < 0.01). Conclusions The down-regulation of the expression of Bim can suppress the apoptosis of H9C2 induced by hypoxia. Its mechanism is associated with the endoplasmic reticulum stress. It is likely to be a new direction for treatment of coronary atherosclerotic heart disease.

ASSA13-16-7 Rosuvastatin Treatment Improved the Composition of Trans-Fatty Acids of Erythrocyte Membrane in Patients with Hypercholesterolemia

Objective This study was to investigate the composition of trans-fatty acid (TFA) of erythrocyte membrane in hypercholesterolemic patients (HC) and the effect of rosuvastatin treatment. Methods The fasting serum lipids and 22 FAs were determined in 30 HC patients and 30 ortholiposis subjects (control group). Eight main cis-fatty acids (CFA) and 3 TFAs were used for analysis. CFAs included 2 saturated FA (SFAs, C16:0 and C18:0); 1 monounsaturated FA (C18:1) and 5 poly-unsaturated FAs (PUFA). TFAs included C16:1–9t, C18:1–9t and C18:1–11t. Repeated examination was performed in the HC group after 4 week rosuvastatin (10mg/day) treatment. The percentage of a FA to the total FA was calculated as the composition of the FA. Results Compared with the control group, the HC group had significantly higher compositions of C18:1, C16:1 and C18:1, lower compositions of 5 PUFAs, as well as higher compositions of 3 TFAs. In the HC group, the ratios of 18:1–9t/18:1–9c and 18:1–11t/18: 1–11c were significantly higher, but the ratio of 16:1–9t/16:1–9c was lower. The rosuvastatin treatment could restore the composition of TFA and the abnormal ratios of trans-/cis- isomer of erythrocyte membrane. Conclusions Our results demonstrated that the compositions of 3 TFAs of erythrocyte membrane obviously increase in hypercholesterolemic patients, but the ratio of the trans- to cis- isomer varies with TFA. Rosuvastatin treatment can effectively normalise the compositions and ratios of the trans- to cis- isomer of TFA in erythrocyte membrane.