Atrial Overexpression of ACE2Improves the Canine Rapid Atrial Pacing-induced Structural Remodeling
|School||Chongqing Medical University|
|Keywords||Atrial fibrillation Angiotensin-converting enzyme2 Genetherapy Atrial remodeling Renin angiotensin system|
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.