【作者】 王春；

【导师】 谷天祥；

【作者基本信息】 中国医科大学 ， 外科学， 2007， 博士

【摘要】 目的心房颤动（房颤）是临床上最常见的慢性心律失常,是影响人口死亡率的独立因素。但临床上始终未发现解决房颤的根本方法,最大的阻碍就是房颤的发生机制始终不明确。自从1995年Wijffels等提出心房重构的概念以来,对其机制进行了大量的动物实验和临床研究,形成比较一致的观点:心房重构是心房颤动发生和持续的分子基础,其核心内容为心肌细胞动作电位时程和绝对不应期缩短,不同因素对重构的影响决定了房颤发生的复杂性。近年研究发现kv1.5通道在人类心房明显表达,而心室没有表达或很少表达且无生理功能,是构成心房肌细胞复极化的主要成分之一,是心房肌细胞超速延迟整流性钾电流（Ikur）的分子基础,对心房的复极以及动作电位时程（APD）起重要作用。多项研究发现,房颤时心房肌存在不同程度的能量代谢失衡导致氧应激损伤。体外细胞培养实验证实氧自由基可以直接影响kv1.5通道的电生理特性,在动作电位复极相增大kv1.5通道的电流幅度,促进通道闸门的快速开放,进而导致APD明显缩短。人类房颤心肌细胞中氧自由基对kv1.5通道影响的机制如何,尚无文献报道。尽管现有的研究结果尚不能够做出肯定的结论,但是心房肌组织的氧应激问题将会是研究和治疗房颤的新靶点。本课题以人类房颤心房肌细胞为研究对象,同时检测kv1.5通道和氧化还原酶在心房肌细胞的表达情况,以及氧自由基对kv1.5通道的影响等,探讨氧应激及其kv1.5通道的影响在心房重构中的内在作用。材料和方法一、研究对象本实验标本取自风湿性心脏病接受心脏瓣膜置换手术患者的右心耳组织,根据患者基本心律将标本分为心房颤动组和窦性心律组。二、研究方法1、RT-PCR研究kv1.5通道和氧化还原酶在心房肌细胞的表达情况采用Trizol法提取心肌组织标本的总RNA,二步法RT-PCR试剂盒首先逆转录合成cDNA,再进行kv1.5通道、含黄素单加氧酶-1（flavin containingmonooxygenase-1,FMO-1）、单胺氧化酶-B（monoamine oxidase B,MAO-B）、谷胱甘肽过氧化物酶-1（glutathione peroxidase-1,GPX-1）和血红素加氧酶-2（hemeoxygenase-2,HO-2）的扩增。扩增产物在1.5%琼脂糖上电泳,以GAPDH作为内参照,图像扫描仪测定电泳条带的光密度值,计算目的基因的相对表达量。2、Western Blot检测kv1.5通道蛋白的表达提取右心耳组织总蛋白,采用考马斯亮蓝法测定蛋白浓度。电泳、转膜、封闭、加用kv1.5和GAPDH一抗、二抗、ECL发光显色、扫描计算kv1.5的相对表达量。3、膜片钳技术研究氧自由基对kv1.5通道的影响（1）人心房肌细胞的分离体外循环插管前取右心房组织0.5cm³,将组织块用预先充氧的无钙液反复冲洗后,洗去血液和Ca²⁺后,组织块放入含150U/mlⅤ型胶原酶、12.5U/mlⅩⅩⅣ型蛋白酶和1mg/ml牛血清白蛋白的无Ca²⁺台式液（100%O₂,36±1℃）5mL中消化30分钟,再用无蛋白酶的上述新鲜液体5mL消化大约10分钟。当镜下细胞明显增多时,采用两步离心法（800转,4分钟）终止消化。离心后的细胞悬液用190目（μm）尼龙网滤掉碎片,留置细胞悬液,无钙液5倍稀释,逐步复钙至0.6mmol/L,备用。（2）膜片钳全细胞记录技术分别记录SR组、AF组、4-氨基吡啶（4-AP）+AF组、过氧化氢（H₂O₂）+4-AP+AF组的Ikur电流密度（pA/pF）和动作电位时程（APD）各15个细胞（n=15）。使用膜片钳放大器（Axopatch 200B）、数模转换装置（Digidata 1200）、Pclamp5.5.1软件采集数据,所得的数据采用Clampfit及ORIGIN（6.0）分析及绘图。所测电流用膜电容标化后以电流密度（pA/pF）表示,绘制电流电压（I/V）曲线,记录动作电位。4、统计学分析所有数据均以均值±标准差（（?）±s）表示,应用SPSS13.0软件采用配对t检验进行统计学处理,P＜0.05为有统计学差异。结果1、心房颤动时心房肌kv1.5通道及细胞氧化还原酶的基因表达水平应用凝胶图像扫描定量分析系统获取GAPDH和目的基因扩增产物的含量发现,与窦性心律组比较,窦性心律组kv1.5/GAPDH比值为0.91±0.17,AF组比值为0.63±0.11,较SR组明显减低,P＜0.05,有统计学差异。慢性房颤组FMO-1或MAO-B/GAPDH比值增高;GPX-1或HO-2/GAPDH比值减低,P＜0.05,有统计学差异。2、心房肌细胞kv1.5通道蛋白表达的水平应用图像灰度定量分析GAPDH和kv1.5蛋白的表达量,结果发现,窦性心律组kv1.5/GAPDH比值为1.31±0.19,AF组比值为0.52±0.17,较SR组明显减低,P＜0.05,有统计学差异。3、氧自由基对kv1.5通道的影响Ikur的I/V关系指令电压为+10～+50 mV时,AF组Ikur密度较SR组明显降低,4-AP+AF组较AF组明显降低,H₂O₂+4-AP+AF组较4-AP+AF组明显升高。其中,在+50mV时,电流密度由SR的（8.91±1.86）pA/pF降为AF组的（4.12±1.37）pA/PF,P＜0.01;4-AP+AF组的电流密度（2.75±0.84）pA/pF较AF组（4.12±1.37）pA/pF明显降低,P＜0.01;H₂O₂+4-AP+AF组的电流密度（3.86±1.18）pA/pF较4-AP+AF组（4.12±1.37）pA/pF明显升高,P＜0.01。AF组的APD（310±21ms）较SR组的APD（425±17ms）明显缩短,4-AP+AF组的APD（387±19ms）较AF组（310±21ms）明显延长,P＜0.01;H₂O₂+4-AP+AF组的APD（297±22ms）较4-AP+AF组的（387±19ms）明显缩短,P＜0.01。讨论近年的临床研究结果显示针对心房重构的治疗在预防房颤方面十分有效,这使我们看到深入研究心房重构的机制以及如何遏止和逆转心房重构对临床治疗房颤的重要意义。多项研究发现,房颤时心房肌存在不同程度的能量代谢失衡导致氧应激损伤。Carnes等研究证实犬经快速心房起搏后,氧自由基生成显著增多,应用抗氧化剂维生素C后,电重构发生的时间明显延后。Shiroshita等的一系列研究显示,具有抗氧化作用的辛伐他汀等药物,可以延缓心房快速起搏诱发的电生理重构及房颤的发生。Ikur是近年来发现的一种仅在心房细胞特异表达的离子通道电流,主要是心房细胞复极2期的外向离子流,能促进动作电位3期复极,是有效不应期和动作电位时限的决定因素之一。本组AF组的Ikur研究结果与Vanwagoner等研究中Ikur有相似程度的下降,与Ikur通道蛋白kv1.5表达下降有关;但Bosch等发现,房颤时Ikur无明显改变。结果不同可能与所选病例的心脏病性质、房颤持续存在时间以及用药方案等有关。有研究表明,慢性房颤使正常时心房潜在的电不均一性的弥散度增大。近年的研究显示Ikur仅存于心房肌,而于心室肌仅有微量表达,无明确功能,对心室的动作电位没有不利影响。数学模型法研究发现,房颤时,ICaL较大程度降低（如60%）时,Ikur 50%下调可能是一种代偿性措施,可使APD恢复至正常范围,可能对3期复极产生影响。提示通过干预Ikur通道下调而延长AERP,改变心房兴奋周期来影响心房传导、复极,将使以Ikur为潜在靶点的抗心律失常药物研究成为可能。所以一些选择性抑制Ikur的研究显示,特异性Ikur抑制剂对AF具有良好的治疗效果,同时不会导致室性心律失常,从而达到安全有效的治疗目标,但是此种治疗并不能完全控制和转复AF。本组结果发现,AF组Ikur密度较SR组明显降低,4-AP+AF组较AF组明显降低,H₂O₂+4-AP+AF组较4-AP+AF组明显升高,P＜0.01。AF组的APD较SR组的明显缩短,4-AP+AF组的APD较AF组明显延长,H₂O₂+4-AP+AF组的APD较4-AP+AF组的明显缩短,P＜0.01。提示H₂O₂具有改变kv1.5通道功能的作用,在H₂O₂超量存在的时候,kv1.5通道开放加快,导致电流密度增加,APD缩短,为房颤的发生和维持提供条件,一定程度上解释了单纯应用特异性Ikur抑制剂不能完全控制AF的部分原因。随着对Ikur认识的深入以及氧自由基对离子通道的影响的进一步明确,应用特异性Ikur抑制剂和调整心肌能量代谢状态治疗AF,具大广阔的临床应用前景。结论（1）心房颤动时kv1.5通道基因和蛋白水平的表达均减低。（2）AF中心房肌细胞存在氧自由基生成和消除之间的失衡,提示氧应激状态在AF的发生发展过程中起到重要作用。（3）氧自由基能够影响AF心房肌细胞的kv1.5通道的开放特性,进一步提示氧应激和能量代谢障碍在AF的发生发展过程中起到重要作用。更多还原

【Abstract】 ObjectiveAtrial fibrillation is the kind of most often clinically observed arrhythmia,which is the independent risk factor of the mortality of population.But there is not any effective methods that could solve atrial fibrillation,and the biggest barrier is the unclear mechanism of atrial fibrillation.Since the concept of atrial remodeling proposed by Wijffels in 1995,a large number of basic and clinical researches were developed.And now there is the consistent opion that atrial remodeling is the molecular basis of the genesis and development of atrial fibrillation,essentially including the shortage of myocardial action potential duration（APD） and absolute refractory period（ARP）.So many factors affecting the atrial remodeling determined the complexity of atrial remodeling.Recently,some studies found kv1.5 was only expressed on the atrium and never or little on the ventricle without physiological function,kv1.5 is one of the main elements of atrial repolarization,which is the molecular basis of atrial ultra rapid delayed rectifier potassium currents（Ikur） playing the important role in repolarization and affecting APD.Some researches showed there were disorders of energy metabolism by different extent resulting in oxidative stress.Oxygen free radicals could directly affect the character of kv1.5 channel proved by cell culture study in vitro,which showed oxygen free radicals increased the currents amplitude during repolar phase and enhanced the opening of the channel with the shortage of APD.How does the oxygen free radicals affect human atrial kv1.5 channel is not reported and unclear.Though it could not be concluded according to forthcoming researches,the oxidative stress of the atrium would be the new target of study and treatment for atrial fibrillation.This study pay attention to human artial myocytes,detect the expression of kv1.5,oxidatases and reductases and influence of oxygen free radicals on kv1.5 channel with the purpose of studying the contribution of this mechanism to the atrial remodeling.Materimals and Methods1.Sample collectionThe right atrial appendages were from the patients who received the valvular replacement with rheumatic heart disease and divided into two groups,one is sinus rhythm group and the other is atrial fibrillation group according to the heart rhythm.2.Methods（1） The mRNA expression of the gene was analyzed by RT-PCR.The total RNA was abstracted from the tissue by the means of Trizol method,and then was reverse transcripted into cDNA by the two-step RT-PCR kit.The amplification of the genes of kv1.5,flavin containing monooxygenase-1,monoamine oxidase B,glutathione peroxidase-1 and heme oxygenase-2 were attained through the kits and the inside comparative gene was GAPDH.The amplificated products were dissociated by agarose gel electrophoresis and calculated the expression.（2） The protein expression of kv1.5 was analyzed by Western Blot.The total protein was abstracted from the tissue by the means of RIPA method and then determined the concentration of the protein.Electrophoresis,transferring, hybridization of antigen and antibody and ECL kit were under use to calculate the protein expression of kv1.5.（3） The effects of oxidative radicals on channel kv1.5 were detected by the patch clamp technique.Myocardium sample was cut into small block of 1mm³ with fine scissors.Single myocytewas isolated with enzymatic dissociation techniquesand the cells with best quality were collected and stored in KB solution.A few drops of isolated myocytes were placed in bath of 1cm³ on microscope.5 min later,column-shaped myocytes with strong membranous reflection and clear were prepared for patch clamp.4-AP and H₂O₂ were added in the bathing solution, respectively.Natium currentwas deactivated with -40mv holding potential.When whole cell recording finished,holding potential was set to -40mv,commanding potential was depolarized from -60mv to +60mv,with step pulse at 10mv,holding duration of 200ms and stimulating interval of 3s.Recorded current was analyed with program of Pclamp 5.5.1,Clampan,Clampex and Clampfit.（4） Statistical analysisStatistical analysis was performed with SPSS13.0.Data were expressed as means±SD（（?）+s）.Inter groups comparison was performed with student t test.When P is less than 0.05,the difference was considered statically significant.Results1.The mRNA expression of the gene was analyzed by RT-PCRThe expression of kv1.5 was significantly lower in AF group as compared with SR group（P＜0.05）.The expression of FMO-1 and MAO-B were significantly higher and the expression of HMOX-2 and GPX-1 significantly lower in AF group as compared with SR group（P＜0.05）.2.The protein expression of kv1.5 was analyzed by Western Blot.The protein expression of kv1.5 was significantly lower in AF group as compared with SR group（P＜0.05）.3.The effects of oxidative radicals on channel kv1.5 were detected by the patch clamp technique.The pA/pF（4.12±1.37） of Ikur about +50mV in AF group was significantly less than that（8.91±1.86） in SR group,P＜0.01;The pA/pF（2.75±0.84） of Ikur in 4-AP+AF group was significantly less than that（4.12±1.37） in AF group,P＜0.01;The pA/pF （3.86±1.18） of Ikur in H₂O₂+4-AP+AF group was significantly larger than that（2.75±0.84） in 4-AP+AF group,P＜0.01.The APD（310±21ms） in AF group was significantly shorter than that（425±17ms） in SR group,P＜0.01;The APD（387±19ms） in 4-AP+AF group was significantly longer than that（310±21ms） in AF group,P＜0.01;The APD（297±22ms） in H₂O₂+4-AP+AF group was significantly shorter than that（387±19ms） in 4-AP+AF group,P＜0.01.DiscussionRecent clinical researches found that the treatment for atrial remodeling was very effective in preventing AF,which makes us to think that studying the mechanism of atrial remodeling in depth and finding how to reverse the remodeling should be most important for clinical treatment on AF.Many studies have showed that there are the dysfunction of energy metabolism in atrial myocytes during AF,which resulting in oxidative stress.Carnes found that oxidative redicals were increased during atrial pacing and Vitamine C could reduced them and delayed the happening of electrical remodeling.The expressions of the oxidases were increased and the expressions of reductases were decresed,so the physiological balance between the production and clearance of oxido-reductase was disturbed.It showed that oxidative stress might play a very vital role in the happening and the pathologic progression of AF and offers novel insight into potential treatment with antioxidants.Just similar to the results of Van wagoner’s research,that the pA/pF of Ikur in AF group was significantly less than thatin SR group was related to that the expression of kv1.5 was significantly lower in AF group.Recently,some studies found kv1.5 was only expressed on the atrium and never or little on the ventricle without physiological function,kv1.5 is one of the main elements of atrial repolarization,which is the molecular basis of atrial Ikur playing the important role in repolarization and affecting APD.The mathematics model found the down regulation of Ikur may recover the APD to the normal,which might be a compensative way.Our study found that the pA/pF of Ikur in AF group was significantly less than thatin SR group;in 4-AP+AF group significantly less than in AF group;in H₂O₂+4-AP+AF group significantly larger than in 4-AP+AF group and the APD in AF group was significantly shorter than thatin SR group;in 4-AP+AF group significantly longer than in AF group;in H₂O₂+4-AP+AF group significantly shorter than in 4-AP+AF group.All of the results suggested that oxidative redicals might change the opening characters of kv1.5 and more oxidative redicals could enhance the open of kv1.5,increase the pA/pF of kv1.5 and reduce the APD,which providing conditions for the happening and persistence of AF.Our research found oxygen free radicals change the character of kv1.5,increase pA/pF and reduce APD,which explaining why simple use of specific blockade of Ikurcan not control the AF totally in some extent.Conclusions（1）The mRNA and protein expresstion of kv1.5 decreased during atrial fibrillation;（2）There was imbalance between the production and clearance of oxygen free radicals in fibrillating atrial myocytes,which implied that oxidative stress played a important role in the happening and perpetuation of atiral fibrillation;（3）Oxygen free radicals could affect the opening character of channel kv1.5,which revealed that the disorders of energy metabolism and oxidative stress is a kind of mechanism of the genesis and development of atrial fibrillation.更多还原