【作者】 许国军；

【导师】 程祖亨； 汤宝鹏；

【作者基本信息】 新疆医科大学 ， 内科学， 2013， 博士

【摘要】 目的：房颤的流行病学特征首先和年龄相关，伴随着人口老龄化等社会进步的表现，房颤的患病人数正迅速增加，房颤、老龄化是21世纪全世界必须面对的医疗与社会问题。因此，关注房颤、关注老年房颤就更具有重要的意义。基于房颤和增龄之间的密切关系，近年来国内外学者提出了增龄性房颤这一概念。本研究旨在探讨增龄性心房颤动心房重构的离子分子机制，以期为增龄性房颤的病因、发病机制和治疗等提供新的思路和理论依据。本研究目的是为探索与论证：1）是否心房肌细胞内钙离子平衡异常与左心房复极化障碍伴随增龄而显现，其是否为老年人易触发和易维持房颤的离子机制；2）是否存在心房肌细胞膜L-型钙通道蛋白和细胞内肌质网钙转运调控蛋白异常表达伴随增龄而显现，致使心肌细胞内钙稳态遭到破坏，其是否为增龄性房颤心房电重构的分子基质；3）是否心房肌细胞钙激活中性蛋白酶（calpain1、calpain2）、钙蛋白酶抑素（calpastatin）的表达变化与这种细胞内可能普遍存在的病理生理改变相关，包括在心房老化和房颤中左心房心肌细胞膜特征性的L-型钙通道表达改变、加速的纤维化和细胞凋亡，以期阐明增龄与房颤时心房肌电重构和结构重构以及心功能下降的分子生物学机制及其在增龄性房颤发生维持中的作用；4）伴随着心房老化或者在房颤这种疾病状态下，是否存在MMP-9与TIMP-1，BCL-2与BAX的表达改变与心房结构重构有关；5）是否可能存在的microRNA-1、microRNA-21、microRNA-29和microRNA-133的异常表达与伴随衰老和房颤疾病状态下加重的纤维化和细胞凋亡是否相关。方法：通过持续快速心房起搏建立持续性房颤犬模型，用全细胞膜片钳的方法记录4组犬（成年和老年窦性心律犬、成年和老年持续性房颤犬）的L-型钙电流（ICa-L）电流密度以及动作电位特征；用实时荧光定量聚合酶反应（Realtime PCR）和蛋白免疫印迹（Western blot）的方法针对四组犬：1）检测左心房心肌细胞膜L-型钙通道蛋白和肌浆网钙转运调控蛋白在mRNA和蛋白质表达水平变化；2）检测左心房肌细胞钙激活中性蛋白酶（calpain1、calpain2）、钙蛋白酶抑素（calpastatin）在mRNA和蛋白质表达水平变化；3）检测左心房肌细胞MMP-9与TIMP-1，BCL-2与BAX在mRNA和蛋白质表达水平变化；4）检测左心房肌细胞microRNA-1、microRNA-21、microRNA-29和microRNA-133的表达变化；5）应用光镜、电镜及TUNEL法检测各组犬左心房心肌组织细胞的超微结构变化、心肌纤维化程度以及细胞凋亡指数等结构重构；6）检测目标基因及蛋白的表达变化与心房电重构及结构重构之间是否存在相关性。结果：1.电生理变化：1）窦性心律时体表心电图数据显示，与成年犬比较，老年犬的心电图P波持续时间显著地延长，P波离散度显著地增大（P＜0.05）；2）在窦性心律时成年犬和老年犬左心房组织细胞动作电位的特征性变化趋势：与成年犬比较，老年犬的心肌细胞膜动作电位平台期（2相）电压显著降低，动作电位持续时间（APD90）显著延长，L-型钙电流密度出现了明显地减低（均P＜0.05）；3）与窦性心律时比较，两组犬在发生房颤时均出现了显著的去极化趋势，均出现动作电位持续时间（APD90）缩短，动作电位振幅显著地增大，平台期电位电势显著地减少，L-型钙电流密度出现了显著地减低（均P＜0.05）。2.分子生物学变化：1）在mRNA和蛋白质表达水平，老年犬左心房肌LVDCCa1c显著地降低，而Ca2+-ATPase显著地升高（均P＜0.05），RYR2，IP3R1和PLN普遍地显示出上调的趋势，但无统计学差异（P＞0.05）；与窦性心律犬比较，房颤犬除受磷蛋白（PLN）之外，LVDCCa1c和肌浆网钙转运调控蛋白均显著地下调，尤其是老年房颤犬这种下调趋势更加明显（（均P＜0.05））；2）与成年犬比较，老年犬左心房肌细胞calpain1，calpain2和calpastatin在mRNA和蛋白质表达水平均普遍地显示出上调的趋势，但是未显示出统计学差异（P＞0.05）。此外，在相同年龄的窦性心律犬和慢性房颤犬之间的比较中显示，房颤犬左心房肌细胞calpain1在mRNA和蛋白质表达水平均显著地增高（均P＜0.05），尤其是老年房颤犬增高最为明显；然而，calpain2和calpastatin在mRNA和蛋白质表达水平均未显示出统计学差异（均P＞0.05），老年房颤犬的LVDCCa1c与calpain1在蛋白质表达水平呈现负相关（r=-0.583，P=0.019）；3）与成年犬比较，老年犬左心房肌细胞MMP-9和BAX在mRNA和蛋白质表达水平表达均显著地增高（均P＜0.05），而TIMP-1和BCL-2表达均显著地下降（均P＜0.05）；与窦性心律犬比较，房颤犬左心房肌细胞MMP-9和BAX在mRNA和蛋白质表达水平均显现出有意义的上调趋势（均P＜0.05），尤其是老年房颤犬最为明显，TIMP-1和BCL-2在mRNA和蛋白质表达水平均显现出有意义的下调趋势（均P＜0.05），尤其是老年房颤犬最为明显；4）与成年犬组相比较，在窦性心律时老年犬左心房肌细胞miR-21，29的表达水平显现出显著的上调趋势（均P＜0.05）；然而，miR-1，133的表达水平呈现出显著的下调趋势（均P＜0.05）；与窦性心律老年犬组相比较，在持续性房颤老年组犬左心房心肌组织的miR-1，21，29的表达水平显现出显著的上调趋势（均P＜0.05）；miR-133的表达水平显现出显著的下调趋势（P＜0.05）。3.组织病理学变化：伴随老龄与房颤，犬心肌纤维化程度、细胞超微结构及凋亡指数均显现出有统计学意义的改变（均P＜0.05）。结论：1）这种伴随增龄与房颤而显现的左房电生理和钙转运调控蛋白特异性改变（适应与不良适应反应）可能是增龄性房颤心房电重构的分子基质之一。2）这种伴随增龄与房颤而显现的钙激活中性蛋白酶特异性生物活性改变可能是增龄性房颤心房电重构与结构重构的分子基质之一。3）这种伴随增龄与房颤而显现的MMP-9与TIMP-1，BCL-2与BAX特异性的生物活性改变可能是增龄性房颤心房纤维化重构的分子基质之一。4）这种伴随增龄与房颤而显现的microRNA-1、microRNA-21、microRNA-29和microRNA-133的异常表达与心房结构重构进程中特征性的加重的纤维化和细胞凋亡相关，其对将来可能的临床诊断、预后评估及基因治疗与干预提供了有意义思路和措施。更多还原

【Abstract】 Objective: This study was to investigate whether or not the dysfunction of atrialrepolarization and abnormality of the intracellular Ca2+handling protein was augmentedwith aging; to investigate the correlation between the change in the expression of atrialcalpains and electrical, molecular, and structural remodeling during aging and AF; toinvestigate whether abnormal expressions of matrix metalloproteinase (MMP)-9/tissueinhibitors of MMPs (TIMP)-1and BCL-2/BAX are correlated with the characteristicaccelerated fibrosis and apoptosis during aging and AF; to investigate whether or not theaberrant expression of microRNA-1, microRNA-21, microRNA-29and microRNA-133show a correlation with structural remodeling during aging and AF, and demonstrate thatthese miRNAs, acting singly or in combination, may be responsible for modulating thetransition from adaption to pathological atrial remodelling. Methods: Four groups ofdogs were studied; younger adult and aged dogs in sinus rhythm (SR) and atrialfibrillation (AF) induced by rapid atrial pacing. We used whole cell patch clamprecording techniques to measure L-type Ca2+current (ICa-L) in cells dispersed from theleft atria. The mRNA and protein expressions of the target proteins in the left atrium weremeasured by quantitative RT-PCR and Western blot analysis. The expressions of thetarget microRNAs were measured by Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR). Pathohistological and ultrastructural changes were tested by light andelectron microscopy. Apoptosis indices of myocytes were detected by TUNEL. Results:In SR groups, atrial cells of the old dogs had longer action potential (AP) duration to90%repolarization (APD90), lower AP plateau potential, and peak ICa-Lcurrent densities(all P＜0.05). In both ages, AF led to a higher maximum diastolic potential, a increase ofAP amplitude, decreases of APD90, AP plateau potential and peak ICa-Ldensities (all P＜ 0.05). Compared to the adult group, the mRNA and protein expressions of LVDCCa1cwere decreased in the aged group, whereas the expressions of calcium adenosinetriphosphatase were increased; the mRNA and protein expressions of MMP-9and BAXand those of TIMP-1and BCL-2exhibited a significant increase and a down-regulationpattern, respectively, in the aged groups compared with the adult groups; compared to theadult group, the expressions of miR-21,29was significantly increased, whereas theexpressions of miR-1,133showed obviously down-regulation tendence in the aged group(all P＜0.05). Compared to SR group, expressions of LVDCCa1c and Ca2+handlingprotein except for phospholamban were significantly decreased in both age groups withAF; the mRNA and protein expressions of calpain1were increased in both the adult andold groups with AF; compared with the control groups, the adult and old groups with AFexhibited significantly increased mRNA and protein expressions of MMP-9and BAXand down-regulated expressions of TIMP-1and BCL-2; compared to the aged group, theexpressions of miR-1,21,29was significantly increased in the old group in AF, incontrast, the expressions of miR-133showed obviously down-regulation tendence (all P＜0.05). Samples of atrial tissue showed abnormal pathohistological and ultrastructuralchanges like accelerated fibrosis and apoptosis with aging and in AF (all P＜0.05).Conclusions: These aging-induced electrophysiological and molecular changes showedthat general pathophysiological adaptations might provide a substrate conducive to AF.Age-related alterations in atrial tissues can be attributed to increased expressions ofcalpain1. MMP-9/TIMP-1and BCL-2/BAX hold potential for use as substratesconducive to AF, and their abnormal expressions play a major role in atrial structuralremodelling. These multiple aberrantly expressed miRNAs may be responsible formodulating the transition from adaption to pathological atrial remodelling with agingand/or in AF.更多还原