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(Angiotensin II,Ang II)不仅在心房结构重构中扮演了重要作用，也对心房肌离子通道和缝隙连接蛋白具有不良效应而表现出促心律失常作用。
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。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。新近，一些研究提示Ang-(1-7)不仅能够预防90%动作电位时程的缩短，也能够改善长期心房快速起搏所致的犬心房肌离子通道重构。然而，ACE2/Ang-(1-7)对心房离子通道和缝隙连接蛋白的影响及其分子机制目前尚不清楚。
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电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)进一步探索过表达ACE2对心房离子通道蛋白和缝隙连接的影响，采用免疫印迹、激光共聚焦免疫组化和实时定量RT-PCR检测相关蛋白的表达与分布。
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。
实时定量RT-PCR显示，与Sham组相比，Control组和Ad-EGFP组心房肌的Cav1.2和Kv4.2的mRNA表达量均显著降低（P <0.01）；与Control组和Ad-EGFP组比较，Ad-ACE2组Cav1.2和Kv4.2的mRNA表达显著上调，但其与Sham组比较无统计学差异。Kv4.3和KChiP2的mRNA表达水平在各组之间无显著差异。
Western blot结果显示，与Sham组相比，Control组和Ad-EGFP组心房肌的Cx40蛋白表达水平明显增加；与后两组相比较，心房过表达ACE2能够显著增加Cx40蛋白表达水平，但其与Sham组比较无明显差异。相反，Ad-ACE2组和Sham组心房肌组织中Cx43蛋白表达水平明显高于Control组和Ad-EGFP组。Ad-ACE2组和Sham组两者之间Cx43和Cx40的蛋白表达水平均无显著差异。此外，实时定量RT-PCR结果表明Cx40和Cx43的mRNA表达水平显著高于Control组和Ad-EGFP组。激光共聚焦免疫组织化学荧光技术结果表明绝大部分Cx43蛋白主要表达于与心肌长轴垂直的心房肌闰盘，仅有少量表达于横向肌膜；相比之下，绝大部分Cx40蛋白主要位于在横向肌膜，但有少量表达于与心肌长轴垂直的心房肌闰盘。
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）心房过表达ACE2明显改善心房快速起搏所致的心肌纤维化；（iv）本研究在国内外首次证明，心房过表达ACE2能够改善心房快速起搏所致的心房肌离子通道蛋白与缝隙连接重构。
我们的研究结果表明，心房外膜转染ACE2能够降低房颤复发率和缩短房颤持续时间，其机制可能涉及调节RAS平衡向保护性效应方向移动，削弱心肌间质纤维化，以及改善长期心房快速起搏所致的心肌离子通道蛋白与缝隙连接重构。
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 not only playan important role in atrial structural remodeling, but also have potenteffects on atrial ion channels and connexins, showing strong proarrhythmiceffects.
Angiotensin-converting enzyme2(ACE2) is a monocarboxypeptidasethat metabolizes vasoconstrictive octopeptide Ang II into vasodilativeheptapeptide angiotensin-(1-7)[Ang-(1-7)], thereby functioning as anegative regulator of the renin-angiotensin system. Recent studies haveshown overexpression of ACE2could suppress Ang II–mediatedmyocardial hypertrophy and fibrosis, and prevent cardiac dysfunction.Recengtly, some researchers found that the product of ACE2, Ang-(1-7), might have some antiarrhythmogenic effects during myocardialischemia-reperfusion. Furthermore, another study suggested that Ang-(1-7)could effectively prevent the shortening of90%action potential duration(APD90) and improve ion channels remodeling induced by chronic rapidatrial pacing in dogs. However, the effects of ACE2/Ang-(1-7) on atrial ionchannels and connexins remodeling and their mechanisms are still notclear.
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 electrical remodeling and their mechanisms 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. After2 weeks, 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 conductionabnormalities 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 further explore the effects of overexpression of ACE2on atrial ionchannel and connexins protein, they were detected by western blot,confocal immunohistochemistry and real time RT-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 increased dramatically 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.
A quantitative analysis of fibrosis showed that the percentage offibrosis in all atrial regions in the Ad-ACE2group was markedly lowerthan 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), and was comparablewith 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 ofimmunohistochemistry. 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.
Real time RT-PCR analysis showed that, compared withSham-operated dogs, both Cav1.2and Kv4.2mRNA abundance werelower in the myocardium of control and Ad-EGFP dogs (P<0.01), but wasup-regulated significantly in Ad-ACE2group. No significant differencewas found in terms of Kv4.3and KChiP2mRNA abundance among thefour groups.
Compared with Sham, the protein expression levels of Cx40wassignificantly increased in the control and Ad-EGFP group, but wassignificantly increased in Ad-ACE2group when compared with the controland Ad-EGFP dogs. In contrast, the protein expression levels of Cx43werehigher in Ad-ACE2group that those in the control and Ad-EGFP dogs. NO significant differences were observed in the expression of Cx40and Cx43between Sham and Ad-ACE2group. Furthermore, the mRNA expressionlevels of Cx40and Cx43were significantly higher in the control andAd-EGFP group than those in Sham and Ad-ACE2group, as shown in. Weevaluated connexin localization using confocal immunohistochemistry. Inlongitudinally sectioned atrial myocytes from all groups, Cx43was locatedmainly at the intercalated discs, with little signal at the lateral sarcolemma;in contrast, Cx40was located predominantly at the lateral sarcolemma, butthere was still a certain amount of signal at the intercalated discs.
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) overexpression of ACE2attenuated cardiacfibrosisour;(iii) the present study demonstrated that, for the first time,overexpression of ACE2could improve ion channels and connexinsremodeling induced by chronic rapid atrial pacing in dogs
In conclusion, our results demonstrate that overexpression of ACE2byhomogeneous transmural atrial gene transfer could decreased theinducibility and duration of AF, the mechanism of which are associatedwith shifting the RAS balance towards the protective axis, attenuateing cardiac fibrosis, and improving ion channels and connexins remodelinginduced by chronic rapid atrial pacing in dogs.
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