氧应激状态与心房kv1.5通道表达及功能改变在心房颤动发生机制中的作用
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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0.5cm~3,将组织块用预先充氧的无钙液反复冲洗后,洗去血液和Ca~(2+)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Ca~(2+)台式液(100%O_2,36±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(H_2O_2)+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,H_2O_2+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;H_2O_2+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;H_2O_2+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,H_2O_2+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,H_2O_2+4-AP+AF组的APD较4-AP+AF组的明显缩短,P＜0.01。提示H_2O_2具有改变kv1.5通道功能的作用,在H_2O_2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。
Objective
Atrial 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 Methods
1.Sample collection
The 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 1mm~3 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 1cm~3 on microscope.5 min later,column-shaped myocytes with strong membranous reflection and clear were prepared for patch clamp.4-AP and H_2O_2 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 analysis
Statistical 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.
Results
1.The mRNA expression of the gene was analyzed by RT-PCR
The 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 H_2O_2+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 H_2O_2+4-AP+AF group was significantly shorter than that(387±19ms) in 4-AP+AF group,P＜0.01.
Discussion
Recent 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 H_2O_2+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 H_2O_2+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.
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0.5cm~3,将组织块用预先充氧的无钙液反复冲洗后,洗去血液和Ca~(2+)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Ca~(2+)台式液(100%O_2,36±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(H_2O_2)+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,H_2O_2+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;H_2O_2+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;H_2O_2+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,H_2O_2+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,H_2O_2+4-AP+AF组的APD较4-AP+AF组的明显缩短,P＜0.01。提示H_2O_2具有改变kv1.5通道功能的作用,在H_2O_2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。
Objective
Atrial 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 Methods
1.Sample collection
The 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 1mm~3 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 1cm~3 on microscope.5 min later,column-shaped myocytes with strong membranous reflection and clear were prepared for patch clamp.4-AP and H_2O_2 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 analysis
Statistical 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.
Results
1.The mRNA expression of the gene was analyzed by RT-PCR
The 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 H_2O_2+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 H_2O_2+4-AP+AF group was significantly shorter than that(387±19ms) in 4-AP+AF group,P＜0.01.
Discussion
Recent 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 H_2O_2+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 H_2O_2+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.
引文
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31 Barhanin J,Lesage F,Guillemare E,et al.KvLQT1 and IsK(minK) proteins associate to form the IKs cardiac potassium current.Nature.1996;384(1):78-80.
32 Yamada M,Inanobe A,Kurachi Y.G protein regulation of potassium ion channels.Pharmacol Rev.1998;50(4):723-757.
33 Wang Z,Yue L,White M,et al.Differential distribution of inward rectifier potassium channel transcripts in human atrium versus ventricle.Circulation.1998;98(22):2422-2428.
34 王晓虎,邓玉莲,张建成等.风湿性心脏病患者重构的分子基础.福建医药杂志.2003;25(2):4-6.
35 Krapivinsky G,Gordon E,Wickman K,et al.The G-protein gated atrial k+ channel Ik.Ach is a heteromultimer of two inwardly rectifying k+ channel proteins.Nature.1995;374(1):135-141.
36 张建成,黄从新,邓玉莲等.心房颤动患者L-钙通道电流改变的分子基础.中华心血管病杂志.2002,30(8):460-463。
37 许春萱,张建成,黄从新等.心房颤动患者瞬间外向钾电流重构的分子基础.中华心律失常学杂志.2002,6(4):228-232.
38 Ausma J,Wijffels M,Thone F,et al.Structural changes of atrial myocardium due to sustained atrial fibrillation in the goat.Circulation.1997;96(9):3157-3163.
39 Tieleman RG,Van Gelder IC,Crijns HJ,et al.Early recurrences of atrial fibrillation after electrical cardioversion:a result of fibrillation-induced electrical remodeling of the atria? J Am Coll Cardiol.1998;31(1):167-173.
40 Hobbs WJ,Van Gelder IC,Fitzpatrick AP,et al.The role of atrial electrical remodeling in the progression of focal atrial ectopy to persistent atrial fibrillation.J Cardiovasc Electrophysiol.1999;10(6):866-870.
41 Fareh S,Benardeau A,Nattel S.Comparative study of the efficacy of T-and L-type calcium channel blockegainst atrial remodeling due to sustained atrial tachycardia.Circulation.1999;100(suppl Ⅰ):Ⅰ-11.Abstract.
42 Fareh S,Benardeau A,Thibault B,et al.The T-type Ca2+ channel blocker mibefradil prevents the development of a substrate for atrial fibrillation by taehycardia-induced atrial remodeling in dogs.Circulation.1999;100(21):2191-2197.
43 Li D,Benardeau A,Nattel S.Contrasting efficacy of dofetilide in differing experimental models of atrial fibrillation.Circulation.2000;102(1):104-112.
44 Satoh T,Zipes DP.Unequal atrial stretch in dogs increases dispersion of refractoriness conductive to developing atrial fibrillation.J Cardiovasc Electrophysiol,1996;60(7):833-842.
45 Sparks PB,Mord HG,Vohra JK,et al.Electrical remodeling of the atria following loss of atrioventricular synchrony:A long-term study in humans.Circulation.1999;100(1):1894-1900.
46 Li D,Fareh S,Leung TK,et al. Promotion of atrial fibrillation by heart failure in dogs: atrial remodeling of a different sort.Circulation.1999; 100(1):87-95.
47 Pederson OD,Bagger H,Kober L, et al. Trandolapril reduces the incidence of atrial fibrillation after acute myocardial infarction in patients with left ventriculardysfunction. Circulation. 1999; 100(4):376-380.
48 Li D,Shinagawa K,Pang L, et al. Effects of angiotensin-converting enzyme inhibition on the development of the atrial fibrillation substrate in dogs with ventricular tachypacing-induced congestive heart failure.Circulation.2001; 104(21):2608-2614.
1 Kopecky SL,Gersh BJ.Dilated cardiomyopathy and myocarditis:natural history,etiology,clinical manifestations,and management.Curr Probl Cardiol.1987;12(10):569-647.
2 Panagiotis,C-reactive protein and oxidative stress in fibrillation.International Journal of Cardiology.2003;88(1):103-104.
3 Kopecky SL,Gersh BJ,et al.The natural history of lone atrial fibrillation.A population based study over three decades.N Engl J Med.1987;317(11):669-674.
4 Benjamin EJ,et al.Impact of atrial fibrillation on the risk of death.The Framingham heart study.Circulation.1998;98(10):946-952.
5 宋振玉,刘耕陶编著.当代药理学.北京:北京医科大学、中国协和医科大学联合出版社1994:418-426.
6 Beckman JS,Koppenol WH.Nitric oxide,superoxide,and peroxynitrite:the good,the bad,and ugly.Am J Physiol.1996;271(5):C1424-37.
7 McConrd JM.Oxygen-derived free radicals in postischemic tissue injury.N Engl J Med.1985;312(3):159-163.
8 Griendling KK,Alexander RW,Oxidative stress and cardiovascular disease.Circulation.1997;96(4):3264-3265.
9 Carvajal K,El Hafidi M,et al.Myocardial damage due to ischemia and reperfusion in hypertriglyceridemic and hypertensive rats:participation of free radicals and calcium overload.J Hypertens.1999;17(11):1607-1616.
10 Fareh S,Benardeau A,Thibault B,et al.The T-type Ca2+ channel blocker mibefradil prevents the development of a substrate for atrial fibrillation by tachycardia-induced atrial remodeling in dogs.Circulation.1999;100(21):2191-2197.
11 Van Wagoner DR,Pond AL,et al.L-type Ca~(2+) currents and human atrial fibrillation.Circ Res.1999;85(5):428-436.
12 Wijffels MC,Kirchhof CJ,et al.Atrial fibrillation begets atrial fibrillation:a study in awake chronically instrumented goats.Circulation.1995;92(7):1954-1968.
13 Van Wagoner DR,Nerbonne JM.Molecular basis of electrical remodeling in atrial fibrillation.J Mol Cell Cardiol. 2000; 32(6): 1101-1117.
14 Thijssen VL, Ausma J, et al. Structural changes of atrial myocardium during chronic atrial fibrillation. Cardiovasc Pathol. 2000; 9(1): 17-28.
15 Wijffels MC, Kirchhof CJ, et al. Electrical remodeling due to atrial fibrillation in chronically instrumented conscious goats: roles of neurohumoral changes, ischemia, atrial stretch, and high rate of electrical activation. Circulation. 1997; 96(10): 3710-3720.
16 Ide T, Tsutsui H. Direct evidence for increased hydroxyl radicals originating from superoxide in the failing myocardium. Circ Res.2000; 86(2): 152-157.
17 Mihm MJ, Yu F. Impaired myofibrillar energetics and oxidative injury during human atrial fibrillation. Circulation.2001; 104(2): 174-180.
18 Carnes CA, Chung MK. Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation. Circ Res.2001;89(6):e32-38.
19 Kirsch M, de Groot H. Ascorbate is a potent antioxidant against peroxynitrite-induced oxidation reactions: evidence that ascorbate acts by re-reducing substrate radicals produced by peroxynitrite. J Biol Chem.2000; 275(22): 16702-16708.
2 Van Wagoner DR,Pond AL,McCarthy PM,et al.Outward K~+ current densities and Kv1.5expression are reduced in chronic human atrial fibrillation.Circ Res.1997;80(6):772-781.
3 Bril A.Recent advances in arrhythmia therapy:treatment and prevention of atrial fibrillation.Curr Opin Pharmacol.2002;2(2):154-159.
4 Brundel BJ,Van Gelder IC,Henning RH,et al.Ion channel remodeling is related to intraoperative atrial effective refractory periods in patients with paroxysmal and persistent atrial fibrillation.Circulation.2001;6:103(5):684-690.
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11 Carnes CA, Chung, MK, Nakayama T, et al. Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation. Circ Res.2001; 89(6):E32-8.
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5 Van Wagoner DR.Pharmacologic relevance of K~+ Channel remodeling in atrial fibrillation.J Mol Cell Cardiol.2000;32(10):1763-1766.
6 Van Wagoner DR,Nerbonne JM.Molecular basis of electrical remodeling in atrial fibrillation.J Mol Cell Cardiol.2000;32(6):1101-1117.
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8 李贵荣,陈一岳.心脏不均一性电生理与电药理学和毒理学的关系.中国药理学与毒理学杂志.2001,15(2):95-104.
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11.Courtemanche M,Ramirez RJ,Nattel S.Ionic targets for drug therapy and atrial fibrillation-induced electrical remodeling:insights from a mathematical model.Cardiovasc Res.1999;42(2):477-489.
1 Kannel WB,Abbot RD,Savage DD,et al.Epidemiologic features of chronicatrial fibrillation:the Framingham study.N Engl J Med.1982;306(I7):1018-1022.
2 Psaty BM,Manolio TA,Kuller LH et al.Incidence of and risk factors for atrial fibrillation in older adults.Circulation.1997;96(7):2455-2461.
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4 Waif PA,Abbott RD,KannelWB.Atrial fibrillation:A major contributor to stroke in the elderly.Arch Int Med.1987;147(9):1561-1564.
5 Waif PA,Abbott RD,KannelWB.Atrial fibrillation as an independent risk factor for stroke:The Framingham Study.Stroke,1991;22(8):983-988.
6 Cabin HS,Clubb KS,Hall C,et al.Risk for systemic embolization of atrial fibrillation without mitral stenosis.Am J Cardiol.1990;65(16):1112-1116.
7 Benjamin EJ,Wolf PA,D'Agostino RB,et al.Impact of atrial fibrillation on the risk of death:the Framingham Heart Study.Circulation.1998;98(10):946-952.
8 Wijffels MC,Kirchhof CJ,Dorland R,et al.Atrial fibrillation begets atrial fibrillation:a study in awake chronically instrumented goats.Circulation,1995,92(7):1954-1968.
9 Morillo CA,Klein GJ,Jones DL,et al.Chronic rapid atrial pacing:structural functional,and electrophysiological characteristics of a new model of sustainedatrial fibrillation.Circulation.1995;91(5):1588-1595.
10 Wijffels MC,Kirchhof CJ,Dorland R,et al.Electrical remodeling due to atrial fibrillation in chronically instrumented conscious goats: roles of neurohumoral changes, ischemia, atrial stretch, and high rate of electrical activation.Circulation.1997; 96(10):3710-3720.
11 Gaspo R, Bosch RF, Talajic M, et al. Functional mechanisms underlying tachycardia-induced sustained atrial fibrillation in a chronic dog model. Circulation. 1997; 96(11):4027-4035.
12 Yue L, Feng J, Gaspo R, Li GR, et al. Ionic remodeling underlying action otential changes in a canine model of atrial fibrillation. Circ Res.1997; 81(4):512-525.
13 Courtemanche M, Ramirez RJ, Nattel S. Ionic targets for drug therapy and atrial fibrillation-induced electrical remodeling: insights from a mathematical model. Cardiovasc Res. 1999; 42(2):477-489.
14 Ramirez RJ, Nattel S, Courtemanche M. Mathematical analysis of canine atrial action potentials: rate, regional factors and electrical remodeling. Am J Physiol.2000; 279(4):H1767-H1785.
15 Gaspo R, Bosch RF, Bou-Abboud E, et al. Tachycardia-induced changes in Na+ current in a chronic dog model of atrial fibrillation. Circ Res.1997; 81(6):1045-1052.
16 Van Wagoner DR, Pond AL, McCarthy PM, et al. Outward K~+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ Res.1997; 80(6):772-781.
17 Bosch RF, Zeng X, Grammer JB, et al. Ionic mechanisms of electrical remodeling in human atrial fibrillation. Cardiovasc Res.1999; 44(1):121-131.
18 Van Wagoner DR, Pond AL, Lamorgese M,et al. Atrial L-type Ca2+ currents and human atrial fibrillation. Circ Res.1999; 85(5):428-436.
19 Grammer JB, Bosch RF, Kuhlkamp V, et al. Molecular remodeling of Kv4.3 potassium channels In human atrial fibrillation. J Cardiovasc Electrophysiol.2000; 11(6):626-633.
20 Balana B, Dobrev D, Wettwer E,et al. Decreased ATP-sensitive K(+) current density during chronic human atrial fibrillation. J Mol Cell Cardiol.2003; 35(12):1399-405.
21 Yue L, Melnyk P, Gaspo R, et al. Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation. Circ Res.1999; 84(7):776-784.
22 Van Gelder IC, Brundel BJ, Henning RH,et al. Alterations in gene expression of proteins involved in the calcium handling in patients with atrial fibrillation. J Cardiovasc Electrophysiol. 1999; 10(4):552-560.
23 Brundel BJ,van Gelder IC,Henning RH,et al.Gene expression of proteins influencing the calcium homeostasis in patients with persistent and paroxysmal atrial fibrillation.Cardiovasc Res.1999;42(2):443-454.
24 Lai LP,Su MJ,Lin JL,et al.Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca2+-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor,calsequestrin and phospholamban:an insight into the mechanism of atrial electrical remodeling.J Am Coll Cardiol.1999;33(5):1231-1237.
25 Grammer JB,Bosch RF,Kuhlkamp V,et al.Molecular and electrophysiological evidence for "remodeling" of the L-type Ca2+ channel in persistent atrial fibrillation in humans.Z Kardiol.2000;89(suppl 4):Ⅳ23-Ⅳ29.
26 Brundel BJ,Isabekke Cm Vab Gelder,Robert,et al.Ion channel remodeling is related to intraoperative atrial effective refractory periods in patients with paroxysmal and persistent atrial fibrillation.Circulation.2001;103(5):684-690.
27 Bianca J.J.M Brundel,Isabelle C,et al.Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation differential regulation of protein and mRNA levels for potassium channels.J Am Coll Cardiol.2001;37(3):926-932
28 张建成,黄从新,邓玉莲等.心房颤动患者离子重构的分子基础.中华心律失常学杂志.2001;6(4):84-88.
29 Wang Z,Fermini B,Nattel,et al.Delayed rectifier outward current and repolarization in human atrial myocytes.Circulation Research.1993;73(2):276-285.
30 Sanguinetti M.C,Jiang C,Curran M.E,et al.A mechanistic link between an inherited and an acquired cardiac arrhythmia:HERG encodes the IKr potassium channel.Cell.1995;81(2):299-307.
31 Barhanin J,Lesage F,Guillemare E,et al.KvLQT1 and IsK(minK) proteins associate to form the IKs cardiac potassium current.Nature.1996;384(1):78-80.
32 Yamada M,Inanobe A,Kurachi Y.G protein regulation of potassium ion channels.Pharmacol Rev.1998;50(4):723-757.
33 Wang Z,Yue L,White M,et al.Differential distribution of inward rectifier potassium channel transcripts in human atrium versus ventricle.Circulation.1998;98(22):2422-2428.
34 王晓虎,邓玉莲,张建成等.风湿性心脏病患者重构的分子基础.福建医药杂志.2003;25(2):4-6.
35 Krapivinsky G,Gordon E,Wickman K,et al.The G-protein gated atrial k+ channel Ik.Ach is a heteromultimer of two inwardly rectifying k+ channel proteins.Nature.1995;374(1):135-141.
36 张建成,黄从新,邓玉莲等.心房颤动患者L-钙通道电流改变的分子基础.中华心血管病杂志.2002,30(8):460-463。
37 许春萱,张建成,黄从新等.心房颤动患者瞬间外向钾电流重构的分子基础.中华心律失常学杂志.2002,6(4):228-232.
38 Ausma J,Wijffels M,Thone F,et al.Structural changes of atrial myocardium due to sustained atrial fibrillation in the goat.Circulation.1997;96(9):3157-3163.
39 Tieleman RG,Van Gelder IC,Crijns HJ,et al.Early recurrences of atrial fibrillation after electrical cardioversion:a result of fibrillation-induced electrical remodeling of the atria? J Am Coll Cardiol.1998;31(1):167-173.
40 Hobbs WJ,Van Gelder IC,Fitzpatrick AP,et al.The role of atrial electrical remodeling in the progression of focal atrial ectopy to persistent atrial fibrillation.J Cardiovasc Electrophysiol.1999;10(6):866-870.
41 Fareh S,Benardeau A,Nattel S.Comparative study of the efficacy of T-and L-type calcium channel blockegainst atrial remodeling due to sustained atrial tachycardia.Circulation.1999;100(suppl Ⅰ):Ⅰ-11.Abstract.
42 Fareh S,Benardeau A,Thibault B,et al.The T-type Ca2+ channel blocker mibefradil prevents the development of a substrate for atrial fibrillation by taehycardia-induced atrial remodeling in dogs.Circulation.1999;100(21):2191-2197.
43 Li D,Benardeau A,Nattel S.Contrasting efficacy of dofetilide in differing experimental models of atrial fibrillation.Circulation.2000;102(1):104-112.
44 Satoh T,Zipes DP.Unequal atrial stretch in dogs increases dispersion of refractoriness conductive to developing atrial fibrillation.J Cardiovasc Electrophysiol,1996;60(7):833-842.
45 Sparks PB,Mord HG,Vohra JK,et al.Electrical remodeling of the atria following loss of atrioventricular synchrony:A long-term study in humans.Circulation.1999;100(1):1894-1900.
46 Li D,Fareh S,Leung TK,et al. Promotion of atrial fibrillation by heart failure in dogs: atrial remodeling of a different sort.Circulation.1999; 100(1):87-95.
47 Pederson OD,Bagger H,Kober L, et al. Trandolapril reduces the incidence of atrial fibrillation after acute myocardial infarction in patients with left ventriculardysfunction. Circulation. 1999; 100(4):376-380.
48 Li D,Shinagawa K,Pang L, et al. Effects of angiotensin-converting enzyme inhibition on the development of the atrial fibrillation substrate in dogs with ventricular tachypacing-induced congestive heart failure.Circulation.2001; 104(21):2608-2614.
1 Kopecky SL,Gersh BJ.Dilated cardiomyopathy and myocarditis:natural history,etiology,clinical manifestations,and management.Curr Probl Cardiol.1987;12(10):569-647.
2 Panagiotis,C-reactive protein and oxidative stress in fibrillation.International Journal of Cardiology.2003;88(1):103-104.
3 Kopecky SL,Gersh BJ,et al.The natural history of lone atrial fibrillation.A population based study over three decades.N Engl J Med.1987;317(11):669-674.
4 Benjamin EJ,et al.Impact of atrial fibrillation on the risk of death.The Framingham heart study.Circulation.1998;98(10):946-952.
5 宋振玉,刘耕陶编著.当代药理学.北京:北京医科大学、中国协和医科大学联合出版社1994:418-426.
6 Beckman JS,Koppenol WH.Nitric oxide,superoxide,and peroxynitrite:the good,the bad,and ugly.Am J Physiol.1996;271(5):C1424-37.
7 McConrd JM.Oxygen-derived free radicals in postischemic tissue injury.N Engl J Med.1985;312(3):159-163.
8 Griendling KK,Alexander RW,Oxidative stress and cardiovascular disease.Circulation.1997;96(4):3264-3265.
9 Carvajal K,El Hafidi M,et al.Myocardial damage due to ischemia and reperfusion in hypertriglyceridemic and hypertensive rats:participation of free radicals and calcium overload.J Hypertens.1999;17(11):1607-1616.
10 Fareh S,Benardeau A,Thibault B,et al.The T-type Ca2+ channel blocker mibefradil prevents the development of a substrate for atrial fibrillation by tachycardia-induced atrial remodeling in dogs.Circulation.1999;100(21):2191-2197.
11 Van Wagoner DR,Pond AL,et al.L-type Ca~(2+) currents and human atrial fibrillation.Circ Res.1999;85(5):428-436.
12 Wijffels MC,Kirchhof CJ,et al.Atrial fibrillation begets atrial fibrillation:a study in awake chronically instrumented goats.Circulation.1995;92(7):1954-1968.
13 Van Wagoner DR,Nerbonne JM.Molecular basis of electrical remodeling in atrial fibrillation.J Mol Cell Cardiol. 2000; 32(6): 1101-1117.
14 Thijssen VL, Ausma J, et al. Structural changes of atrial myocardium during chronic atrial fibrillation. Cardiovasc Pathol. 2000; 9(1): 17-28.
15 Wijffels MC, Kirchhof CJ, et al. Electrical remodeling due to atrial fibrillation in chronically instrumented conscious goats: roles of neurohumoral changes, ischemia, atrial stretch, and high rate of electrical activation. Circulation. 1997; 96(10): 3710-3720.
16 Ide T, Tsutsui H. Direct evidence for increased hydroxyl radicals originating from superoxide in the failing myocardium. Circ Res.2000; 86(2): 152-157.
17 Mihm MJ, Yu F. Impaired myofibrillar energetics and oxidative injury during human atrial fibrillation. Circulation.2001; 104(2): 174-180.
18 Carnes CA, Chung MK. Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation. Circ Res.2001;89(6):e32-38.
19 Kirsch M, de Groot H. Ascorbate is a potent antioxidant against peroxynitrite-induced oxidation reactions: evidence that ascorbate acts by re-reducing substrate radicals produced by peroxynitrite. J Biol Chem.2000; 275(22): 16702-16708.
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