Hong Kui

GW24-e0353 A novel mutation R397Q in KCNQ1 gene caused dilated cardiomyopathy with ventricular tachycardia and prolonged QT interval

Objectives Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death with lethal ventricular arrhythmias. So far, the genetic mutations of DCM are mainly related to the sarcomeric protein, and mutations in SCN5A which encoding μ-subunit of sodium channel also have been involved. However, KCNQ1 gene encoding in potassium channel has not been found in DCM patients. The purpose of this study was to demonstrate if DCM with ventricular tachycardia and prolonged QT interval is caused by mutation in the KCNQ1 gene. Methods Clinical features and DNA samples of DCM patients with lethal ventricular arrhythmia were collected. The candidate genes including SCN5A, KCNQ1, KCNE1 and KCNE2 were screened by the direct sequencing to look for the mutations. Whole cell patch clamp analysis of HEK293 cells expressing Wild-type and mutation KCNQ1 channel was used to investigate the channels biophysical properties. The current-voltage relationships, peak current, current density, the voltage dependence of steady-state activation and inactivation of mutated channel were investigated. Results Twelve unrelated DCM patients with lethal ventricular arrhythmia were included and clinical features were available in medical records. Three patients (25%) presented cardiac conduction system abnormality, and nine patients (75%) showed QT prolongation in ECG recordings before amiodarone therapy. A G to A missense mutation in KCNQ1 gene was identified in a 60-year-old man with concomitant DCM and incessant VT and prolonged QT interval, which is at nucleotide site 1190 that resulted in a amino-acid substitution of arginine to glutamine at amino-acid site 397 (R397Q) in the C-terminal region of KCNQ1 gene. Whole cell patch clamp analysis of HEK293 cells expressing WT and mutant KCNQ1 was used to investigate the biophysical properties of the channel. It showed that R397Q (n = 19) decreased Iks current density dramatically compared with WT (14.34 ± 1.73 vs 23.25 ± 2.31, P < 0.01). However, R397Q didn’t influence on the V0.5 and slope factor for the voltage dependence of activation comparing to WT. Meanwhile, this patient experienced recurrent VT only 20 months post radio frequency ablation (RFCA). Finally an implantable cardioverter defibrillator (ICD) was successfully implanted and he had been without complaints up to the 3 years of follow-up visit. Conclusions It was firstly reported that a loss of function in KCNQ1 gene may cause the overlapping phenotype of DCM, ventricular tachycardia and prolonged QT interval. The reduction of Iks in R397Q did not significantly change the properties of gate. It seems that RFCA is the assistant to improve the incessant ventricular arrhythmia in the patients with abnormal structure heart disease and genetic background, ICD is necessary.

MicroRNA-223 prevents cardiomyocyte hypertrophy by targeting cardiac troponin I-interacting kinase

Objectives Our study was designed to investigate the role of microRNA-223 (miR-223) and its direct target gene, cardiac troponin I-interacting kinase (TNNI3K), in regulating cardiomyocyte hypertrophy. Methodology Neonatal rat cardiomyocytes (CMs) were cultured from one to two days old Sprague–Dawley rats. Cardiomyocyte hypertrophy was induced by endothelin-1 (ET-1). Expression of miR-223 in CMs was detected by real-time PCR. miR-223 mimics transfection was performed to achieve overexpression of miR-223 in CMs. Cell size was measured via surface area calculation under fluorescence microscopy after anti-α-actinin staining. Expression levels of ANP, α-actinin, Myh6, Myh7, as cardiac hypertrophy related marker genes, were detected by RT-PCR. The expression of TNNI3K protein was analysed by western blot. Luciferase assay was performed to confirm the direct binding of miR-223 to the 3′UTR of TNNI3K mRNA. Results In ET-1 induced hypertrophic CMs, expression of miR-223 was lower than that in normal CMs (Normal CMs: 1.00±0.08 vs Hypertrophic CMs: 0.62±0.16, p<0.05). Under stimulation of ET-1, miR-223 overexpressed CMs showed alleviated hypertrophic phenomenon, which characterised by less cell surface area (miR-223 group: 2590±781 mm2 vs ET-1 only group: 4680±1040 mm2, p<0.01) and lower expression of ANP, α-actinin, Myh6, Myh7, when compared with ET-1 stimulation only CMs. In miR-223 overexpressed CMs, the expression of TNNI3K protein was significantly decreased (miR-223 group: 0.39±0.05 vs Control: 0.03±0.01). Co-transfection of a miR-223 expression vector with pMIR-TNNI3K led to the reduced activity of luciferase in a dual-luciferase reporter gene assay, suggesting that TNNI3K is a direct target gene of miR-223. Conclusion All these results suggest that TNNI3K, a novel cardiac-specific kinase gene, is a direct target of miR-223. miR-223 plays a important role as suppressor in cardiomyocyte hypertrophy and could be used in clinical treatment of hypertrophy in future.

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-e2441 SCN5A mutation with lethal ventricular arrhythmias and invalidation of lidocaine in acute myocardial infarction

Objectives Patients are at high risk for potentially fatal ventricular tachycardia (VT)/ventricular fibrillation (VF) when suffering from acute myocardial infarction (AMI), due to myocardial ischaemia/reperfusion injury or scar formation. Recently, it was reported that SCN5A gene mutations may also contribute to electrical storm complicating AMI. The purpose of this study was to investigate potential SCN5A mutation in patients developing VT/VF during AMI, and reveal underlying cellular electrophysiological mechanism. Methods DNA samples of ten unrelated AMI patients with VT/VF were collected and clinical features were available in medical records. Candidate gene SCN5A was screened by direct sequencing. In expressed mutants, whole cell patch-clamp analysis was used to define the electrophysiologic properties. Current-voltage relationships, peak current, current density, voltage dependence of steady-state activation and inactivation of mutated channels were investigated. Results A missense mutation A1427S located at the S5-S6 extracellular linker of domain III (DIII) in SCN5A gene was identified in a 56-year-old female, which was not observed in 400 healthy control chromosomes of the same ethnic background. In addition, we identified three previously reported SNPs in our subjects, including A29A, H558R and D1818D. Electrophysiological analysis showed obvious reduction of sodium current in A1427S mutant without the shape change of I-V curve. Retrospective analysis of the A1427S carrier’s medical records indicated that lidocaine infusion exacerbated episodes of VT/VF inversely and she finally died of refractory VT/VF complicating AMI. Conclusions We identified a novel SCN5A mutation A1427S that resulted in refractory VT/VF complicating AMI. Loss-of-function of the sodium channel indicates cautiously use of prophylactic lidocaine. Genetic screening could ideally identify the patients at high risk and aid in guiding the optimal therapy.

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.

Acute effect of circumferential pulmonary vein isolation by catheter radiofrequency ablation on P-wave dispersion

Objective To observe the acute effect of successful circumferential pulmonary vein isolation per se by catheter radiofrequency ablation on P-wave dispersion. Methods 38 patients who were underwent three- dimensional electroanatomical mapping system (Ensite 3000 or Carto) guided circumferential pulmonary vein isolation by radiofrequency catheter ablations for paroxysmal atrial fibrillation from August 2007 to April 2010 in our hospital were included in this study. There were 25 males and 13 females, aged from 34 to 74 years with a mean (59.60±8.35), in which 23 patients with hypertension, 8 with diabetes while all being NYHA class I. The maximum P-wave intervals (Pmax) and minimum P-wave intervals (Pmin) on ECG were respectively measured preoperation and the third day postoperation in all patients with sinus rhythm. P-wave dispersion (Pd) was defined as the difference between Pmax and Pmin and calculated in all cases. Results The Pmin and Pmax were respectively 57.8±15.8 ms, 128.9±25.8 ms and 60.1±14.3 ms, 115±20.6 ms before and after operation (p<0.001 for Pmax). The P-wave dispersion were significantly shortened from 71.1±17.5 ms to 54.7±15.6 ms after operation (p<0.001). There were no correlation between left atrial volume and preoperative Pd (r=0.122, p=0.437) or ΔPd (the change of Pd between postoperation and preoperation) (r=−0.209, p=0.208). Conclusions Even there were no observable correlation between preoperative and ΔPd, successful circumferential pulmonary vein isolation by catheter radiofrequency ablation for paroxysmal atrial fibrillation can immediately reduce maximum P-wave intervals (Pmax) and P-wave dispersion (Pd) suggest that this approach may have acute improvemental effect on heterogeneity of atrial electrical activity.

Heme oxygenase-1 downregulate the activity of nuclear factor NF-κB and protect the damage of cardiocyte induced by lipopolysaccharide in rats

Objective To determine whether HO-1 played a important role in the protection of cardiocyte in the lipopolysaccharide-induced acute cardiocyte injury model. Method Cardiocyte was isolated from SD neonate rat, and was enrolled into C group (normal culture), L group (with stimulation of LPS), L+H group (with stimulation of Hemin (inducer of HO-1), followed by LPS), and L+Z group (with stimulation of ZnPP (inhibitor of HO-1), followed by LPS). The levels of LDH, malondialdehyde (MDA), and superoxide dismutase (SOD) were measured. The cell heart rhythm, survival rate and the apoptosis rate were also examined. The expression of nuclear factor κB (NF-κB), HO-1, and tumour necrosis factor α (TNFα) were measured with RT-PCR, Western blotting and flow cytometry. Results The level of LDH and MDA were significantly higher in L group than those in C group (113±15 vs 69±10 U/L, p<0.05, and 1.88±0.36 vs 0.87±0.25 mmol/l, p<0.05, respectively), and decreased in L+H groups (p<0.05). The level of SOD in L, L+H, and L+Z groups was significantly lower than that in control group (p<0.05). The level of SOD in L+H group is higher than that in L group (p<0.05). The rate of apoptosis and cardiocyte heart rhythm increased significantly and survival rate was significantly lower in L, L+H, and L+Z groups than those in C group (p<0.05). The level of HO-1 mRNA was higher in L, L+H, and L+Z groups than that in C group (p<0.01), among which L+H group was the highest. The level of HO-1, TNFα and NF-κB in L, L+H, and L+Z groups was also higher than those in C group (p<0.05), among which the level of HO-1 protein in L+H group was the highest, the level of TNFα and NF-κB was the highest in L+Z group. Conclusion These data therefore suggested that HO-1 provide critical protection against LPS-induced cardiocyte injury. The protection seemed to be mediated through down-regulation of the activity of NF-κB.

e0432 An essential role of serum B-type natriuretic peptide in patients with acute inferior myocardial infarction

Objective To investigate the relationship between the level of serum B-type natriuretic peptide (BNP) and right ventricular infarction in patient s with acute inferior myocardial infarction (AIMI). Method The serum BNP level was measured in 213 consecutive patient s with AIMI who were admitted in the coronary care unit (CCU) and in the normal control with 98 cases from October 2006 to May 2009. The patients were divided into five groups in accordance with the coronary angiographic findings and ECG: Acute inferior myocardial infarction group; acute inferior myocardial infarction and right ventricular infarction group; control group; left anterior circumflex (LXC) group; proximal, middle and distal segment of right coronary artery (RCA)group. All patient s were performed directly percutaneous coronary intervention (PCI) within 24 h after the onset of AIMI. The incidence of major adverse cardiac events (MACE), including arrhythmia, heart failure, cardiac shock and mortality, had been observed during hospital, 30 days and 3 months. Result BNP level in the acute inferior myocardial infarction and right ventricular infarction group was significantly higher than that in acute inferior myocardial infarction group (p<0.101). The level in the proximal2mid segment of RCA group was higher than that in the LXC group (p<0.101). Additionally, logistic regression analysis showed that the level of BNP was an independent predictor of MACE in the 30 days and 3 months in acute inferior myocardial infarction patient s (r=0.701 0, 95 % CI <0.01 to 0.615, p<0.101). Conclusion We demonstrated the level of BNP in patients with acute inferior myocardial infarction or/and right ventricular infarction, and BNP could be a good predictor for patients with acute inferior myocardial infarction and right ventricular infarction.

e0183 Livin protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway

Introduction Although anoxic preconditioning (APC) in the myocardium has been investigated for many years, its physiological mechanism is still not completely understood. Increasing evidence indicates that transiently increased resistance to ischaemic damage following APC is dependent on de novo protein synthesis. However, the key effector pathway(s) associated with APC still remains unclear. Livin, a member of the inhibitor of apoptosis protein (IAP) family, since IAP-mediated activation of JNK1, as well as protection against TNF-β and ICE-induced apoptosis. The detailed mechanism underlying its antiapoptotic function in cardiomyocytes has not yet been fully characterised. Objective To investigate whether Linvin expression might be aberrantly induced in cardiomyocytes that were subjected to anoxia/reoxygenation (A/R) injury and to investigate whether Linvin might also contribute to cardio-protection after APC. Methods We cloned a Linvin expression vector, transfected it into rat cardiomyocytes, and examined Linvin expression in rat cardiomyocytes that were subjected to A/R injury. Moreover, we studied the role of three major MAPK pathways, for example, p38 MAPK, JNK, and ERK1/2, in order to evaluate the molecular mechanism underlying Linvin up-regulation and A/R induced cardiomyocyte injury. Results APC induced an up-regulation of Linvin and the transfection of Linvin gene into the cardiomyocytes attenuated A/R injury. The inhibition of p38 MAPK by SB203580 abolished both the Linvin up-regulation and the cardio-protection provided by APC. Conclusion APC could act to protect the heart from A/R injury with cooperation from the Linvin in addition, it up-regulates Linvin expression through a p38 MAPK signalling pathway.