【作者】 贾晓萌；

【导师】 侯应龙；

【作者基本信息】 山东大学 ， 内科学， 2014， 博士

【摘要】 研究背景心房颤动(房颤AF)是指心房活动不协调,导致规则有序的心房电机械功能恶化,在心电图上的表现为P波消失,代之以不规则的基线波动,心室率极不规则,是最严重的心房电活动紊乱之一,具有较高的致病率、致残率,并导致了沉重的社会经济负担。无序的颤动使心房泵血功能丧失或恶化,加之房室结对快速无序的心房激动的递减传导,可引起心室极不规则的兴奋,心房收缩力丧失,血流瘀滞易形成栓子。因此心功能受损、心室率(律)紊乱、心房附壁血栓形成等是AF患者的主要病理生理特点。过去很多实验室或临床研究已经表明,心房电重构(ER)和结构重构(SR)是发生AF的两大主要机制。心房ER主要包括心房有效不应期(AERP)及动作电位时程的缩短,动作电位传导速度减慢,是AF或快速心房率所产生的有利于AF维持和发生的心房电生理特性的改变。ER发生在AF过程的较早期,而SR发生的相对较晚。近来对编码特异离子通道蛋白基因的mRNA表达异常的研究引起了越来越多的关注,它可以引起离子通道蛋白数量、结构、性质的改变,尤其是钾离子通道,从而成为ER和心律失常的潜在分子机制。其中转录后水平对mRNA的调节也成为了人们关注的焦点。目前药物依然是AF治疗的重要方法,药物能恢复和维持窦性心律,控制心室率。但是药物治疗的副作用较多如恶心、头晕、疲劳甚至室性心律失常。AF的非药物治疗包括电转复、射频消融治疗和外科迷宫手术治疗。电复律是指用两个电极片放置在病人胸部的适当部位,通过除颤仪发放电流,重新恢复窦性心律的方法。电复律不是一种根治AF的方法,AF往往会复发,而且部分患者仍需要继续服用抗心律失常药物维持窦性心律。导管消融治疗及外科迷宫手术都属于有创治疗,易并发感染及血栓形成等。因此,要找到一个高效、安全的新策略用于治疗AF是非常重要的。MicroRNA(miRNA)是小RNA中的一个大家族,最早是Lee等人在线虫中发现,其在果蝇、植物和哺乳动物等真核生物中均有所发现。因为它在动植物体内发挥着重要且广泛的调控作用,日渐成为人们新的研究热点。miRNA为一类内源性非编码的单链小RNA,通过与其5’端“种子序列”与目的基因mRNA的3’非翻译区完全或不完全性互补配对,对目的基因mRNA的碱基进行切割或抑制翻译,指导RNA诱导沉默复合体(RISC)来调节目的基因表达,介导转录后基因调控。新进的研究表明miRNA不仅调控发育,而且在造血、细胞增殖和死亡等方面发挥作用,另外对心肌肥厚、心力衰竭、动脉粥样硬化和心律失常等心血管系统疾病的发生发展有重要的影响。MiRNA作为转录后调控的重要环节,可以对编码特异离子通道蛋白基因的mRNA表达产生影响,进而使离子通道蛋白及连接蛋白过度表达或低表达,致使离子通道平衡失调,诱发心律失常。miRNA的表达具有组织特异性,如在心肌富含miR-1,let-7,miR-133,miR-126-3p,miR-30c,而动脉平滑肌富含miR-145, let-7,miR-125b,miR-125a,miR-23和miR-143,但miR-1与miR-133在其中也有表达。miR-1与miR-133被认为是肌肉特异性miRNA,在成人的心脏及骨骼肌中优先表达。现在的研究认为miR-1在许多心脏疾病中扮演重要角色,特别是心律失常的病理生理过程。Yang等研究发现：梗死小鼠心脏内过量表达miR-1可以导致心律失常。miR-1通过作用于基因GJA1和KCNJ2减少心肌Cx43,Kir2.1蛋白的表达,使减慢传导,降低了心肌内向整流钾离子电流(IKl),导致细胞膜去极化,促使发生快速性心律失常,心电图中表现为QRS波增宽,QT间期延长。而miR-1通过反义寡核苷酸(AMO-1)被移除后,心律失常的发生明显减少。由此可知miR-1通过引起心肌缺血时离子通道失衡而达到其致心律失常作用。近来在Terentyev等人的研究中发现miR-1还可以通过调节钙离子流来参与心律失常的发生。因此,miR-1被认为是治疗心律失常的新靶点。虽然miR-1在心律失常病理生理过程中的作用已经有所研究,但是miR-1在AF早期ER中所发挥的机制至今还没有报道。目前miRNA-1在体转染的研究中大部分选小鼠为动物模型,转染后将心肌细胞进行体外培养,检测其对应的离子通道蛋白含量的变化,但在大型动物模型中进行miRNA-1在体转染的研究较为少见,而大型动物如犬、兔等AF的发生发展过程更接近于人类,且在体转染miRNA对动物模型AF的维持与诱发等观察更直接,因此,我们选择新西兰大白兔做为AF模型来探讨相关miRNA-1的参与机制,有助于我们更好理解人类AF发生的机制。虽然新西兰大白兔的miRNA序列还没有纳入miRbase数据库中,但我们在Ensembl数据库(http://asia.ensembl.org/Oryctolagus_cuniculus/Info/Index)中找到新西兰大白兔的基因序列草图,可用软件分析出其miR-1的序列。据此,本课题组拟以快速心房起搏兔为研究模型,以miR-1为候选miRNA,活体心房内注射转染miR-1及AMO-1,检测miR-1在快速心房起搏兔模型心房肌中的表达及钾离子通道蛋白含量及其编码mRNA的变化,以阐明miR-1参与AF的发展与维持,为AF的治疗提供新的靶点。研究目的1.评价miR-1在快速起搏1周后在兔心房肌中表达水平的变化；2.明确miR-1的异常表达与快速起搏兔心房早期ER的关系；3.探究miR-1促进心房早期ER的潜在分子机制。材料和方法1.动物模型建立1.1动物分组选购健康新西兰大白兔24只,雌雄不拘,体重1.5-2.5kg,随机分为4组,各组6只。其中第1组设为假手术,第2组设为快速起搏组,第3组设为起搏+miRNA-1转染组,第4组设为起搏+AMO-1转染组。①假手术(第1组,n=6),埋置起搏器不予起搏,1周后开胸,右房(RA)心肌内注射对照病毒,1周后处死；②心房快速起搏组(第2组,n=6),埋置起搏器以起搏频率600bpm连续起搏1周。开胸,RA心肌内注射对照病毒,1周后处死；③心房快速起搏+miR-1转染组(第3组,n=6),埋置起搏器以起搏频率600bpm连续起搏1周。开胸,RA心肌内注射miR-1,1周后处死；④心房快速起搏+miR-1转染组(第4组,n=6),埋置起搏器以起搏频率600bpm连续起搏1周。开胸,RA心肌内注射AMO-1,1周后处死。1.2快速心房起搏模型建立先用3%戊(苯)巴比妥钠(30-35mg/kg)经耳缘静脉将新西兰大白兔麻醉,继以氟烷(2-3%)及一氧化二氮(60-75%)维持麻醉状态(28),仰卧位固定动物,气管插管,呼吸机机械通气,调节呼吸频率30-50bpm,每分通气量15-40ml,吸呼比为2：1。胸颈部用利多卡因局麻并脱毛、常规消毒、铺巾,切开右颈部皮肤、分离皮下组织,暴露右颈内静脉,送入“J”形心房单电极,在X线透视下将电极头端固定于右心耳,整个过程都遵守无菌操作。2.电生理指标检测电生理检查通过多通道程序刺激仪分别于起搏前、转染前、转染1周三个时间点进行,并由电生理记录系统记录下RA心内心电图。AERP的测定采用S1S2反向扫描刺激法,S1S2呈8：1,步长5ms,基础起搏刺激周长分别为150、130ms,AERP定义为S2不引起心房激动的最长S1S2间期。AF由S1S2程序刺激或者burst刺激诱发。3.构建和生产慢病毒新西兰大白兔miR-1和AMO-1的前体序列由TELEBIO公司构建,慢病毒滴度为1×109TU/ml。4.在体基因转染在快速起搏1周后,右胸第4肋间打开胸腔,充分暴露心脏并剥离心包,将慢病毒复合物多点斜行注射到快速心房起搏兔RA前壁,关胸。一周后处死,立即取出心脏用PBS液由主动脉根部逆行灌注,然后分离出RA,拭干后保存在-80。C有待下一步使用。5.实时定量RT-PCR实时荧光定量RT-PCR反应检测miR-1和KCNE1/KCNB2mRNA表达水平。6. Western blot检测Western blot检测KCNE1和KCNB2所编码的离子通道蛋白表达水平。7.荧光素梅报告分析通过荧光素梅报告分析证实KCNE1和KCNB2是miR-1的靶基因。结果1.在快速起搏1周后,兔RA可发现早期ER；2.在快速起搏1周后,miR-1的表达水平发生上调；3.通过转染外源基因使miR-1过表达,促进了心房ER的发生,应用AMO-1拮抗miR-1后可逆转上述现象；4. KCNE1和KCNB2是miR-1的靶基因,他们的表达水平呈负相关。结论MiR-1通过作用于其靶基因KCNE1和KCNB2来调节后者编码的钾离子通道蛋白,从而影响心脏钾离子通道及电流,参与了由快速起搏引起的早期心房ER,表明miR-1可能在AF的发生过程中起到重要的作用。因此,miR-1作为治疗AF的潜在靶点具有重要的临床意义。创新性1.用新西兰大白兔制作快速心房起搏动物模型并检测miR-1在心房细胞中的表达变化；2.心房肌内活体注射miR-1及AMO-11周后体外检测离子通道蛋白及其编码基因的变化,探讨miR-1对钾离子通道及心房ER的影响及意义。局限性1.迄今为止,已有数千个microRNA分子先后在生物体内被鉴定,我们只进行了miR-1对心房早期ER的研究。2.同一种miRNA在不同的AF类型或在AF的不同时期发挥作用的不尽相同,我们只研究了miR-1对快速起搏兔右心房早期ER的影响。更多还原

【Abstract】 BackgroundAtrial fibrillation (AF) is one of the most common arrhythmia with substantial morbidity, mortality, and socioeconomic burden. Experimental and clinical studies have shown that electrical remodeling (ER) and structure remodeling (SR) are two major mechanisms involved in the AF. ER occurred early in the course of AF and led to characteristic changes in action potential duration (APD) and effective refractory period (ERP), whereas SR starts later and proceeds more slowly. Recently, abnormal expression of gene encoding the ion-channel protein, especially potassium (κ) channel, has attracted the researchers’interest in the molecular mechanism for ER and arrhythmia. Also, the difference between the level of messenger ribonucleic acid (mRNA) and that of the correspondent protein, frequently observed in gene expression studies, has aroused researcher’s interest to investigate the regulatory mechanisms at the post-transcriptional level. Although traditional pharmacological therapies were effective in maintaining normal sinus rhythm, they were associated with numerous adverse side reactions such as nausea, dizziness, fatigue and even ventricular arrhythmias, while non-pharmacological therapies, such as current cardioversion, surgery, radiofrequency ablation, for instance, may lead to complications such as infection and thrombosis. Therefore, it is important to find an efficient and safe new strategy for the treatment of the AF. MicroRNAs (miRNA), a group of endogenous single-stranded non-protein-coding small RNAs (-22nucleotides long) were initially described in1993. MiRNA interacts with the3’untranslated region (3’UTR) of its target mRNA via exact complementarity with the7-8nt at its5’ end, the so-called’seed sequence’, which is critical for miRNA actions to guide RNA induced silencing complex (RISC) to down-regulate the expression of its target mRNA at the post-transcriptional level.MicroRNA-1(miR-1) was known to be muscle-specific miRNAs preferentially expressed in adult cardiac and skeletal muscle tissues, which was the top20abundant miRNAs in human heart. Current studies indicated that miR-1is involved in many heart diseases, especially in cardiac arrhythmias and its expression is associated with cardiac arrythmogenic potential in ischemic heart diseases. Delivery of miR-1into normal or infarcted rat hearts induces significantly widened QRS complex and prolonged QT interval in electrocardiograms, and AMO-1(anti-miR-1inhibitor oligonucleotides) could rescue this effect. The upregulation of miR-1could increase conduction time and depolarize the membrane potential through repressing the levels of Kir2.1and Connexin43, which may be partly responsible for its arrhythmogenic potential. Recently, one study by Terentyev et al suggested that miR-1may also participate in arrhythmia by deteriorates Ca2+handling.The miRNA of rabbit is absent in the miRbase until now. Though miR-1has been studied in the pathogenesis of cardiac arrhythmia, its possible role in the early stage of atrial ER has not yet been reported. Therefore, we performed the present study, to provide the first evidence that miR-1played a significant part in the early stage of atrial ER through a rabbit model with1-week right atrial tachypacing (A-TP). We used in this work established animal techniques for electrophysiology measurements and lentiviral vectors (LV) to deliver the genes of interest. The advantage of LV is their low immunogenicity and they can be easily transferred into cells and tissues and lead to much higher gene expression than that achieved by using adenovirus vectors in previous similar experiments.Objectives1. To evaluate the expression of miR-1in right A-TP rabbit model by a pacemaker for 1-week.2. To definite the relationship between the abnormal expression of miR-1and the early stage of atrial ER in AF.3. To elucidate the potential molecular mechanisms of miR-1accelerates the early stage of atrial ER in AF.Methods1. Establishment of animal model1.1Animal preparationAdult New Zealand White rabbits (regardless of their gender;1.5-2.5kg) were randomly allocated into4groups:1:control group (Ctl, n=6):with no pacing and transfected with control LV;2:right A-TP group (Pacing, n=6):submitted to pacing at600beats per minute (bpm) for1week, then transfected with control LV;3:right A-TP group transfected with miR-1(P+miR-1, n=6), recombinant LV carrying miR-1were injected into right atrial after right A-TP;4:right A-TP with AMO-1(P+AMO-1, n=6), where animals, after right A-TP, were injected into right atrial with recombinant LV carrying AMO-1.1.2Establishment of right atrial tachypacing (A-TP) modelRabbits were anesthetized with pentobarbital sodium (30-35mg/kg) and were ventilated by tracheostomy with a volume-regulated respirator. Halothane and N2O were supplemented to maintain a constant level of anesthesia for all procedures. Ventilator settings were adjusted to maintain physiological arterial blood gases. After administrating local anesthesia with lidocaine in the neck skin, right jugular vein was isolated and ligated by skin incision. A pacemaker was implanted in a subcutaneous pocket and attached to an electrode-lead in the RA appendage via the right jugular vein under the guidance of X-ray. All surgical procedures were performed under sterile conditions.2. Electrophysiological monitorElectrophysiological examination was performed at3time points (before pacing, before transfection and1-week after transfection), by using a programmable multichannel stimulator and intracardiac electrograms (ECG) were measured by using electrophysiological recording system, by placing the catheter into the right atrial (RA). Atrial effective refrractory period (AERP) was measured with S1-S2programmed electrical stimulation [PES]. AF was induced by PES with burst stimulation.3. Construction and production of Lentiviral vectorThe miR-1and AMO-1sequences of New Zealand White rabbit precursor were synthesized by TELEBIO (Shanghai, PR China). The titer of Lentiviral vector was1×109TU/ml.4. In vivo gene transferAfter1week of A-TP, the heart was exposed and the RAs were fixed. Then recombinant LVs were directly injected into RAs. After that, the heart was placed back into the thoracic cavity. One week after lentiviral injection, the rabbits were killed and the heart was immediately excised and washed by ice-cold PBS retroperfusion via the aortic root, then the RA was dissected, blotted dry, frozen in liquid nitrogen, and stored at-80℃.5. Real-time qRT-PCR analysisTotal RNA was extracted from rabbits RAs, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) was performed to detect the level of miR-1and KCNE1and KCNB2genes.6. Western blot analysisWestern blot analysis was used to determine the protein expression of KCNE1and KCNB2.7. Luciferase reporter assaysKCNE1and-KCNB2as the target genes for miR-1were confirmed by luciferase activity assay.Results1. The early stage of atrial ER in AF was found in right A-TP rabbit model by a pacemaker for1-week; 2. The expression of miR-1was increased with the right atrial ER;3. Over-expression of miR-1via in vivo transfections of recombinant LV accelerated right atrial ER after A-TP meanwhile AMO-1could rescue it;4. The level of miR-1and KCNE1/KCNB2appear to be negatively correlated, indicating thus that miR-1could accelerate the atrial ER through KCNE1and KCNB2target genes.ConclusionsMiR-1accelerates the atrial ER induced by A-TP via target K+channel genes, indicating that miR-1may play an important role in the initiation of AF. Thus, miR-1has great clinical relevance as a potential therapeutic target for AF.更多还原