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。
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-β1
In 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.
引文
[1]. Zhang S. Atrial fibrillation in mainland China: epidemiology and currentmanagement. Heart,2009.95(13):1052-5.
[2]. Zhou Z, Hu D. An epidemiological study on the prevalence of atrial fibrillationin the Chinese population of mainland China. J Epidemiol,2008.18(5):209-16.
[3]. Ball J, Carrington MJ, McMurray JJ. et al. Atrial fibrillation: Profile andburden of an evolving epidemic in the21st century. Int J Cardiol,2013.
[4]. McManus DD, Rienstra M, Benjamin EJ. An update on the prognosis ofpatients with atrial fibrillation. Circulation,2012.126(10): e143-6.
[5]. Lafuente-Lafuente C, Longas-Tejero MA, Bergmann JF. et al. Antiarrhythmicsfor maintaining sinus rhythm after cardioversion of atrial fibrillation. CochraneDatabase Syst Rev,2012.5: CD005049.
[6]. Lee G, Sanders P, Kalman JM. Catheter ablation of atrial arrhythmias: state ofthe art. Lancet,2012.380(9852):1509-19.
[7]. Shah RU, Freeman JV, Shilane D. et al. Procedural complications,rehospitalizations, and repeat procedures after catheter ablation for atrial fibrillation. JAm Coll Cardiol,2012.59(2):143-9.
[8]. Casaclang-Verzosa G, Gersh BJ, Tsang TS. Structural and functionalremodeling of the left atrium: clinical and therapeutic implications for atrial fibrillation.J Am Coll Cardiol,2008.51(1):1-11.
[9]. Bunch TJ, Munger TM, Friedman PA. et al. Substrate and proceduralpredictors of outcomes after catheter ablation for atrial fibrillation in patients withhypertrophic cardiomyopathy. J Cardiovasc Electrophysiol,2008.19(10):1009-14.
[10]. Cohen M, Naccarelli GV. Pathophysiology and disease progression of atrialfibrillation: importance of achieving and maintaining sinus rhythm. J CardiovascElectrophysiol,2008.19(8):885-90.
[11]. Casaclang-Verzosa G, Gersh BJ, Tsang TS. Structural and functionalremodeling of the left atrium: clinical and therapeutic implications for atrial fibrillation.J Am Coll Cardiol,2008.51(1):1-11.
[12]. Teh AW, Kistler PM, Lee G. et al. Long-term effects of catheter ablation forlone atrial fibrillation: progressive atrial electroanatomic substrate remodeling despitesuccessful ablation. Heart Rhythm,2012.9(4):473-80.
[13]. Dobrev D, Nattel S. New insights into the molecular basis of atrial fibrillation:mechanistic and therapeutic implications. Cardiovasc Res,2011.89(4):689-91.
[14]. Ehrlich JR, Hohnloser SH, Nattel S. Role of angiotensin system and effects ofits inhibition in atrial fibrillation: clinical and experimental evidence. Eur Heart J,2006.27(5):512-8.
[15]. Goette A, Lendeckel U. Electrophysiological effects of angiotensin II. Part I:signal transduction and basic electrophysiological mechanisms. Europace,2008.10(2):238-41.
[16]. Kostenis E, Milligan G, Christopoulos A. et al. G-protein-coupled receptorMas is a physiological antagonist of the angiotensin II type1receptor. Circulation,2005.111(14):1806-13.
[17]. Dias-Peixoto MF, Santos RA, Gomes ER. et al. Molecular mechanismsinvolved in the angiotensin-(1-7)/Mas signaling pathway in cardiomyocytes.Hypertension,2008.52(3):542-8.
[18]. Costa MA, Lopez VM, Gomez KA. et al. Angiotensin-(1-7) upregulatescardiac nitric oxide synthase in spontaneously hypertensive rats. Am J Physiol HeartCirc Physiol,2010.299(4): H1205-11.
[19]. Zhong J, Basu R, Guo D. et al. Angiotensin-converting enzyme2suppressespathological hypertrophy, myocardial fibrosis, and cardiac dysfunction. Circulation,2010.122(7):717-28,18p following728.
[20]. Gwathmey TM, Pendergrass KD, Reid SD. et al.Angiotensin-(1-7)-angiotensin-converting enzyme2attenuates reactive oxygen speciesformation to angiotensin II within the cell nucleus. Hypertension,2010.55(1):166-71.
[21]. Mercure C, Yogi A, Callera GE. et al. Angiotensin(1-7) blunts hypertensivecardiac remodeling by a direct effect on the heart. Circ Res,2008.103(11):1319-26.
[22]. Giani JF, Munoz MC, Mayer MA. et al. Angiotensin-(1-7) improves cardiacremodeling and inhibits growth-promoting pathways in the heart of fructose-fed rats.Am J Physiol Heart Circ Physiol,2010.298(3): H1003-13.
[23]. Oudit GY, Kassiri Z, Patel MP. et al. Angiotensin II-mediated oxidative stressand inflammation mediate the age-dependent cardiomyopathy in ACE2null mice.Cardiovasc Res,2007.75(1):29-39.
[24]. Santos RA, Castro CH, Gava E. et al. Impairment of in vitro and in vivo heartfunction in angiotensin-(1-7) receptor MAS knockout mice. Hypertension,2006.47(5):996-1002.
[25]. Alenina N, Xu P, Rentzsch B. et al. Genetically altered animal models for Masand angiotensin-(1-7). Exp Physiol,2008.93(5):528-37.
[26]. Pan CH, Lin JL, Lai LP. et al. Downregulation of angiotensin convertingenzyme II is associated with pacing-induced sustained atrial fibrillation. FEBS Lett,2007.581(3):526-34.
[27]. Liu E, Yang S, Xu Z. et al. Angiotensin-(1-7) prevents atrial fibrosis and atrialfibrillation in long-term atrial tachycardia dogs. Regul Pept,2010.162(1-3):73-8.
[28]. Muller OJ, Katus HA, Bekeredjian R. Targeting the heart with genetherapy-optimized gene delivery methods. Cardiovasc Res,2007.73(3):453-62.
[29]. Katz MG, Swain JD, White JD. et al. Cardiac gene therapy: optimization ofgene delivery techniques in vivo. Hum Gene Ther,2010.21(4):371-80.
[30]. Su CH, Wu YJ, Wang HH. et al. Nonviral gene therapy targetingcardiovascular system. Am J Physiol Heart Circ Physiol,2012.303(6): H629-38.
[31].崔坤、范晋奇、考国营等,经犬心房外膜涂布实现外源基因在心房肌(细胞)的高效转染.中国生物化学与分子生物学报,2011,12:1167-1173.
[32]. Kikuchi K, McDonald AD, Sasano T. et al. Targeted modification of atrialelectrophysiology by homogeneous transmural atrial gene transfer. Circulation,2005.111(3):264-70.
[33]. Igarashi T, Finet JE, Takeuchi A. et al. Connexin gene transfer preservesconduction velocity and prevents atrial fibrillation. Circulation,2012.125(2):216-25.
[34]. Soucek R, Thomas D, Kelemen K. et al. Genetic suppression of atrialfibrillation using a dominant-negative ether-a-go-go-related gene mutant. Heart Rhythm,2012.9(2):265-72.
[35]. Zhong J, Guo D, Chen CB. et al. Prevention of Angiotensin II-Mediated RenalOxidative Stress, Inflammation, and Fibrosis by Angiotensin-Converting Enzyme2.Hypertension,2011.57(2):314-22.
[36]. Ferreira AJ, Santos RA, Almeida AP. Angiotensin-(1-7): cardioprotectiveeffect in myocardial ischemia/reperfusion. Hypertension,2001.38(3Pt2):665-8.
[37]. De Mello WC, Ferrario CM, Jessup JA. Beneficial versus harmful effects ofAngiotensin (1-7) on impulse propagation and cardiac arrhythmias in the failing heart. JRenin Angiotensin Aldosterone Syst,2007.8(2):74-80.
[38]. De Mello WC. Angiotensin (1-7) reduces the cell volume of swollen cardiaccells and decreases the swelling-dependent chloride current. Implications for cardiacarrhythmias and myocardial ischemia. Peptides,2010.31(12):2322-4.
[39]. Liu E, Xu Z, Li J. et al. Enalapril, irbesartan, and angiotensin-(1-7) preventatrial tachycardia-induced ionic remodeling. Int J Cardiol,2011.146(3):364-70.
[40]. Donoghue M, Wakimoto H, Maguire CT. et al. Heart block, ventriculartachycardia, and sudden death in ACE2transgenic mice with downregulated connexins.J Mol Cell Cardiol,2003.35(9):1043-53.
[41]. Aldhoon B, Melenovsky V, Peichl P. et al. New insights into mechanisms ofatrial fibrillation. Physiol Res,2010.59(1):1-12.
[42]. Gu J, Liu X, Wang QX. et al. Angiotensin II increases CTGF expression viaMAPKs/TGF-beta1/TRAF6pathway in atrial fibroblasts. Exp Cell Res,2012.318(16):2105-15.
[43]. Willems R, Sipido KR, Holemans P. et al. Different patterns of angiotensin IIand atrial natriuretic peptide secretion in a sheep model of atrial fibrillation. JCardiovasc Electrophysiol,2001.12(12):1387-92.
[44]. Li D, Shinagawa K, Pang L. et al. Effects of angiotensin-converting enzymeinhibition on the development of the atrial fibrillation substrate in dogs with ventriculartachypacing-induced congestive heart failure. Circulation,2001.104(21):2608-14.
[45]. Tsai CT, Lin JL, Lai LP. et al. Membrane translocation of small GTPase Rac1and activation of STAT1and STAT3in pacing-induced sustained atrial fibrillation.Heart Rhythm,2008.5(9):1285-93.
[46]. Michael G, Xiao L, Qi XY. et al. Remodelling of cardiac repolarization: howhomeostatic responses can lead to arrhythmogenesis. Cardiovasc Res,2009.81(3):491-9.
[47]. Li Y, Li WM, Gong YT. et al. The effects of cilazapril and valsartan on themRNA and protein expressions of atrial calpains and atrial structural remodeling inatrial fibrillation dogs. Basic Res Cardiol,2007.102(3):245-56.
[48]. Iwai M, Horiuchi M. Role of the ACE2/angiotensin1-7/Mas axis in thecardiovascular system. Hypertens Res,2010.33(11):1108-9.
[49]. Castro-Chaves P, Cerqueira R, Pintalhao M. et al. New pathways of therenin-angiotensin system: the role of ACE2in cardiovascular pathophysiology andtherapy. Expert Opin Ther Targets,2010.14(5):485-96.
[50]. Grobe JL, Mecca AP, Lingis M. et al. Prevention of angiotensin II-inducedcardiac remodeling by angiotensin-(1-7). Am J Physiol Heart Circ Physiol,2007.292(2):H736-42.
[51]. Ferreira AJ, Shenoy V, Qi Y. et al. Angiotensin-converting enzyme2activation protects against hypertension-induced cardiac fibrosis involvingextracellular signal-regulated kinases. Exp Physiol,2011.96(3):287-94.
[52]. Liu E, Yang S, Xu Z. et al. Angiotensin-(1-7) prevents atrial fibrosis and atrialfibrillation in long-term atrial tachycardia dogs. Regul Pept,2010.162(1-3):73-8.
[53]. Liu E, Xu Z, Li J. et al. Enalapril, irbesartan, and angiotensin-(1-7) preventatrial tachycardia-induced ionic remodeling. Int J Cardiol,2011.146(3):364-70.
[54]. Kourliouros A, Savelieva I, Kiotsekoglou A. et al. Current concepts in thepathogenesis of atrial fibrillation. Am Heart J,2009.157(2):243-52.
[55]. Schotten U, Verheule S, Kirchhof P. et al. Pathophysiological mechanisms ofatrial fibrillation: a translational appraisal. Physiol Rev,2011.91(1):265-325.
[56]. Lin CS, Pan CH. Regulatory mechanisms of atrial fibrotic remodeling in atrialfibrillation. Cell Mol Life Sci,2008.65(10):1489-508.
[57]. Iwasaki YK, Nishida K, Kato T. et al. Atrial fibrillation pathophysiology:implications for management. Circulation,2011.124(20):2264-74.
[58]. Ferreira AJ, Bader M, Santos RA. Therapeutic targeting of theangiotensin-converting enzyme2/Angiotensin-(1-7)/Mas cascade in therenin-angiotensin system: a patent review. Expert Opin Ther Pat,2012.22(5):567-74.
[59]. Iwata M, Cowling RT, Yeo SJ. et al. Targeting the ACE2-Ang-(1-7) pathwayin cardiac fibroblasts to treat cardiac remodeling and heart failure. J Mol Cell Cardiol,2011.51(4):542-7.
[60]. Tikellis C, Bernardi S, Burns WC. Angiotensin-converting enzyme2is a keymodulator of the renin-angiotensin system in cardiovascular and renal disease. CurrOpin Nephrol Hypertens,2011.20(1):62-8.
[61]. Der Sarkissian S, Grobe JL, Yuan L. et al. Cardiac overexpression ofangiotensin converting enzyme2protects the heart from ischemia-inducedpathophysiology. Hypertension,2008.51(3):712-8.
[62]. Der Sarkissian S, Grobe JL, Yuan L. et al. Cardiac overexpression ofangiotensin converting enzyme2protects the heart from ischemia-inducedpathophysiology. Hypertension,2008.51(3):712-8.
[63]. Gu J, Liu X, Wang QX. et al. Angiotensin II increases CTGF expression viaMAPKs/TGF-beta1/TRAF6pathway in atrial fibroblasts. Exp Cell Res,2012.318(16):2105-15.
[64]. Ma F, Li Y, Jia L. et al. Macrophage-stimulated cardiac fibroblast productionof IL-6is essential for TGF beta/Smad activation and cardiac fibrosis induced byangiotensin II. PLoS One,2012.7(5): e35144.
[65]. He X, Gao X, Peng L. et al. Atrial fibrillation induces myocardial fibrosisthrough angiotensin II type1receptor-specific Arkadia-mediated downregulation ofSmad7. Circ Res,2011.108(2):164-75.
[66]. Pan CH, Lin JL, Lai LP. et al. Downregulation of angiotensin convertingenzyme II is associated with pacing-induced sustained atrial fibrillation. FEBS Lett,2007.581(3):526-34.
[67]. Kitamura K, Shibata R, Tsuji Y. et al. Eicosapentaenoic acid prevents atrialfibrillation associated with heart failure in a rabbit model. Am J Physiol Heart CircPhysiol,2011.300(5): H1814-21.
[68]. Li D, Shinagawa K, Pang L. et al. Effects of angiotensin-converting enzymeinhibition on the development of the atrial fibrillation substrate in dogs with ventriculartachypacing-induced congestive heart failure. Circulation,2001.104(21):2608-14.
[69]. Li D, Shinagawa K, Pang L. et al. Effects of angiotensin-converting enzymeinhibition on the development of the atrial fibrillation substrate in dogs with ventriculartachypacing-induced congestive heart failure. Circulation,2001.104(21):2608-14.
[70]. Ferreira AJ, Murca TM, Fraga-Silva RA. et al. New cardiovascular andpulmonary therapeutic strategies based on the Angiotensin-converting enzyme2/angiotensin-(1-7)/mas receptor axis. Int J Hypertens,2012.2012:147825.
[71]. Huang CY, Tan TH. DUSPs, to MAP kinases and beyond. Cell Biosci,2012.2(1):24.
[72]. Dauletbaev N, Eklove D, Mawji N. et al. Down-regulation of cytokine-inducedinterleukin-8requires inhibition of p38mitogen-activated protein kinase (MAPK) viaMAPK phosphatase1-dependent and-independent mechanisms. J Biol Chem,2011.286(18):15998-6007.
[73]. Olson AK, Protheroe KN, Segar JL. et al. Mitogen-activated protein kinaseactivation and regulation in the pressure-loaded fetal ovine heart. Am J Physiol HeartCirc Physiol,2006.290(4): H1587-95.
[74]. Valente AJ, Yoshida T, Gardner JD. et al. Interleukin-17A stimulates cardiacfibroblast proliferation and migration via negative regulation of the dual-specificityphosphatase MKP-1/DUSP-1. Cell Signal,2012.24(2):560-8.
[75]. McCollum LT, Gallagher PE, Tallant EA. Angiotensin-(1-7) abrogatesmitogen-stimulated proliferation of cardiac fibroblasts. Peptides,2012.34(2):380-8.
[76]. Choudhary R, Palm-Leis A, Scott RR. et al. All-trans retinoic acid preventsdevelopment of cardiac remodeling in aortic banded rats by inhibiting therenin-angiotensin system. Am J Physiol Heart Circ Physiol,2008.294(2): H633-44.
[77]. Palm-Leis A, Singh US, Herbelin BS. et al. Mitogen-activated protein kinasesand mitogen-activated protein kinase phosphatases mediate the inhibitory effects ofall-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem,2004.279(52):54905-17.
[1]. Tatemoto K, Hosoya M, Habata Y. et al. Isolation and characterization of a novelendogenous peptide ligand for the human APJ receptor. Biochem Biophys ResCommun,1998.251(2):471-6.
[2]. Maguire JJ, Kleinz MJ, Pitkin SL. et al.[Pyr1]apelin-13identified as thepredominant apelin isoform in the human heart: vasoactive mechanisms andinotropic action in disease. Hypertension,2009.54(3):598-604.
[3]. Chen MM, Ashley EA, Deng DX. et al. Novel role for the potent endogenousinotrope apelin in human cardiac dysfunction. Circulation,2003.108(12):1432-9.
[4]. O'Dowd BF, Heiber M, Chan A. et al. A human gene that shows identity with thegene encoding the angiotensin receptor is located on chromosome11. Gene,1993.136(1-2):355-60.
[5]. Tatemoto K, Hosoya M, Habata Y. et al. Isolation and characterization of a novelendogenous peptide ligand for the human APJ receptor. Biochem Biophys ResCommun,1998.251(2):471-6.
[6]. Kawamata Y, Habata Y, Fukusumi S. et al. Molecular properties of apelin: tissuedistribution and receptor binding. Biochim Biophys Acta,2001.1538(2-3):162-71.
[7]. Hosoya M, Kawamata Y, Fukusumi S. et al. Molecular and functionalcharacteristics of APJ. Tissue distribution of mRNA and interaction with theendogenous ligand apelin. J Biol Chem,2000.275(28):21061-7.
[8]. Japp AG, Newby DE. The apelin-APJ system in heart failure: pathophysiologicrelevance and therapeutic potential. Biochem Pharmacol,2008.75(10):1882-92.
[9]. Quazi R, Palaniswamy C, Frishman WH. The emerging role of apelin incardiovascular disease and health. Cardiol Rev,2009.17(6):283-6.
[10]. Ishida J, Hashimoto T, Hashimoto Y. et al. Regulatory roles for APJ, aseven-transmembrane receptor related to angiotensin-type1receptor in bloodpressure in vivo. J Biol Chem,2004.279(25):26274-9.
[11]. Burrell LM, Johnston CI, Tikellis C. et al. ACE2, a new regulator of therenin-angiotensin system. Trends Endocrinol Metab,2004.15(4):166-9.
[12]. Kalea AZ, Batlle D. Apelin and ACE2in cardiovascular disease. Curr Opin InvestigDrugs,2010.11(3):273-82.
[13]. Vickers C, Hales P, Kaushik V. et al. Hydrolysis of biological peptides by humanangiotensin-converting enzyme-related carboxypeptidase. J Biol Chem,2002.277(17):14838-43.
[14]. Akcilar R, Turgut S, Caner V. et al. Apelin Effects on Blood Pressure and RAS inDOCA-Salt-Induced Hypertensive Rats. Clin Exp Hypertens,2013.
[15]. Barnes GD, Alam S, Carter G. et al. Sustained Cardiovascular Actions of APJAgonism During Renin-Angiotensin System Activation and in Patients with HeartFailure. Circ Heart Fail,2013.
[16]. Kazemi-Bajestani SM, Patel VB, Wang W. et al. Targeting the ACE2and ApelinPathways Are Novel Therapies for Heart Failure: Opportunities and Challenges.Cardiol Res Pract,2012.2012:823193.
[17]. Ishida J, Hashimoto T, Hashimoto Y. et al. Regulatory roles for APJ, aseven-transmembrane receptor related to angiotensin-type1receptor in bloodpressure in vivo. J Biol Chem,2004.279(25):26274-9.
[18]. Ashley E, Chun HJ, Quertermous T. Opposing cardiovascular roles for theangiotensin and apelin signaling pathways. J Mol Cell Cardiol,2006.41(5):778-81.
[19]. Iwanaga Y, Kihara Y, Takenaka H. et al. Down-regulation of cardiac apelin systemin hypertrophied and failing hearts: Possible role of angiotensin II-angiotensin type1receptor system. J Mol Cell Cardiol,2006.41(5):798-806.
[20]. Fukushima H, Kobayashi N, Takeshima H. et al. Effects of olmesartan onApelin/APJ and Akt/endothelial nitric oxide synthase pathway in Dahl rats withend-stage heart failure. J Cardiovasc Pharmacol,2010.55(1):83-8.
[21]. Chun HJ, Ali ZA, Kojima Y. et al. Apelin signaling antagonizes Ang II effects inmouse models of atherosclerosis. J Clin Invest,2008.118(10):3343-54.
[22]. Siddiquee K, Hampton J, Khan S. et al. Apelin protects against angiotensinII-induced cardiovascular fibrosis and decreases plasminogen activator inhibitortype-1production. J Hypertens,2011.29(4):724-31.
[23]. Sun X, Iida S, Yoshikawa A. et al. Non-activated APJ suppresses the angiotensin IItype1receptor, whereas apelin-activated APJ acts conversely. Hypertens Res,2011.34(6):701-6.
[24]. Siddiquee K, Hampton J, McAnally D. et al. The apelin receptor inhibits theangiotensin II type1receptor via allosteric trans-inhibition. Br J Pharmacol,2013.168(5):1104-17.
[25]. Lee DK, Cheng R, Nguyen T. et al. Characterization of apelin, the ligand for theAPJ receptor. J Neurochem,2000.74(1):34-41.
[26]. Tatemoto K, Takayama K, Zou MX. et al. The novel peptide apelin lowers bloodpressure via a nitric oxide-dependent mechanism. Regul Pept,2001.99(2-3):87-92.
[27]. Cheng X, Cheng XS, Pang CC. Venous dilator effect of apelin, an endogenouspeptide ligand for the orphan APJ receptor, in conscious rats. Eur J Pharmacol,2003.470(3):171-5.
[28]. Katugampola SD, Maguire JJ, Matthewson SR. et al.[(125)I]-(Pyr(1))Apelin-13isa novel radioligand for localizing the APJ orphan receptor in human and rat tissueswith evidence for a vasoconstrictor role in man. Br J Pharmacol,2001.132(6):1255-60.
[29]. Berry MF, Pirolli TJ, Jayasankar V. et al. Apelin has in vivo inotropic effects onnormal and failing hearts. Circulation,2004.110(11Suppl1): II187-93.
[30]. Szokodi I, Tavi P, Foldes G. et al. Apelin, the novel endogenous ligand of theorphan receptor APJ, regulates cardiac contractility. Circ Res,2002.91(5):434-40.
[31]. Farkasfalvi K, Stagg MA, Coppen SR. et al. Direct effects of apelin oncardiomyocyte contractility and electrophysiology. Biochem Biophys Res Commun,2007.357(4):889-95.
[32]. Kuba K, Zhang L, Imai Y. et al. Impaired heart contractility in Apelingene-deficient mice associated with aging and pressure overload. Circ Res,2007.101(4): e32-42.
[33]. Ashley EA, Powers J, Chen M. et al. The endogenous peptide apelin potentlyimproves cardiac contractility and reduces cardiac loading in vivo. Cardiovasc Res,2005.65(1):73-82.
[34]. Scimia MC, Hurtado C, Ray S. et al. APJ acts as a dual receptor in cardiachypertrophy. Nature,2012.488(7411):394-8.
[35]. Falcao-Pires I, Goncalves N, Gavina C. et al. Correlation between plasma levels ofapelin and myocardial hypertrophy in rats and humans: possible target for treatment?Expert Opin Ther Targets,2010.14(3):231-41.
[36]. Foldes G, Horkay F, Szokodi I. et al. Circulating and cardiac levels of apelin, thenovel ligand of the orphan receptor APJ, in patients with heart failure. BiochemBiophys Res Commun,2003.308(3):480-5.
[37]. Chong KS, Gardner RS, Morton JJ. et al. Plasma concentrations of the novelpeptide apelin are decreased in patients with chronic heart failure. Eur J Heart Fail,2006.8(4):355-60.
[38]. Przewlocka-Kosmala M, Kotwica T, Mysiak A. et al. Reduced circulating apelin inessential hypertension and its association with cardiac dysfunction. J Hypertens,2011.29(5):971-9.
[39]. van Kimmenade RR, Januzzi JJ, Ellinor PT. et al. Utility of amino-terminalpro-brain natriuretic peptide, galectin-3, and apelin for the evaluation of patientswith acute heart failure. J Am Coll Cardiol,2006.48(6):1217-24.
[40]. Francia P, Salvati A, Balla C. et al. Cardiac resynchronization therapy increasesplasma levels of the endogenous inotrope apelin. Eur J Heart Fail,2007.9(3):306-9.
[41]. Pchejetski D, Foussal C, Alfarano C. et al. Apelin prevents cardiac fibroblastactivation and collagen production through inhibition of sphingosine kinase1. EurHeart J,2012.33(18):2360-9.
[42]. Chandrasekaran B, Dar O, McDonagh T. The role of apelin in cardiovascularfunction and heart failure. Eur J Heart Fail,2008.10(8):725-32.
[43]. Lee DK, George SR, O'Dowd BF. Unravelling the roles of the apelin system:prospective therapeutic applications in heart failure and obesity. TrendsPharmacol Sci,2006.27(4):190-4.
[44]. Li L, Zeng H, Chen JX. Apelin-13increases myocardial progenitor cells andimproves repair postmyocardial infarction. Am J Physiol Heart Circ Physiol,2012.303(5): H605-18.
[45]. Katugampola SD, Kuc RE, Maguire JJ. et al. G-protein-coupled receptors in humanatherosclerosis: comparison of vasoconstrictors (endothelin and thromboxane) withrecently de-orphanized (urotensin-II, apelin and ghrelin) receptors. Clin Sci (Lond),2002.103Suppl48:171S-175S.
[46]. Hashimoto T, Kihara M, Imai N. et al. Requirement of apelin-apelin receptorsystem for oxidative stress-linked atherosclerosis. Am J Pathol,2007.171(5):1705-12.
[47]. Chun HJ, Ali ZA, Kojima Y. et al. Apelin signaling antagonizes Ang II effects inmouse models of atherosclerosis. J Clin Invest,2008.118(10):3343-54.
[48]. Erdem G, Dogru T, Tasci I. et al. Low plasma apelin levels in newly diagnosed type2diabetes mellitus. Exp Clin Endocrinol Diabetes,2008.116(5):289-92.
[49]. Tasci I, Erdem G, Ozgur G. et al. LDL-cholesterol lowering increases plasma apelinin isolated hypercholesterolemia. Atherosclerosis,2009.204(1):222-8.
[50]. Kadoglou NP, Sailer N, Kapelouzou A. et al. Effects of atorvastatin on apelin,visfatin (nampt), ghrelin and early carotid atherosclerosis in patients with type2diabetes. Acta Diabetol,2012.49(4):269-76.
[51]. Tasci I, Dogru T, Naharci I. et al. Plasma apelin is lower in patients with elevatedLDL-cholesterol. Exp Clin Endocrinol Diabetes,2007.115(7):428-32.
[52]. Pitkin SL, Maguire JJ, Kuc RE. et al. Modulation of the apelin/APJ system in heartfailure and atherosclerosis in man. Br J Pharmacol,2010.160(7):1785-95.
[53]. Kadoglou NP, Fotiadis G, Kapelouzou A. et al. The differential anti-inflammatoryeffects of exercise modalities and their association with early carotid atherosclerosisprogression in patients with Type2diabetes. Diabet Med,2013.30(2): e41-50.
[54]. Ellinor PT, Low AF, Macrae CA. Reduced apelin levels in lone atrial fibrillation.Eur Heart J,2006.27(2):222-6.
[55]. Kallergis EM, Manios EG, Kanoupakis EM. et al. Effect of sinus rhythm restorationafter electrical cardioversion on apelin and brain natriuretic Peptide prohormonelevels in patients with persistent atrial fibrillation. Am J Cardiol,2010.105(1):90-4.
[56]. Falcone C, Buzzi MP, D'Angelo A. et al. Apelin plasma levels predict arrhythmiarecurrence in patients with persistent atrial fibrillation. Int J ImmunopatholPharmacol,2010.23(3):917-25.
[57]. Cheng CC, Weerateerangkul P, Lu YY. et al. Apelin regulates theelectrophysiological characteristics of atrial myocytes. Eur J Clin Invest,2013.43(1):34-40.
[58]. Galanth C, Hus-Citharel A, Li B. et al. Apelin in the control of body fluidhomeostasis and cardiovascular functions. Curr Pharm Des,2012.18(6):789-98.
[59]. Barnes G, Japp AG, Newby DE. Translational promise of the apelin--APJ system.Heart,2010.96(13):1011-6.
[60]. Falcao-Pires I, Ladeiras-Lopes R, Leite-Moreira AF. The apelinergic system: apromising therapeutic target. Expert Opin Ther Targets,2010.14(6):633-45.
