【作者】 徐先增；

【导师】 刘唐威；

【作者基本信息】 广西医科大学 ， 心血管内科学， 2011， 博士

【摘要】 1背景和目的心房颤动（atrial fibrillation,AF）是临床上最常见的心律失常,见于各种器质性心脏病,我国通常并发于风湿性心脏瓣膜病（rheumatic heart disease,RHD）,特别是二尖瓣病变。部分患者则无明确病因。AF发生后可导致心功能进一步受损,卒中发病率上升,病死率成倍增加。AF的预防和治疗均不理想,原因是AF的发生和维持机制尚不完全明了。AF发生后心房组织可发生一系列的结构和功能改变,致使AF易于复发和维持,发生心房重构,其中电生理方面表现为动作电位和有效不应期时程的缩短,频率适应性降低,即所谓的电重构。电重构使得房内多子波折返更易于维持,而不易终止。电重构牵涉到一系列的离子通道的改变,各种离子通道在电重构中所起的作用也成为近年来研究的热点,其中那些心房表达多于心室的离子通道有可能成为预防和治疗AF的潜在理想靶点。钙激活钾通道分布非常广泛,被细胞内微量钙离子（<1.0μM）激活,按电导大小可以分为大电导（big conductance,BK）、中电导（intermediate conductance,IK）和小电导（small conductance ,SK）三种类型,SK2则属于SK的一种亚型,α亚基是通道的功能亚基,为KCNN2基因所编码;钙调蛋白（calmodulin,CaM）是通道的辅助亚基。SK2可被极低浓度（100 pM¹0 nM）的蜂毒明肽（apamin）特异性阻断。研究证明心房的SK2表达明显多于心室,短时间快速刺激肺静脉即可见SK2表达增加,蛋白膜向转移增强,全细胞水平电流密度加大,并且这些变化与动作电位的缩短关系明显,因此被认为可能是治疗AF的另一个理想靶点。近期的一个动物实验证明,应用SK2阻断剂能够预防和终止AF的发作。但以上研究均为模拟短期AF的动物实验,在人类患持续性AF后SK2的结构和功能的改变尚不为人知晓。本研究主要涉及心房与心室之间、左右心房之间SK2基因与蛋白表达量的差别。并探讨在人类患持续性AF后SK2各亚基的基因蛋白表达水平和SK2功能水平的改变,以及这一改变在电重构所起的作用,为寻找防治持续性AF的可能理想靶点提供理论依据。2内容和方法2.1 SK2在心脏各部位的分布特性2.1.1选择RHD行二尖瓣置换术的患者,在体外循环前取左右心耳组织,并分成两份用于免疫组织化学（immunohistochemistry,IHC）和逆转录聚合酶链反应（reverse transcription-polymerase chain reaction,RT-PCR）,检测SK2基因和蛋白的表达水平。2.1.2选择先天性心脏病（congenital heart disease,CHD）存在右心室流出道肥厚并行畸形矫正术的患者,在体外循环前取右房耳组织,术中取右室流出道组织,每份标本分成两份用于IHC和RT-PCR,检测SK2基因和蛋白的表达水平。2.1.3应用Langendoff灌流装置急性分离获得SD大鼠的心房和心室肌细胞,应用全细胞模式记录Ca²⁺激活K+电流（Ik,ca）,也被称为apamin敏感性K+电流。2.2人患持续性AF后SK2的改变2.2.1选择RHD行二尖瓣置换术的患者,分为是否存在持续性AF（持续时间大于7天）分为AF和窦性心律（normal sinus rhythm,NSR）两组。在体外循环前取右心耳组织,进行常规HE染色和Masson染色,观察细胞、细胞外基质等一系列心房结构重构的变化,并通过RT-PCR和IHC,检测SK2α亚单位和辅助亚单位基因蛋白的表达水平。2.2.2选择行二尖瓣置换术的RHD的患者,分为是否存在持续性AF分为AF和NSR两组,在体外循环前同时取右心耳组织,两步酶解法急性分离获得心房肌细胞,应用全细胞模式,记录apamin敏感性K+电流的电流密度。2.2.3应用Langendoff心脏灌流系统可以获得约50%的结构功能良好且耐钙的心肌细胞。结构功能良好的心肌细胞胞膜完整,折光性强,有明显的横纹,可用于膜片钳实验。3结果3.1 SK2的心脏各部位分布3.1.1左右心房对照合并持续性AF的RHD患者8例, SK2通道在左右心房组织的基因与蛋白表达均未见差别。3.1.2心房心室对照6例先天性心脏病患者,法洛氏四联症（tetralogy of Fallot,TOF）3例,室间隔缺损（ventricular septal defect ,VSD）2例,肺动脉瓣狭窄（pulmonary stenosis,PS）1例,全部为NSR。KCNN2基因相对表达量在人心房和心室间无差别,但SK2蛋白表达量心房比心室明显增加。3.1.3 SD大鼠心房心室肌细胞Ik,ca电流的比较应用数字相减方法可记录到大鼠心房和心室肌细胞的Ik,ca。Ik,ca呈内向整流特性,对apamin敏感,翻转电位-60mV,接近Nernst方程。心房心室肌细胞均可记录到Ik,ca。脉冲电压为-50～40mV时,心房肌细胞Ik,ca电流密度显著大于心室肌细胞;脉冲电压为-90～-40mV时,心房心室肌细胞Ik,ca电流密度无差别。心房肌细胞和心室肌细胞的膜电容测定值分别为48.25±4.15pF和21.36±3.23pF,差异有统计学意义。3.2人类持续性AF后SK2的改变3.2.1两组患者基线资料与NSR患者相比,持续性AF患者二尖瓣狭窄（mitral stenosis,MS）比例更大,左心房内径以及心胸比率增加更显著,P<0.05。持续性AF患者年龄偏大,肺动脉收缩压偏高,但与NSR患者比较,差别未达到统计学意义。在男女性别比例、左心室舒张径、左室收缩径、心功能NYHA分级等方面,持续性AF患者与NSR患者差别不大。3.2.2HE染色和Masson染色心房肌细胞纵向呈现条杆状,横向呈现类圆形,内有肌丝,细胞核多为一个,位于细胞中央,多个细胞呈密集束状排列,有分支相连。持续性AF患者的心房肌细胞增大,排列紊乱,持续性AF患者的心房组织内胶原组织增生明显增加。3.2.3 AF组SK2α亚单位基因和蛋白表达的变化AF和NSR两组患者KCNN2基因相对表达量分别为0.70±0.30和1.25±0.52,差别有显著统计学意义（P<0.01）。AF组SK2蛋白表达减少,IOD分别为2 525 900±772 807.4和2 206 917±1 170 312,下调幅度13%,但未达到统计学意义（P>0.05）,细胞内分布特点变化不明显。3.2.4 AF组SK2辅助亚单位钙调蛋白（calmodulin,CaM）基因和蛋白表达的变化持续性AF发生后心房组织的CaM mRNA水平无明显变化。CaM蛋白的阳性表达呈棕黄色,主要在心房肌细胞的胞膜上分布,呈相对浓然的环状;胞浆也有少量分布,表达主要在核周;胞核内无分布。分布特点与SK2蛋白呈现重叠性。持续性AF组与NSR组CaM蛋白表达差别未达到统计学意义。3.2.5持续性AF患者心房肌细胞Ik,ca电流的变化AF患者与NSR患者比较心房肌细胞动作电位时程显著缩短。应用数字相减方法可获得NSR和持续性AF患者心房肌细胞的apamin敏感性K⁺电流,即Ik,ca。总体来讲,与NSR患者相比,持续性AF患者心房肌细胞均Ik,ca电流密度上调,在脉冲电压在-10⁺40mV时,差别达到显著统计学意义。持续性AF患者心房肌细胞的膜电容显著增大。4讨论研究离子通道一般从基因、蛋白以及功能等方面入手,研究方法多种多样,而较为经典的实验方法包括RT-PCR,IHC和膜片钳3种。我们在实验中,正是通过RT-PCR,IHC和膜片钳3种经典实验方法对持续性AF后SK2的变化进行研究。为探讨SK2在心脏各部位分布的差异性,发现左右心房间SK2的基因和蛋白表达均不存在差异,右心耳组织SK2表达的变化亦可反映左心房的变化。另外还发现心室组织比较心房组织基因表达不减少,但蛋白表达明显减少,这一结果和以往的动物实验结果不一致,说明在人的心室组织中SK2表达可能存在某种目前尚不明了的翻译后调节机制。以SD大鼠心房心室肌细胞为实验对象,发现心房肌细胞的Ik,ca的电流密度显著大于心室肌细胞,这一结果提示应用SK2通道特异性阻断剂治疗房性心律失常,可以主要影响心房肌细胞的电生理特性,而对心室肌细胞的电生理特性影响很小,因此SK2通道将来可作为防治房性心律失常的的理想靶点。采集RHD二尖瓣病变患者的右心耳组织,并从基因、蛋白、功能多个层面应用经典实验方法第一次探究人类持续性AF发生后SK2通道的变化。本组RHD二尖瓣病变行二尖瓣置换术（mitral valve replacement,MVR）时病情均较重,均为持续性AF,因此未再另列一组,而仅以持续性AF与NSR患者进行比较。与NSR患者相比,持续性AF组更多为MS患者,左心房增大更明显。MS患者的心房重构的特征既是左心房增大,增大的心房可为多个折返的存在提供足够的空间,利于AF的维持。除左心房增大外,HE和Masson染色的病理结果还发现,持续性AF组心房胶原组织增生严重,心肌细胞排列紊乱。目前认为胶原组织增生严重导致心房间质纤维化是AF基质的重要组成部分,间质纤维化可打乱心肌细胞规则的排列顺序,使细胞间电信号传导减慢,有利于折返的维持。检测NSR和AF两组患者右心耳组织的mRNA水平,发现两组患者心房组织SK2α亚单位基因KCNN2均有表达,但与既往动物实验中短期刺激心房模仿阵发性AF的结果不同,本实验发现持续性AF患者心房组织的KCNN2 mRNA水平下调。另外,NSR与持续性AF患者右心房肌细胞SK2蛋白均呈现阳性表达,其蛋白分布主要在细胞膜。两组患者心房肌细胞的SK2分布特点为一致,持续性AF患者SK2通道蛋白表达偏低,这与此前的动物实验结果也不相同。NSR和持续性AF两组患者右心耳组织均有CaM基因表达,但持续性AF发生后右心耳组织的CaM mRNA水平未改变。NSR与持续性AF患者右心房肌细胞CaM蛋白均呈现也都呈阳性表达,两组患者心房肌细胞的CaM分布特点为一致,蛋白表达量也未见有变化。以上结果说明,持续性AF未对CaM的基因和蛋白表达造成影响,其表达水平与持续性AF apamin敏感性K⁺电流密度上调无关。应用apamin前后电流相减可记录到单纯apamin敏感性电流,持续性AF患者心房肌细胞上的apamin敏感性电流密度更大。通道电流的密度可能受很多因素影响,其中一个重要因素是通道基因蛋白表达水平,持续性AF患者KCNN2表达下调,一般情况下可以导致apamin敏感性电流密度下调。但由于AF发生后,心房肌细胞胞内Ca²⁺显著超负荷,胞内Ca²⁺增加可以使SK2功能水平上调,因此可以认为此时胞内Ca²⁺增加对于SK2功能水平的影响超过了SK2基因和蛋白表达下调带来的影响。5结论综上所述,右心房SK2基因蛋白表达的水平可以反映左心房SK2基因蛋白表达的水平。SK2通道心房心室间基因表达无差异,但心房SK2通道蛋白表达以及apamin敏感性K⁺电流均较心室增强。与以往动物实验不同,人类持续性AF后,SK2通道α亚单位基因蛋白表达下调,辅助亚单位CaM的基因和蛋白表达未发生变化,但功能水平水平显著上调。apamin敏感性电流密度在除受基因蛋白表达水平影响外,主要受心房肌细胞内Ca²⁺水平超负荷影响。SK2通道可以作为治疗人持续性AF的理想靶点,SK2通道阻断剂作为新一代抗心律失常药物治疗心房颤动的效果值得期待。更多还原

【Abstract】 Background and objectiveAtrial fibrillation（AF）, the most common clinical arrhythmia, is seen in patients with structural heart disease, most commonly rheumatic heart disease （RHD） in China, but some patients suffer from AF with no obvious cause. AF can lead further damage to heart function, increased incidence of stroke and multiplied mortality.By now, Prevention and treatment of AF are all not ideal, mainly because the mechanisms of incidence and maintenance of AF are not fully clear, but in recent years, mechanisms of electrical remodeling of AF have undoubtedly become a major hot spot. After AF, a series of atrial structural and functional changes called atrial remodeling occur, which show electrophysiologically shorten action potential duration and effective refractory period, reduced adaptation for frequency. The electrophysiological changes after AF are also called electrical remodeling. Electrical remodeling makes multiwavelet reentry easy to maintain and difficult to terminate and is involved with a series of changes of various ion channels. Ion channels more expressed in atrium than in ventricle may be potential ideal target for the prevention and treatment of AF.Calcium-activated potassium channels are widely distributed in many kinds of cells, activated by intracellular calcium（<1.0μM） and divided into three types--large conductance （BK）, intermediate conductance （IK） and small conductance （SK） by electrical conductivity. SK2 is a subtype of SK, whoseαsubunit is the functional subunit of the channel coded by KCNN2 gene. SK2 can specifically blocked be very low concentration（100 pM 10 nM） of apamin, a kind of peptide from bee venom. Studies have shown that SK2 expression was more significantly in atrium than in ventricle. Short-term rapid pacing in pulmonary vein can result in increased SK2 expression, more SK2 protein transferring to membrane and increased current density in the whole-cell mode, which are in relationships with significant shortening of action potential duration, and SK2 is considered a possible ideal target for treatment of AF. Another recent animal experiments have indeed proved that application of SK2 blockers can prevent the onset of AF. However, above conclusions were all from animal studes and changes SK2 in patients suffering from persistent AF are unknown.This study involving differences of SK2 gene and protein expression between left and right atrium and between atrium and ventricle between in patients suffering from persistent AF, changes of gene and protein expression of SK2α-subunit and functional SK2 in people with persistent AF, as well as changes of the SK2 accessory subunit of calmodulin （CaM） in persistent AF patients. The purpose is our study to explore SK2 change in the role of electrical remodeling in people suffering from persistent AF and to find if SK2 is ideal target for prevention and treatment of persistent AF.Methods and results1 SK2-specific distribution of various parts of heart1.1 RHD patients undertaken mitral valve replacement were selected. parts of right atrial appendage and left atrial appendage were gotten at the same time before cardiopulmonary bypass （CPB）, and each specimen was divided into two parts for immunohistochemistry （IHC） and reverse transcription polymerase chain reaction （RT-PCR）, to observe if there were different SK2 gene and protein expression between left and right atrium.1.2 Patients with congenital heart disease（CHD） and hypertrophy right ventricular outflow who undertaken deformity correction were selected. Parts of right atrial appendage and right ventricular outflow were gotten during the operations and each specimen was divided into two parts for IHC and RT-PCR to observe if there were different SK2 gene and protein expression between atrium and ventricle. 1.3 As it’s difficult to isolate human atrial and ventricular myocytes, we isolated atrial and ventricular myocytes from SD rats by Langendoff perfusion apparatus and recoded Ik,ca current （or Apamin-sensitive potassium current） in the whole cell mode in order to observe whether there were differences in SK2 channel functionally between atrium and ventricle.2 SK2 changes after suffering from persistent AF1.1 According to the existence of persistent AF（ >7days）, RHD patients who undertaken mitral valve replacement surgery were divided into two groups, AF group and normal sinus rhythm （NSR） group. Parts of right atrial appendage of these patients were obtained before CPB to discover a series of changes in atrial structural remodeling by routine HE staining and Masson staining and to observe gene and protein expression changes of SK2αsubunit and CaM after suffering from persistent AF by IHC and RT-PCR1.2 Also according to the existence of persistent AF, RHD patients who undertaken mitral valve replacement surgery were divided into two groups, AF group and NSR group. Parts of right atrial appendage of these patients were obtained before CPB to isolate atrial myocytes enzymatically, which to be used to record action potentials and apamin sensitive K⁺ current in whole cell mode in order to record Ik,ca. About 50% of calcium-resistant cardiacites with undamaged structure could be gotten by application of Langendoff perfusion system. Myocardial cells with undamaged structure were shown membrane integrity, strong refraction and clear cross striations, which could be used for patch clamp experiments.Results1 SK2 in various parts of heart1.1 left atrium vs right atrium8 cases of RHD patients with persistent AF were selected, and there was no difference in SK2 gene and protein expression between left atrium and right atrium.1.2 atrium vs ventricle6 cases of CHD patients, including 3 cases of Tetralogy of Fallot（TOF）, 2 Ventricular septal defect （VSD）, 1 Pulmonary stenosis（PS）, all of them were NSR. no difference in relative expression of KCNN2 gene expression was found between human atrium and ventrile, but expression of SK2 protein in atrium increased more significantly than in ventricle.1.3 Comparison of Ik,ca between atrial myocytes and ventricular myocytes of SD ratIk,ca, also called apamin sensitive K⁺ current, of rat atrial and ventricular muscle cells was obtained by digital subtraction. Ik,ca is an apamin ensitive K⁺ current with inward rectifying properties, whose reversal potential was about -60mV, close to the Nernst equation. Ik,ca could be recorded in atrial and ventricular myocytes . When pulse voltage was during -50 40mV, Ik,ca current density was significantly more greater than in atrial muscle myocytes than in ventricular ones. measured values of membrane capacitance were 21.36±3.23pF and 48.25±4.15pF in atrial and ventricular muscle cells respectively, and the difference was statistically significant.2 SK2 changes after suffering from persistent AF2.1 Baseline clinical data of the two groups18 patients with NSR and 23 patients with persistent AF were selected in our study. Comparing with NSR group, patients in AF group sustained a greater proportion of mitral stenosis, had larger left atrial diameters and more increased heart-chest rate significantly （P<0.05）. Patients in AF group were older with a higher pulmonary artery systolic pressure than patients in NSR group, but the difference did not reach statistical significance. In the respect of male-female ratio, left ventricular diastolic diameter, left ventricular systolic diameter and NYHA classification in cardiac function, there were also no difference between NSR and AF group statistically.2.2HE staining and Masson stainingAtrial myocytes showed rod-like shape at longitudinal section and round-like shape at horizontal section, there were filaments shown in cytoplasm and a nucleus In most cases located in the central of cell. Multiple cells were arranged in dense bundles, with branches connected. Atrial myocytes of patients in AF group, whose arrangement were in disorder, were larger in the size and also were surrounded by more collagen tissue than in NSR group .2.3 SK2αsubunit gene and protein expression changes after suffering from persistent AFRelative KCNN2 gene expression in atrium of patients in AF group and NSR group groups of were 0.70±0.30 and 1.25±0.52, the difference was statistically significant （P <0.01）. SK2 protein expression in atrium of patients in AF group also decreased, down by 13% compared with NSR group, and IOD were 2525900±772807.4 in NSR group and 2206917±1170312 in AF group respectively, but the difference in SK2 protein expression between the two group did not reach statistical signific level（P>0.05）. The intracellular distribution of SK2 protein did not change significantly between the two group.2.4 Changes in CaM （SK2 channel auxiliary subunit） of patients with persistent AFAfter persistent AF CaM mRNA levels of atrial tissue did not change. CaM protein expression in IHC was brown, mainly in the atrial muscle cell membrane, looked like relatively strong staining rings; CaM protein also expressed cytoplasm in a small amount, but did not expressed in the nucleus. Distribution of CaM overlaped with the SK2 protein. Difference in CaM protein expression from atrium between the two groups did not reach statistical significance, and CaM intracellular distribution did not change after suffering from persistent AF.2.5 Ik,ca current changes in patients with persistent AF atrial myocytes Compared with the NSR group, action potential duration of atrial myocytes in AF group were was significantly shortened. Apamin sensitive K⁺ current or Ik,ca could be obtained by digital subtraction in atrial myocytes from patients with NSR and persistent AF. In general, compared with the NSR group, Ik,ca current density of atrial myocytes from in AF group upregrated , and when pulse voltage at -10 +40 mV, the difference of Ik,ca current density of atrial myocytes between the two groups were significative statistically. Membrane capacitance of atrial muscular cells from patients with persistent AF patients increased significantly.DiscussionA variety of research methods are used to study ion channels in general from gene, protein and function, but RT-PCR, IHC and patch clamp are the 3 most classical experimental methods. In Our study, RT-PCR, IHC and patch clamp, the three kinds of classical experimental methods, were used to study SK2. To investigate the distribution of SK2 in different parts of heart found around the heart, we found no differences of SK2 gene and protein expression existed between left and right atrium, and SK2 expression level of right atrial appendage could represent the level of overall atrium. Ventricular tissue was also found a compared KCNN2 gene expression level in atrial tissue, but SK2 protein expression in ventricular tissue was significantly reduced, this result was inconsistent to previous results of animal experiments, indicating that SK2 expression in human ventricular tissue may exist an unclear protranslation mechanism. Ik,ca current density of atrial myocytes from SD rat was found was significantly greater than ventricular myocytes, and the results suggest that specific blocker SK2 channel mainly affect electrophysiological properties of atrial muscle cells, so SK2 channels may be used as target of new ideal antiarrhythmic drugs for prevention and treatment of atrial arrhythmias in the future.SK2 changes after persistent AF in atrium from people suffering from RHD patients mainly involving mitral valve were to explore in various levels including gene, protein, function by classical experimental methods for the first time. Because paroxysmal AF is relatively rare when RHD patients accept MVR , so there were only patients with persistent AF compared with those with NSR. Compared with NSR group, Patients in AF group were suffering more from MS and displayed more obvious left atrial enlargement. characteristics of atrial remodeling in MS patients were obvious left atrial enlargement, providing more space for reentry and benefiting maintenance of AF. In addition to left atrial enlargement, HE staining and Masson staining of atrium specimens from patients also showed that more collagen tissue proliferated in atrium in AF group and cardiac muscle cells arranged in disorder. Interstitial fibrosis causing by collagen tissue proliferation is an important part of substrate leading to AF. Interstitial fibrosis can disrupt the order of myocardial cells, so that signal transmission between cells slow down, which is conducive to the maintenance of reentry.KCNN2 mRNA levels testing of right atrial appendage from patients with NSR and persistent AF were found KCNN2 expression in all atrium in the two groups, but unlike previous animal studies ,this research shown that KCNN2 mRNA in right atrial appendage from patients with persistent AF was at the lower level. In addition, right atrial myocytes from patients with either NSR or persistent AF showed a positive protein expression of SK2 protein, and SK2 protein expressed mainly on membrane of atrial myocytes. SK2 distribution is consistent in atrial myocytes of two groups of patients. Persistent AF patients had a low protein expression level of SK2 channel, which was not the same results with previous animal experiments.Right atrial appendage tissue of patients with NSR and persistent AF were also used to test CaM gene expression, but after the occurrence of persistent AF, CaM mRNA levels of right atrium did not change. Right atrial myocytes in NSR and AF group showed positive CaM protein expression, CaM protein distributionin the two groups had not difference. These results suggest that persistent AF do not affect on gene and protein expression level of CaM, and upregrated apamin sensitive K⁺ current density has nothing to do with CaM.Apamin sensitive current could be recorded by digital subtraction, and apamin sensitive current density of atrial myocytes in NSR group was greater. Current density may be affected by many factors and one important factor is gene expression level of the channel. Decreased KCNN2 gene expression in atrium of patients with persistent AF can result in reduced density of apamin sensitivity K⁺ current. However, intracellular Ca2+ overload significantly after AF and increased intracellular Ca²⁺ can upregrate SK2 channel functionally, so we can say increased Ca2+ in atrial mycardiocyte more impacted functional level of SK2 than downregulated SK2 gene and protein expression.ConclusionIn summary, SK2 gene and protein expression were the same in right and let atrium, so SK2 gene and protein expression of right atrium could reflect SK2 gene and protein expression of left atrium. SK2 gene expression in ventricule shown no difference with that in atrium, but SK2 protein expression and apamin sensitive K⁺ current in atrium increased significantly compared with ventricule. Different from previous animal experiments, after suffering from persistent AF, gene expression and protein expression of SK2αsubunit downregrated, gene expression and protein expression of CaM （auxiliary subunit of SK2） unchanged, and functional level of SK2 upregrated, suggesting that apamin sensitive current density were not only effected by SK2 subunits gene and protein expression level, but also by Ca2+ concentration in atrial myocytes. SK2 channel can be looked as an ideal target treatment for patients persistent AF, and it is promising for SK2 channel blockers as a new generation of antiarrhythmic drugs to treat atrial fibrillation.更多还原