【作者】 范晋奇；

【导师】 殷跃辉；

【作者基本信息】 重庆医科大学 ， 内科学， 2013， 博士

【摘要】 背景：心房颤动(Atrial Fibrillation,简称房颤)是临床上最常见的心律失常，具有较高的致残率和致死率，严重影响患者生活质量。既往研究表明肾素-血管紧张素系统(Renin-Angiotensin System, RAS)的过度激活，尤其是血管紧张素II(Ang II)心房结构重构中扮演了重要作用。丝裂原活化的蛋白激酶(Mitogen-activated protein kinase, MAPK)直接介导了AngII对心房组织的不良重构；MAPK磷酸酶-1(MAPK phosphatase-1,MKP-1)作为双特异性磷酸酶家族的重要成员，能够通过其去磷酸化作用直接导致MAPK失活。MAPK和MKP-1的动态平衡在房颤发病机制中的作用尚不清楚。血管紧张素转换酶2(ACE2)是一种羧肽酶，其能够将具有血管收缩效应的AngII转化为扩血管七肽血管紧张素-(1-7)[ANG-(1-7)]，从而起到负性调节RAS的作用。最近的研究表明，过表达ACE2能够拮抗血管紧张素Ⅱ介导的心肌肥厚和间质纤维化，防止心功能不全。目的：既往研究已经证明心房外膜涂染腺病毒具有较高靶向性和高转染效率。本研究拟通过心外膜涂染血管紧张素转化酶2(Angiotensin-converting enzyme2, ACE2)基因的方法探讨其预防和改善心房快速起搏模型所致结构重构的影响及其分子机制。方法：入选成年健康杂种犬(雌雄不分，20-30Kg)28只，随机分为Sham组（假手术组）、Control组(对照组)、Ad-EGFP组(心房快速起搏+心房外膜涂染Ad-EGFP)和Ad-ACE2组(心房快速起搏+心房外膜涂染Ad-ACE2)，每组7只。其中假手术组和对照组为空腺病毒，Ad-EGFP组和Ad-ACE2组分别涂抹携带有EGFP和ACE2的腺病毒。所有对照组、Ad-EGFP组和Ad-ACE2组的实验犬均接受心房快速起搏(450次/分)，假手术组不给予心房起搏。2周后，所有实验犬接受开胸手术操作和心外膜电生理研究(Electrophysiological study, EP)，然后完成心外膜基因涂染。基因转染后21天，所有动物完成第二次心脏电生理检查后处死，取动物标本进行组织学和分子生物学研究，如下：1)为探讨心房快速起搏所致的组织病理学改变，采用苏木精-伊红(HE)和天狼猩红染色评价。2)为探讨过表达ACE2对RAS的影响，本研究采用了实时荧光定量RT-PCR、免疫印迹、ELISA和免疫组化等方法评价了RAS系统成员的表达，如ACE2，Ang II和Ang-(1-7)。3)为探讨心房外膜过表达ACE2改善心房结构重构可能涉涉及的分子机制，本研究采用蛋白印迹技术评价了MAPK与MKP-1的表达水平及其动态平衡；采用蛋白印迹技术和实时荧光定量PCR技术评价了转化生长因子-β1(Transforming growth factor-β1, TGF-β1)和胶原蛋白的表达。结果：3周后第二次手术与第一次手术时相比较，Control组、Ad-EGFP组和Ad-ACE2组左、右心房的有效不应期(Atrial effective refractoryperiod, AERP)均有显著性降低，且(AERP350-AERP250)/100(反应AERP频率适应性)亦显著降低，Sham组基因转染前后左、右心房的AERP无显著改变。在房性心律失常的诱发率与持续时间方面，Sham组仅能诱发出短暂房性心律失常；与Sham组和基线相比，Control组和Ad-EGFP组房性心律失常诱发率显著增加，且房性心律失常的持续时间明显延长；然而，Ad-ACE2组基因转染前后房性心律失常的诱发率和持续时间无明显改变。HE染色未见各组犬发生严重的心包炎症、渗出和出血。Sham组犬心房肌细胞纤维排列有序，细胞之间的间隙适中。Control组和Ad-EGFP组犬心房肌细胞纤维出现挛缩、断裂、排列紊乱，天狼星染色提示心房肌间质广泛纤维化，心内膜和心外膜纤维组织增厚。相反，与Control组和Ad-EGFP组相比，上述病理异常在Ad-ACE2组明显减轻，心房肌纤维分布与排列大致有序，心肌间质纤维化显著减轻。纤维化定量分析进一步显示，Ad-ACE2组心房肌纤维化百分比显著低于Control组和Ad-EGFP组(5.8±2.4%vs.11.9±2.3%vs.14.3±3.4%，P<0.001)，但与Sham组相比无明显差异(P=0.614)。Western blot半定量分析结果：与Sham组相比，Control组和Ad-EGFP组ACE2蛋白表达水平显著降低；然而，Ad-ACE2组ACE2表达水平为Sham组的2倍。进一步地，Relatime RT-PCR结果显示ACE2基因的mRNA表达水平在各组之中呈现出与其蛋白表达相一致的趋势（图5）。采用免疫组化半定量分析方法评价Ang II和Ang-(1-7)在心房组织中的相对表达水平。结果表明Control组和Ad-EGFP组心房组织中AngII的表达水平显著高于Sham组和Ad-ACE2组，而Control组和Ad-EGFP组Ang-(1-7)的表达水平较Sham组和Ad-ACE2组更低。ELISA的结果显示与上述免疫组化的结果一致。与Sham组犬相比，Control组和Ad-EGFP组心房组织的p38MAPK、ERK1/2和p-ERK1/2的蛋白表达水平显著增加；但与Control组和Ad-EGFP组比较，Ad-ACE2组犬p38MAPK、ERK1/2和p-ERK1/2的蛋白表达水平显著降低(P <0.01)。相反，与Sham组和Ad-ACE2组相比较，Control组和Ad-EGFP组心房组织MKP-1的蛋白表达量显着降低，且Sham和Ad-ACE2两组之间心房组织MKP-1的蛋白表达量无显著差异。Western blot定量分析显示与Sham组相比，Control组和Ad-EGFP组心房肌组织中TGF-β1和Col III的蛋白表达显著增加(P <0.01)；而与后两组比较，过表达ACE显著降低TGF-β1和Col III的蛋白表达水平。相对应的，与Sham组相比，心房肌组织TGF-β1、I和IV型胶原蛋白的mRNA表达水平在Control组和Ad-EGFP组显著增加，而与后两组相比较，过表达ACE2能够降低TGF-β1和胶原蛋白的mRNA表达水平（P<0.01）结论：本研究主要发现：（i）心房过表达ACE2降低房颤诱发率和缩短房颤持续时间；（ii）心房过表达ACE2导致心房肌组织局部内源性Ang II水平的降低和Ang-(1-7)水平的增加，将ACE2/Ang(1-7)/Mas轴与ACE/Ang II/AT1R轴之间的平衡向保护性的ACE2/Ang(1-7)/Mas轴移动；（iii）本研究在国内外首次证明，心房过表达ACE2显著降低MAPKs的表达，增加负性调节的MAPKs的磷酸酶MKP-1的表达；（iv）心房过表达ACE2明显改善心房快速起搏所致的心肌纤维化，降低胶原蛋白和促纤维化因子TGF-β1的表达。总之，我们的研究结果表明，心房外膜转染ACE2可能通过调节RAS平衡向保护性效应方向移动，上调MKP-1表达的同时抑制MAPK的激活，降低TGF-β1和胶原蛋白的表达等机制改善心房结构，以及最终降低房颤诱发率和持续时间。更多还原

【Abstract】 Background:Atrial fibrillation (AF) is the most common clinical arrhythmia and isassociated with cardiovascular morbidity and excessive mortality. Previousstudies have demonstrated activation of the local renin angiotensin system(RAS), especially angiotensin II (Ang II) have been found to play animportant role in atrial structural remodeling. Mitogen-activated proteinkinases (MAPKs) are important mediators of Ang II effects on tissuestructure. MAPK phosphatase1(MKP-1) is an important member of thedual-specificity phosphatase family that is expressed in the heart, where itregulates inactivation of MAPKs by dephosphorylation. The dynamicbalance of MAPKs and MKP-1in atrial fibrillation has not beenestablished.Angiotensin-converting enzyme2(ACE2) is a monocarboxypeptidasethat metabolizes vasoconstrictive octopeptide Ang II into vasodilativeheptapeptide angiotensin-(1-7)[Ang-(1-7)], thereby functioning as a negative regulator of the renin-angiotensin system. Recent studies haveshown overexpression of ACE2could suppress Ang II–mediatedmyocardial hypertrophy and fibrosis, and prevent cardiac dysfunction.Objective:Previous studies have shown that epicardial gene painting causeshomogeneous and complete transmural atrial gene transfer. Therefore, thepurpose of this study was to investigate whether atrial overexpression ofACE2by homogeneous transmural atrial gene transfer can help to reverseAF induced atrial structural remodeling and their mechanism in a canineatrial pacing model.Method:Twenty-eight mongrel dogs of either gender, weighing20to30Kg,were randomly divided into4groups: Sham-operated (Sham), control, genetherapy with Ad-EGFP (Ad-EGFP group) and gene therapy with Ad-ACE2(Ad-ACE2group)(n=7per subgroup). All dogs in the control, Ad-EGFPand Ad-ACE2group were paced at450beats per minute for a period of14days. The dogs in Sham group were instrumented without pacing. After2weeks, all dogs underwent thoracotomy operation and an invasiveelectrophysiology (EP) study, then receveid epicardial gene painting. Onpostgene transfer day21, animals underwent electrophysiology study,histology, and molecular studies, as described follow: 1) To identify the potential pathologic substrate underlying conductionbnormalities in rapid-pacing dogs, histologic studies were performed.Atrial tissue sections was stained with Hematoxylin and eosin (H-E) orPicrosirius Red staining by traditional methods.2) To study the effect of ACE2overexpression on other RAS components,we measured the expression levels of ACE2, Ang II and Ang-(1-7) byreal-time PCR, western blot, ELISA and immunohistochemistry.3) To investigate the potential mechanism of ACE2improving atrialstructural remodeling, the balance regulation of between MAPKs andMKP-1was evaluated using immunoblot. The expression levels oftransforming growth factor-beta1(TGF-β1) and collagen were assayedusing immunoblot and real-time PCR.Results:In addition to sham group, after5weeks atrial tachypacing, AERP atall BCLs at either site decreased significantly,(AERP350-AERP250)/100became significantly smaller, suggesting a reduction of rate adaptation ofAERP. After3weeks of gene transfer, AF became inducible in all controland Ad-EGFP dogs, the inducibility and duration of AF increaseddramatically compared with the baseline and the Sham group (P<0.01),whereas the inducibility and duration of AF were found to be markedlylower in the Ad-ACE2group than those in the control and Ad-EGFP group. Serious pericardial inflammation, effusion, and hemorrhage were notobserved in any of the dogs. Atrial myocyte from sham dogs showed anormal composition of sarcomeres distributed throughout the cell, and theintra-cellular space also appeared normal. In contrast, atrial myocytes ofcontrol and Ad-EGFP dogs showed a loss of some contractile materials andabnormal sarcomeres. In addition, extensive interstitial fibrosis, evidencedby Picrosirius Red stain was found in these tissues. Thick layers of fibroustissue were observed in the endocardium and epicardium. In contrast, thesepathologic abnormalities of atrial tissues were attenuated in the Ad-ACE2group.The percentage of fibrosis in all atrial regions in the Ad-ACE2groupwas markedly lower than that in the Sham and Ad-EGFP group (5.8±2.4%vs.11.9±2.3%and14.3±3.4%at the right atrial appendage, p<0.001), andwas comparable with that in the Sham group (p=0.614).ACE2protein expression in the control and Ad-EGFP group wassignificantly decreased compared with that in the Sham subjects; comparedwith the later, it was increased two folds in the Ad-ACE2group. Similarly,ACE2gene expression showed the same statistical trend as its proteinexpression.The relative expression levels of Ang II and Ang-(1-7) in the atrialtissue were evaluated by semi-quantitative analysis of immunohistochemistry. In comparison with the Sham and Ad-ACE2group,the expression levels of Ang II were significantly higher in the Ad-EGFPand control group, but the expression levels of Ang-(1-7) was lower.Corresponding to that, the changing trend of both Ang II and Ang-(1-7) inatrial tissue detected by ELISA was similar to the results ofsemi-quantitative analysis of immunohistochemistry.The amount of p38, ERK1/2and p-ERK1/2protein were significantlyincreased in the control and Ad-EGFP dogs compared with Sham dogs, butwas reduced in the Ad-ACE2group when compared with control andAd-EGFP groups(P<0.01). In contrast, the amount of MKP-1protein wassignificantly lower in control and Ad-EGFP group dogs in comparison withthe sham and Ad-ACE2dogs.Both TGF-β1and Col III protein expression by Western blot analysisincreased dramatically in the control and Ad-EGFP dogs (P<0.01); whereascompared the latter two groups, they were significantly decreased inAd-ACE2group. Corresponding to that, compared to the Sham group, themRNA expression levels of TGF-β1, Col I and Col IV were alsosignificantly higher in the control and Ad-EGFP dogs, while they weresignificantly lower in the Ad-ACE2group in comparisons with latter twogroups (P<0.01) Conclusion:The salient findings of this study are:(i) overexpression of ACE2leadto a significant reduction of the endogenous Ang II level and a significantincrease of the endogenous Ang-(1-7) level, thus shifting the RAS balancetowards the protective axis;(ii) our study demonstrated that, for the firsttime, overexpression of ACE2decreased expression of MAPKs andincreased expression of negative regulators for MAPKs, MKP-1, resultingin attenuation of atrial fibrosis marker, Col III and TGF-β1In conclusion, our results demonstrate that overexpression of ACE2byhomogeneous transmural atrial gene transfer could shift the RAS balancetowards the protective axis, attenuate cardiac fibrosis remodelingassociated with up-regulation of MKP-1to reduce MAP kinase activities,and subsequently decreased the inducibility and duration of AF.更多还原