Overexpression Of ACE2 Effects The Canine Atrial Remodeling Induced By Atrial Rapid Pacing

Background:Atrial fibrillation(AF) is the most commonly encountered arrhythmia in clinical practice, which is difficult to treat and easy to recur, becoming a pandemic in human cardiovascular disease. Various disorders, such as ischemic, valvular, inflammatory, degenerative heart diseases and hypertension predispose to atrial fibrillation. However, the current treatment of atrial fibrillation is not satisfactory.A plenty of studies have demonstrated that the renin angiotensin system(RAS) was activated in different degrees in the occurrence and maintenance of atrial fibrillation. Angiotensin II(Ang II), the most important effector of RAS, with the functions of contracting vessels, promoting fibrosis and cardiac hypertrophy, can be converted into angiotensin-(1-7) [Ang-(1-7)] by angiotensin converting enzyme 2(ACE2), which is considered as a protective effector. Recent studies showed that overexpression of ACE2 could antagonize the effect of Ang II-induced atrial remodeling. Atrial remodeling is the basic mechanism of the occurrence and maintenance of atrial fibrillation, thus improving atrial matrix becomes the basic link to prevent and treat atrial fibrillation. Studies have shown that Rac1 might conduce to cardiac fibrosis and atrial remodeling by up-regulating CTGF, N-cadherin and connexin 43. Rad(Ras associated with diabetes) GTPase could inhibite the transcription of CTGF in the heart, and modulate cytoskeleton remodeling through the Rho/Rhokinase pathway. However, the dynamic balance of Rac1 and Rad GTPase in atrial fibrillation has not been established, and the mechanism of ACE2 overexpression on how to improve atrial remodeling needs further investigate. Objective:This study was to establish canine rapid atrial pacing models to investigate whether overexpression of ACE2 by transferring gene to atrial epicardium could improve the rapid atrial pacing induced atrial remodeling and their molecular mechanism. Method:Twenty-eight mongrel healthy canines(weighing 22 ± 2 Kg, either male or female), were divided randomly into four groups: Sham group(sham-operated), AF-Control group(rapid pacing), AF-EGFP group(rapid pacing and gene therapy with Ad-EGFP) and AF-ACE2 group(rapid pacing and gene therapy with Ad-ACE2)(n=7 per group). Only the dogs in the AF-Control, AF-EGFP and AF-ACE2 groups were instrumented with a pacemaker and paced at 450 bpm. Two weeks later, all dogs underwent the first thoracotomy operation and invasive electrophysiology(EP) study. Then only the dogs in the AF-EGFP and AF-ACE2 groups received the gene-painting procedure as previously described. After three weeks, canines received the second thoracotomy operation and EP study. Atrial tissue was harvested for histology and molecular studies as following: Atrial tissue was stained by hematoxylin-eosin and picrosirius red to investigate the potential pathologic matrix abnormalities in rapid-pacing canines. To evaluate the effect of ACE2 overexpression on RAS components, the expression levels of AT1 R, AT2 R, Apelin and Aplnr were detected by western blot analysis. Further, to estimate the mechanism of ACE2 overexpression improving rapid-pacing induced atrial fibrosis and atrial remodeling, the expression levels of Rac1、Rad GTPase and Gem were measured by western blot and immunohistochemistry. The expression of connective tissue growth factor(CTGF) 、 α-Smooth muscle actin(α-SMA) and N-Cadherin were assayed by western blot, and the location and expression of α-SMA were also detected using immunohistochemistry fluorescence method. c-jun、c-myc and c-fos mRNA expression were measured by real-time RT-PCR. Results:Compared with the different data observed in twice electrophysiology studies, the result showed that in the second EP study, the atrial effective refractory period(AERP) was decreased marketly in the AF-Control, AF-EGFP and AF-ACE2 groups, and the rate adaptation of AERP was also reduced significantly. Whereas, the AERP of Sham group dogs had no significant change. Via gene transferring for three weeks, the duration and the inducibility of AF increased marketly in the AF-Control and AF-EGFP groups, while those in the AF-ACE2 group were not increased significantly compared with the Sham group.Hematoxylin-eosin staining was used to observe cellular morphologic change. There was no obvious hemorrhage, pericardial inflammation or effusion in canines. In the Sham group, atrial myocytes presented a normal composition of sarcomeres distributed and the integrality of collagen throughout the cell. On the contrary, myocytes in the AF-Control and AF-EGFP groups presented abnormal sarcomeres, a loss of contractile materials and the intra-cellular space also appeared abnormal. However, the abnormalities were lightened in the AF-ACE2 group dogs. Meanwhile, the images of picrosirius red staining showed that extensive interstitial fibrosis was observed in the AF-Control and AF-EGFP group dogs, and fibrosis was significantly stronger than the Sham and AF-ACE2 group dogs. Quantitative analysis demonstrated that the area of collagen in the AF-ACE2 group was significantly lower than the AF-Control group and AF-EGFP groups(P<0.0001), and had no significant difference compared with the Sham group(P = 0.234).The expression levels of RAS components were measured by western blot. The results showed that AT1 R protein expression was significant higher in the Ad-EGFP and AF-Control groups compared with the Sham and AF-ACE2 groups, while AT2 R protein expression was decreased markedly in the Ad-EGFP and AF-Control groups. The expression levels of Apelin and Aplnr were similar in each group. They both decreased dramatically in the AF-EGFP and AF-Control groups, but ACE2 overexpression reversed the trend.The amount of Rac1 protein was significantly increased in the AF-Control and AF-EGFP groups compared with it in the Sham group, but it was decreased in the AF-ACE2 group when compared with the the AF-Control and AF-EGFP groups by western blot analysis(P<0.05). In contrast, the amount of Rad GTPase protein was dramatically higher in the AF-ACE2 group(P<0.0001), and the amount of Gem protein,who also belonging to the RKG family as Rad, was markedly lower in the AF-Control and AF-EGFP groups(P<0.0001). The result of semi-quantitative immunohistochemical analysis showed the protein expression levels of Rac1 and Gem were similar to the date of western blot analysis. α-SMA expression was reduced significantly in the Sham and AF-ACE2 groups compared with the AF-EGFP and AF-Control groups by western blot analysis(P<0.05). This tendency was also observed in the protein expression levels of CTGF and N-Cadherin(P<0.05, P<0.001, respectively). Further, the location and expression of α-SMA measured by immunohistochemistry fluorescence assay showed that α-SMA was mainly distributed in the cytoplasm, and a large number of green fluorescent cells were visible in the AF-EGFP and AF-Control groups compared with those in the Sham and AF-ACE2 groups. This was consistent with the result of western blot analysis. Meanwhile, compared to the Sham and AF-ACE2 groups, the mRNA expression levels of c-jun, c-myc and c-fos were also markedly higher in the AF-Control and AF-EGFP groups(P<0.05). Conclusion:Our study mainly demonstrated:(1) overexpression of ACE2 reduced the inducibility of atrial fibrillation and shortened its duration;(2) overexpression of ACE2 reduced the expression of AT1 R dramitically, and increased the expression of AT2 R, Apelin and Aplnr significantly, thus reversing the imbalance of RAS induced by atrial rapid pacing;(3) overexpression of ACE2 regulated the dynamic balance of Rac1 and Rad GTPase by down-regulating Rac1 expression and up-regulating Rad GTPase expression, inhibited the expression levels of fibrosis related factors as CTGF, α-SMA and N-Cadherin, reduced the mRNA expression of c-fos, c-jun and c-myc, so subsequently improved atrial fibrosis and remodeling.In conclusion, the results of our study manifested that overexpression of ACE2 on atrial epicardium could adjust the imbalance of RAS induced by atrial rapid pacing to the protective direction, down-regulate Rac1 expression and up-regulate Rad GTPase expression, and inhibit the expression levels of fibrosis related factors. Accordingly, this might ameliorate atrial remodeling and improve atrial matrix so as to effectively reduce the inducibility and shorten the duration of atrial fibrillation, ultimately benefit the prevention and treatment of atrial fibrillation.