【作者】 董炳庆；

【导师】 闫素华；

【作者基本信息】 山东大学 ， 内科学， 2012， 博士

【摘要】 研究背景：糖尿病和冠心病是目前临床常见的两种疾病,糖尿病合并心肌梗死患者室性心律失常发生率明显增加,死亡率也随之增加。国内外研究发现,心肌梗死患者并发室性心律失常与梗死后细胞和组织水平的电重构即离子通道重塑有关。离子通道重塑包括离子通道或转运体的表达、调控和相关蛋白伴侣的改变。这些病理生理重塑主要发生于钠通道、钾通道、钙通道、钙转运体、缝隙连接蛋白、超极化激活的非选择性阳离子通道等。上述变化促使动作电位时程延长,复极离散度增加,传导异常,使得心肌电生理特性发生改变而出现电学的失稳态,最终导致致命性心律失常和心脏性猝死的发生。糖尿病是一组由于胰岛素分泌缺陷和(或)胰岛素作用缺陷导致的以慢性血糖水平增高为特征的代谢异常综合征。其中,2型糖尿病约占90%,其病因未明,多数患者确诊时已存在慢性并发症,如心、脑、肾、眼、神经等部位病变,这也是2型糖尿病致残、致死的主要原因。目前公认高血糖是糖尿病各种慢性并发症的主要诱发因素,但高血糖并非直接参与并发症形成,而是高血糖先引起体内多种生化指标的改变,进而损害各个脏器导致并发症的发生。既往国内外研究高血糖引起的生化改变机制复杂多样,如非酶类糖化过程增强、氧化还原应激反应、醛糖还原酶激活、二酰基甘油-蛋白激酶C通路活性增加等。但糖尿病并发症发生机制十分复杂,其具体的病理生理及分子机制至今尚未完全阐明。糖尿病的心血管系统并发症严重影响糖尿病患者的临床预后,而冠心病及自主神经病变则为引起糖尿病患者心力衰竭和猝死发生率增加的重要因素。除了血管病变外,心肌细胞功能本身也存在明显异常,表现为心肌细胞收缩力下降及电生理特性的改变,后者又表现为心肌细胞动作电位时程显著延长。钾通道电流是引起心肌细胞动作电位复极的重要电流,糖尿病对心肌细胞动作电位的影响,部分是通过对钾离子通道的影响而导致。人们应用动物模型来研究糖尿病对心肌的影响,至今已比较成熟的方法是链脲佐菌素或四氧嘧啶所导致的胰岛素缺乏而诱发糖尿病,最常用的模型为链脲佐菌素模型,链脲佐菌素通过选择性破坏胰岛β细胞而诱发糖尿病。在这一模型,心脏很快出现电生理和机械特性的改变,其电生理改变主要表现为动作电位形态和时程的改变,在心电图上则表现为QT间期延长及T波低平。对心肌细胞离子流研究的一个重要发现则为瞬间外向性钾电流密度降低,这一变化在心室的心外膜心肌较心内膜心肌明显。另有动物实验研究也显示糖尿病心肌细胞动作电位时程延长部分系由于外向复极钾电流特别是瞬间外向钾离子电流降低所致。现已明确,高血糖和急性心肌梗死后,心脏局部肾素-血管紧张素系统激活,其中心环节是血管紧张素Ⅱ识别其特异性受体,通过一定的信号途径激活G蛋白,进而激活磷脂酶C,使细胞内三磷酸肌酸和二酯酰甘油的合成增加,进而激活心肌蛋白激酶C,过度表达蛋白激酶C激活丝裂素活化蛋白激酶,将信号导入细胞核内,激活转录基因,引起多种生长刺激因子表达异常如C-fOS等,磷酸化非常广泛的蛋白质底物,导致细胞骨架蛋白合成障碍、心肌细胞受损、心肌异常生长、心肌重塑、心功能减低。缬沙坦是一种特异的AT1受体竞争性拮抗剂,具有剂量依赖性,其通过与AT1跨膜区氨基酸作用,阻止血管紧张素Ⅱ与AT1受体结合,阻断血管紧张素Ⅱ诱导的生物学效应。动物研究已证实,缬沙坦可通过拮抗AT1受体减轻或改善心房纤维化所致的局部传导异常,减少去甲肾上腺素的释放,改善心房复极的不均一性,以及抑制心房电重构。最新NAVIGATOR研究发现缬沙坦能有效预防新发糖尿病达14%,显著降低空腹血糖、餐后2小时血糖。国内对心肌梗死大鼠模型的研究证实缬沙坦可改善心肌梗死后心室肌的电重构。但缬沙坦对糖尿病合并心肌梗死的心室肌电重构影响国内外尚未见报道。糖尿病心肌梗死后是否进一步影响心室肌电重构也未见报道。本研究通过构建糖尿病合并心肌梗死兔动物模型,运用实时荧光定量PCR的方法和Western blot方法,对糖尿病合并心肌梗死后心室肌细胞瞬间外向钾离子通道蛋白及基因表达的变化特点进行了探讨,并应用不同剂量的缬沙坦对其进行不同阶段干预研究,以期发现其与室性心律失常的关系,以便指导临床,改善糖尿病合并心肌梗死患者的预后。目的：探讨糖尿病兔心肌梗死后心室肌瞬间外向钾离子通道蛋白及基因表达的变化以及缬沙坦对此变化的影响。方法：1.选择健康新西兰兔作为研究对象,通过高脂高糖喂养2个月后由耳缘静脉注射四氧嘧啶(80mg/kg)制作糖尿病模型,成模后改为普通饲料继续单笼喂养4个月,并随机分为糖尿病+心肌梗死组(DM+MI组)、糖尿病假手术组(DM组)和非糖尿病假手术组(对照组),通过结扎冠状动脉前降支制作心肌梗死模型,最终每组14只。手术后所有入选成活兔均常规喂养2个月,在3%戊巴比妥钠(40mg/kg)麻醉下迅速开胸取出心脏,剪取左心室梗死周围区心肌组织,用冷生理盐水冲洗后,DEPC水漂洗,分装于冻存管置于液氮中保存备用。采用实时荧光定量PCR方法和Western blot方法观察心室肌钾离子通道Kv4.2、Kv4.3和Kv1.4的基因及蛋白表达变化。2.选择造模成功的新西兰兔随机分为6组：安慰剂组(placebo group, Group P)、低剂量组(low dose Group, Group L)、中等剂量组(moderate dose Group, Group M)、高剂量服药6周组(high dose of medication6weeks Group, Group H,)、高剂量服药8周组(high dose of medication8weeks Group, Group H2)和高剂量服药10周组(high dose of medication10weeks Group, Group H3)每组10只。GroupL组给予5mg/(kg·d)缬沙坦灌胃6周；Group M组给予10mg/(kg·d)缬沙坦灌胃6周；Group H1组给予30mg/(kg·d)缬沙坦灌胃6周；GroupH2组给予30mg/(kg·d)缬沙坦灌胃8周；Group H3组给予30mg/(kg·d)缬沙坦灌胃10周。Group P组给予生理盐水代替缬沙坦灌胃6周。分别在6周、8周或10周后,在3%戊巴比妥钠麻醉下迅速开胸取出心脏,剪取左心室梗死周围区心肌组织,用冷生理盐水冲洗后,DEPC水漂洗,分装于冻存管置于液氮中保存备用,采用实时荧光定量PCR方法和Western blot方法观察心室肌钾离子通道Kv4.2、Kv4.3和Kv1.4的基因及蛋白表达变化。结果：1. DM+MI组和DM组兔心室肌Kv4.2和Kv4.3mRNA水平均显著低于对照组(P<0.05),而Kv1.4显著高于对照组(P<0.05)；DM+MI组兔心室肌Kv4.2和Kv4.3mRNA水平显著低于DM组(P<0.05),而Kv1.4并无显著升高(P>0.05)。2. DM+MI组和DM组兔心室肌Kv4.2和Kv4.3蛋白表达量均显著低于对照组(P<0.05),而Kv1.4显著高于对照组(P<0.05)；DM+MI组兔心室肌Kv4.2和Kv4.3蛋白表达量显著低于DM组(P<0.05),而Kv1.4并无明显变化(P>0.05)。3.分别与Group P比较,各治疗组兔心室肌Kv4.2和Kv4.3mRNA水平均显著升高(P<0.05),而Kv1.4反而显著降低(P<0.05)；Group M, Group H1、Group H2和Group H3兔心室肌Kv4.2和Kv4.3mRNA水平均显著高于Group L(P<0.05),而Kv1.4并无显著升高(P>0.05)；与Group H1比较,Group H3兔心室肌Kv4.2和Kv4.3mRNA水平均显著升高(P<0.05),而Kv1.4无显著变化(P>0.05)；不论是Kv4.2、Kv4.3还是Kv1.4,在Group H1和GroupH2间mRNA水平并无明显变化(P>0.05)。4.分别与Group P比较,各治疗组兔心室肌Kv4.2和Kv4.3蛋白表达水平均显著升高(P<0.05),而Kv1.4反而显著降低(P<0.05)；Group M、Group H1、Group H2和Group H3兔心室肌Kv4.2和Kv4.3蛋白表达水平显著高于Group L(P<0.05),而Kv1.4并无显著升高(P>0.05)；与GroupH1比较,GroupH3组兔心室肌Kv4.2和Kv4.3蛋白表达均显著升高(P<0.05),而Kv1.4无显著变化(P>0.05)；不论是Kv4.2、Kv4.3还是Kv1.4,在Group H1和GroupH2间蛋白表达量并无明显变化(P>0.05)。5.分别与Group P比较,各治疗组在实验的终末期,其空腹血糖水平明显下降(P<0.05)。结论：1.糖尿病兔心肌梗死后心室肌瞬间外向钾离子通道基因及蛋白表达发生了改变即电重构现象,可能是心肌梗死后室性心律失常易感性增加机制之一。2.缬沙坦可改善糖尿病兔心肌梗死后心室肌瞬间外向钾离子通道基因及蛋白表达,修复糖尿病合并心肌梗死心室肌电重构,这可能是降低室性心律失常发生率的重要原因。3.缬沙坦对实验糖尿病心肌梗死兔的空腹血糖有一定改善作用。4.缬沙坦疗效呈时间和剂量依赖性,长期治疗效果更明显。更多还原

【Abstract】 BACKGROUND:Diabetes and coronary heart disease are common diseases. And the incidence of ventricular arrhythmias and mortality in diabetic patients with myocardial infarction (MI) increase dramaticly. But their mechanisms are unclear. Electrical remodeling was gradually recognized related with incidence of ventricular arrhythmia in patients with MI. Electrical remodeling includes ion channel or transporter related protein expression, regulation and partner changes, which causes disturbances of heart rhythm and induces various arrhythmias. These pathophysiological remodeling occurs mainly in sodium channels, potassium channels, calcium channels, transporters, gap junction proteins, hyperpolarization activated nonselective cation channels, and so on. All these changes have prompted the extension of action potential duration, increased the dispersion of repolarization, induced abnormal conduction and loss of steady-state of cardiac electrophysiology, and then lead to fatal arrhythmias and sudden cardiac death.Diabetes mellitus is a group of chronic metabolic syndrome because of defect of insulin secretion and (or) defects of insulin, characterized by elevated blood sugar levels. Among them, type2diabetes accounts for about90%. Hyperglycemia has injured multiple organs before diabetes being diagnosed, and caused high incidence of disability and mortality. Those chronic complications are not directly caused by high blood sugar but by many biochemical changes in high blood glucose environment. The nuclear mechanism of the complications is very complicated and has not yet been fully elucidated. Cardiovascular complications of diabetes are complex and diversity. Such as complying with coronary heart disease and autonomic neuropathy in diabetic patients cause increasing incidence of sudden death and congestive heart failure and seriously affect clinical prognosis. Additionally, hyperglycemia also impairs cardiac contractility and causes electrophysiological changes, which significantly delay duration of action potential. Potassium channel current is the major current during repolarization. The impact of diabetes on action potential duration may be caused by the effects of potassium ion channel. Diabetic animal models which induced by streptozotocin or alloxan through selective destruction of pancreatic beta cells is skillful. Streptozotocin inducing diabetic model is the usual insulin deficiency model. It leads to the change of cardiac electrical and mechanical properties, which manifests as changes in action potential morphology and duration, QT interval prolongation and T wave flat on the ECG. Moreover, diabetes reduces density of transient outward potassium current and obviously changes outer membrane of endocardial myocardium. Previous study showed that the prolongation of action potential duration partly dued to outward repolarization potassium current and transient outward potassium current was reduced in diabetic cardiac myocytes.High blood glucose and MI both activate local cardiac rennin angiotensin aldosterone system (RAAS). Angiotensin Ⅱ (Ang Ⅱ) is very important in this pathological process. It can identify specific receptors through some signaling pathways, such as activating G protein, activating phospholipids enzyme C (PLC), and activating the myocardial protein kinase C (PKC). Excessive expression of PKC activates mitogen-activated protein kinase, then signals into the cell nucleus, activates the transcription gene, causes a variety of growth stimulating factor expression abnormalities like C-fos, which hinders cell protein synthesis, damages myocardial cell and leads to myocardium abnormal growing, myocardial remodeling, and cardiac function reducing. In short, Ang Ⅱ may be an important initiator of arrhythmia by affecting the function of ion channels. Valsartan, as a kind of specific AT1receptor competitive antagonist, prevents Ang Ⅱ through AT1transmembrane amino acids and then blocks the Ang Ⅱ-induced biological negative effects. A few studies have identified that valsartan can improve local abnormal conduction of atrial fibrosis, reduce the release of norepinephrine, improve atrial repolarization heterogeneity, and inhibit electrical remodeling. Moreover, it also can dramatically reduce the incidence of type2diabetes. Whether valsartan impact ventricular electrical remodeling in diabetic patients with MI or not is unknown. In this study, we evaluate the expression profile following MI in a diabetic model, to explore the effects of valsartan on the expression of transient outward potassium channel (Kv1.4, Kv4.2, Kv4.3) in the left ventricle of diabetic rabbits after experimental MI.AIM:To explore the expression of the transient outward potassium channel in diabetic rabbits after experimental MI and valsartan intervention study.METHODS:1. New Zealand rabbits were randomly divided into diabetes+MI group(Group DM+MI, n=14), sham operation diabetic group(Group DM, n=14) and non-diabetic sham operation group(Group sham, n=14). And then diabetic rabbit model was produced. High fat and high calorie mixed diet were feeded for2months and then ALX (80mg/kg, Alloxan) was injected through auricular vein. The standards of diabetic animals are the concentrations of fasting blood glucose>14mmol/L by2consecutive analyses72h and7d after injection. If not done, then repeat injection ALX (50mg/kg) above methods. After diabetic model was established, general feed was given for4months in individual cages. MI model was created by ligature of the left anterior descending coronary artery. It was confirmed by regional cyanosis and electrocardiographic change (more than two ST segment elevations of0.1mV or higher and7days later the Q wave appears). After routine feeding for2months, the animals were sacrificed and heart was isolated. Left ventricle tissue around infarction aera was rinsed in saline and DEPC water to remove excess blood, snap-frozen in liquid nitrogen, and stored at-80℃. Real-time quantitative PCR and western blot were used to observe the expression of Kv4.2, Kv4.3, and Kv1.4in ventricle.2. Sixty successful models of New Zealand rabbits were randomly divided into six groups:the placebo Group (Group P, n=10), low dose Group (Group L, n=10), moderate dose Group (Group M, n=10), high dose of medication6weeks Group (Group H1, n=10), high dose of medication8weeks Group (Group H2, n=10) and high dose of medication10weeks (Group H3, n=10).The treatment Groups were intragastricly administrated valsartan for6to10weeks. Among them, the Group L give5mg/(kg-d) valsartan for6weeks; Group M give10mg/(kg·d) valsartan for6weeks; Group H1give3Omg/(kg-d) valsartan for6weeks; Group H2give30mg/(kg·d) valsartan for8weeks; Group H3give30mg/(kg·d) valsartan for10weeks. Group P give normal saline instead of valsartan for6weeks. All rabbits were sacrificed6weeks,8weeks or10weeks respectively after the procedure. Then heart was isolated and left ventricle tissue around infarction aera was carefully excised and rinsed in saline and DEPC water to remove excess blood, snap-frozen in liquid nitrogen, and stored at-80℃. Real-time quantitative PCR and western blot were used to observe the expression of Kv4.2, Kv4.3, and Kv1.4in ventricle.RESULTS:1. Compared with Group sham, the expression levels of Kvl.4mRNA in both Group DM+MI and Group DM were increased significantly, while the expression levels of Kv4.2and Kv4.3mRNA were decreased significantly (P<0.05). The expression levels of Kv4.2and Kv4.3mRNA in Group DM+MI rabbit ventricle were significantly lower than the Group DM (P<0.05). But the expression of Kvl.4mRNA did not change significantly (P>0.05).2. Compared with Group sham, the expression levels of Kvl.4protein in both Group DM+MI and Group DM were increased significantly, while the expression levels of Kv4.2and Kv4.3protein were decreased significantly (P<0.05). The expression levels of Kv4.2and Kv4.3protein in Group DM+MI rabbit ventricle were significantly lower than the Group DM (P<0.05). But the expression of Kv1.4protein did not change significantly (P>0.05).3. Compared with Group P, the expression levels of Kv1.4mRNA of each treament group were decreased significantly and the expression levels of Kv4.2and Kv4.3mRNA were increased significantly (P<0.05). The expression levels of Kv4.2and Kv4.3mRNA in Group M、Group H1. Group H2or Group H3were significantly higher than Group L (P<0.05), while Kv1.4did not significantly change (P>0.05). The expression levels of Kv4.2and Kv4.3mRNA in Group H3were significantly higher than Group H1(P<0.05), and Kvl.4did not significantly change (P>0.05). But the expression of Kv4.2, Kv4.3and Kv1.4mRNA levels between Group H1and Group H2showed no significant change (P>0.05).4. Compared with Group P, the expression levels of Kv1.4protein of each treament group were decreased significantly and the expression levels of Kv4.2and Kv4.3protein were increased significantly (P<0.05). The expression levels of Kv4.2and Kv4.3protein in Group M、Group H1. Group H2or Group H3were significantly higher than Group L (P<0.05), while Kv1.4did not significantly change (P>0.05). The expression levels of Kv4.2and Kv4.3protein in Group H3were significantly higher than Group H1(P<0.05), and Kv1.4did not significantly change (P>0.05). But the expression of Kv4.2, Kv4.3and Kv1.4protein levels between Group H1and Group H2showed no significant change (P>0.05).5. Compared with group P respectively, the fasting glucose levels in valsartan treatment groups at the end-stage of the experiment decreased significantly (P<0.05).CONCLUSIONS:1. The expression of transient outward potassium channel changed obviously in diabetic rabbits after experimental MI. And these suggested there existed electrical remodeling, which probably increased the susceptibility of ventricular arrhythmias after MI with diabetes.2. Valsartan treatment partly improved expression of transient outward potassium channel and inhibited electrical remodeling, which was likely to reduce the incidence of ventricular arrhythmias in diabetes after experimental MI.3. Polonged valsartan treatment was helpful to decrease fasting blood glucose in diabetic rabbits with experimental MI.4. The role of valsartan presented dose and dependent. In addition to these, long-term treatment was more effective.更多还原