Effects Of Angiotensin1-9and Caveolin-1on Atrial Remodeling

Backgroud:The angiotensin-converting enzyme2(ACE2) system, a newly discovered and important rennin-angiotensin system (RAS) member, counterbalances the effect of the traditional RAS system and plays a protective role in heart disease pathophysiology. ACE2is the homolog of ACE which hydrolyzes angiotensin (Ang) II and Ang I to Angl-7and Ang1-9, respectively. Several reports demonstrated that the ACE2/Angl-7/Mas axis exerts protective actions in the pathophysiology of heart diseases and inhibits many detrimental cardiovascular disease phenotypes.In contrast to Angl-7, there has been scarce research on the effect of Ang1-9on the cardiovascular system, because it was initially thought to have no biological activity and to be active only after conversion to Angl-7. However, recent studies subverted the above point and clarified that, as Ang1-7, Ang1-9is a counter-regulator of the RAS system. Ang1-9was reported to block left cardiac hypertrophy in a rat myocardial infarction model and to attenuate ventricular fibrosis in the stroke-prone spontaneously hypertensive rat, indicating that Ang1-9is an active endogenous factor with cardiovascular protective effects. To date, the role of Ang1-9on atrial remodeling remains poorly understood. The present study therefore investigated the effect of Angl-9on atrial remodeling in the Ang II-induced hypertension rat model.Aims:1. To study the effect of angiotensin-(1-9) on the atrial remodeling in vivo.2. To study the effect of angiotensin-(1-9) on the atrial caridomycytes--HL-1cells and atrial fibroblasts in vitro.3. To study the mechanism of angiotensin-(1-9) affecting the atrial remodeling.Materials and Methods:1. Animal Experimental Protocols Eighty male Wistar rats (213.3±7.6g) were housed in cages with ad libitum access to rat chow and water. Rats were randomly assigned to sham, Ang Ⅱ, Angl-9and A779groups. There were no differences in body weight and systolic blood pressure among the four groups before treatment onset. Hypertension and cardiac remodeling were induced by chronic subcutaneous infusion of Ang Ⅱ (100ng/kg per min) delivered for4weeks by osmotic minipump (model2ML4, Alzet, Cupertino, CA). In the sham, Angl-9and A779groups, osmotic minipumps contained0.9%saline, Ang Ⅱ (100ng/kg per min)+Angl-9(450ng/kg per min), or a combination of Ang Ⅱ (100ng/kg per min), Ang1-9(450ng/kg per min) and A779(100ng/kg per min), respectively. Each treatment group included6rats, and no rodents died during the experimental treatment. Indirect blood pressure was recorded before and after4weeks treatment by tail-cuff plesthymography. Echocardiography was performed after4weeks treatment under anesthesia. Images were obtained from two-dimensional, M-mode, pulsed-wave Doppler and tissue Doppler imaging. After echocardiography, hearts were removed by blunt dissection; the atria were separated from the ventricles and discarded, and the heart ventricles including both the LV and right ventricle (RV) as well as the interventricular septum were weighed. A section of left atrial tissue from each rat was immediately frozen in liquid nitrogen for RNA and protein isolation and an adjacent cross section was fixed in10%neutralized formalin for histopathology analysis.2. Cell cultureHL-1atrial myocytes were kindly donated by Dr. Claycomb. Rat atrial fibroblasts were derived from8week-old Wistar rats, and cells of passages2-4were used. For fibroblasts and HL-1cells, serum was withdrawn24h before preincubation with1μM Angl-9for90min, after which100nM Ang II was added. The effects of A779[a mas antagonist for Angl-7] and PD123319[an angiotensin type2(AT2) receptor antagonist for Ang1-9] were evaluated by preincubating the cells with A779(500nM) or PD123319(500nM) for30min before Angl-9was added. The cultures were incubated for24h for qRT-PCR analysis or for48h for western blot assays.Results:1. Angl-9attenuates Ang Ⅱ-induced atrial remodeling in vivoChronic infusion of Ang Ⅱ caused a significant increase in systolic blood pressure and heart beat in rats compared with saline infused animals, which was unchanged by co-infusion of Ang1-9and A779. And after4weeks treatment, body weight increased in all four groups compared with baseline weight; the latter increase was mumerically lower in the Ang Ⅱ group, however, differences were not significantly different among treatments (P=0.197). Additionally, ventricular weights were similar among all four groups (P=0.663). The calculated heart weight-to-body weight ratio was significantly higher in the Ang Ⅱ than in the sham group, while the ratio was lower in the Angl-9and A779groups compared to the Ang Ⅱ group. Left atrial diameter in rats in the Ang Ⅱ, Ang1-9, and A779groups was not different from that in saline-infused animals.Infusion of Ang Ⅱ markedly increased ANP and BNP relative atrial gene expression compared with atria of animals infused with saline (P=0.007and0.015, respectively). Concomitant treatment with Angl-9significantly attenuated the increase in both ANP and BNP mRNA levels compared with infusion of Ang II alone (P=0.007and0.029, respectively). Co-treatment with A779had no significant effect on relative ANP or BNP mRNA expression in vivo compared with that in rats infused with Ang1-9+AngⅡ.Masson-trichrome-stained left-atrium cross sections showed chronic infusion of Ang II caused a marked increase in atrial interstitial fibrosis compared with saline-infused animals. The increase in interstitial fibrosis with Ang Ⅱ infusion was significantly attenuated by the co-administration of Ang1-9. Consistent with the result of masson-trichrome stained atrial tissue, the amount of fibrotic markers (TGF-β1and collagen I) in atrial tissue of Ang II-treated rats was markedly greater than that of rats in the sham group. The amount of fibrotic markers was lower in Angl-9-treated rats compared to Ang Ⅱ-treated rats, and A779did not affect the suppressive effect on atrial fibrosis of Angl-9in vivo.2. Angl-9attenuates effects of Ang Ⅱ on HL-1cells and atrial fibroblatsAng II elevated ANP expression by2.9-fold compared to the control group in HL-1cells (P=0.009). This effect was significantly prevented by Angl-9. A779incubation had no effect on the Ang1-9-dependent suppression of ANP expression while co-administration of PD123319with Angl-9eliminated the suppressive effect.Isolated primary cultures of neonatal atrial fibroblasts were used to confirm direct effects of Angl-9on atrial fibroblasts. Co-administration of Angl-9(1μM) inhibited Ang Ⅱ-induced increases in TGF-β1and collagen I production.However, the addition of PD123319to Ang1-9-stimulated fibroblasts reversed the anti-fibrotic effect of Angl-9while A779did not, which indicates that Angl-9mediates antiproliferative effects on atrial fibroblasts via the AT2R. Meanwhile, Angl-9inhibited the Ang Ⅱ-induced activation of ERK signal pathway.To evaluate collagen degradation, we assessed the effects of Angl-9on expression of two principal matrix metalloproteinase collagenases (MMP-1and MMP-9), which degrade the extracellular matrix. The results showed that Angl-9attenuated the ability of Ang Ⅱ to downregulate MMP-1and MMP-9. PD123319abolished the effect of Angl-9on upregulation of MMPs expression, whereas A779did not.Conclusion1. Angiotensin-(1-9) is an endogenous active factor.2. Angiotensin-(1-9) could reversed angiotensin Ⅱ-induced adverse atrial structural remodeling in vivo and in vitro.3.Angiotensin-(1-9) plays its protective roles via AT2R and MARK-ERK signal pathway. Backgroud:Caveolae are50-to100-nm omega-shaped invaginations of the cytoplasmic membrane, which were first reported as early as the middle of the last century. Over the past decade, the study on caveolae has blossomed into a rapidly expanding field, and caveolae have been identified as an important member participating in the transcytosis of macromolecules, cholesterol transport and signal transduction in various types of cells. Caveolin-1is the first member of the caveolae gene family consisting of three structurally related proteins:caveolin-1(Cav-1), caveolin-2(Cav-2), and caveolin-3(Cav-3). Cav-1is the principal structural component of caveolae organelles cells.In the cardiovascular system, Cav-1and Cav-2are co-expressed in a variety of cells and tissue types but are most abundantly present in fibroblasts and endothelial cells, whereas Cav-3is strictly expressed in cardiomyocytes.Atrial fibrillation(AF), the most common cardiac arrhythmia, is frequently accompanied by atrial interstitial fibrosis. A plethora of studies in animal models of atrial fibrillation (AF) and clinical AF have verified that AF is associated with progressive atrial structural and electrical remodeling. During the development of cardiac fibrosis, the transforming growth factor-β1(TGF-β1) is considered to be the key profibrotic cytokine. Cav-1-knockout animals displayed enhanced TGF-β1signaling activities, as reflected by more widespread collagen deposition accompanied by reduced expression of matrix metalloproteinases MMP-8and MMP-13mRNAs in the heart. This would imply that Cav-1can cause subordinate alterations in cardiac structure and function by regulating cardiac fibrosis.In view of all these findings, we were prompted to propose a hypothesis that Cav-1might produce an anti-AF effect by participating in the atrial structural remodeling process through its anti-fibrotic action. The carboxyl tail of Cav-1, or the Cav-1scaffolding domain (CSD; residues82-101in Cav-1), is the primary structure that interacts with other molecules. The peptides derived from CSD (the CSD peptide) are able to elicit the same cellular functions as Cav-1, which is fully cell permeable and has been widely used as a mimic of the full-length Cav-1in studies of variety cellular functions associated with Cav-1, indicating the peptide is a superior gain-of-function tool for studying the function of Cav-1. Therefore, basing on results of our research on changes in the atrial tissue of AF, an in vitro study was conducted to examine our hypothesis whether Cav-1could reverse the pathological atrial structural remodeling in patients with AF by using a gain-of-function approach with the CSD peptide and a loss-of-function approach with siRNA.Aims:1. To study changes of Cav-1in atrial fibrosis during atrial fibrillation2. To study the mechanism of changes of Cav-1in patients with AF.3. To study the effect of CSD petides on atrial fibriosis.Materials and Methods:1. PatientsThe subjects included102patients,54females and48males, with rheumatic heart disease undergoing mitral/aortic valve replacement from January11,2012to June30,2013, with the age of52.58±11.0(range from29to76years old). Fifty-four were diagnosed with AF (AF group) and48with no history of AF (sinus rhythm (SR) group). After written informed consent was obtained, atrial tissue samples from the right atrial appendage were obtained from all subjects. The samples were then used for primary culturing of human atrial fibroblasts, or frozen and stored at-80℃for RNA and protein extraction for qRT-PCR and Western blot, respectively, or fixed by10%neutralized formalin for histopathology analysis.2. Cell cultureHuman atrial fibroblasts (HAFs) were derived from biopsies of the right atrial appendage. Cells of passages2-4were plated in6-well plates. Serum was withdrawn24h before incubation with100ng/ml recombinant human TGF-|31for48h. HAFs were also pretreated for30min with the CSD peptide (5μM) or the scrambled peptide (Scr peptide;5μM), and subsequently incubated for48h with or without100ng/ml TGF-β1. Cells were then harvested from each experimental group for Western blot analysis or qRT-PCR. 3. siRNA transfectionHAFs were plated in6-well plates with80%final density, and transfected with caveolin-1siRNA (sense5’-GCCGUGUCUAUUCCA UCUA-3’; antisense5’-UAGAUGGAAU AGACACGGC-3’) or a non-silencing negative control siRNA (sense5’-UUCUCCGAACGUGU CACGU-3’; antisense5’-ACGUGACACGUUC GGAGAA-3’) using Hiperfect transfection reagent (Introgen) following protocols provided by the manufacturer. After48h of incubation the cells were either harvested for protein extraction or treated with TGF-β1as described above.4. Statistical analysisResults were presented as mean±SE. Between group differences were determined using one-way ANOVA followed by Fisher’s protected least significant difference (Fisher’s PLSD) tests. The statistical tests were two-tailed, with a p-value of<0.05for significance. The statistical analysis was performed with SPSS16.0software (SPSS, Chicago, IL).Results:1. Downregulation of atrial Cav-1and increase of atrial fibrosis in AF patientsThere were no significant differences between the SR and AF group in sex, age, types of valve disease, ejection fraction and degrees of cardiac function. Left atrial diameter in AF group was bigger than that in SR group (59.4±10.5vs.47.8±4.9P=0.004).Western blot and qRT-PCR analyses showed the protein level of Cav-1was approximately30%lower in AF subjects than in the SR group (P<0.001). The Cav-1mRNA was also downregulated, even to a greater extent, in AF relative to SR (P<0.001).The protein level of transforming growth factor beta1(TGF-β1) was significantly elevated (-2.5fold) in AF patients relative to SR subjects (P<0.001). And the statistics showed a negative correlation between TGF-beta1level and Cav-1level (r=0.75, P=0.012). Meanwhile, there was much higher percentage of fibrotic tissues, as indicated by the more widespread areas stained with Masson-trichrome, in AF than in SR tissues. Furthermore, the level of collagen I, the main component of the extracellular matrix in cardiac fibrosis, and collagen III were markedly greater in atrial tissues from AF patients than from SR subjects.2. Downregulation of Cav-1by TGF-β1in HAFsHAFs were incubated with TGF-β1of a fixed concentration of100ng/ml for48h, the results showed that Cav-1was reduced by42%(P=0.004).3. Enhanced TGF-β1induced-Smad signal pathway and collagen production by knock-down caveolin-1with siRNA in HAFsThe siRNA of caveolin-1could suppressed the expression of caveolin-1. The downregulation of caveolin-1subsequently increased the amount of phosphorylated Smad2and Smad3and the expression of collagens in HAFs.4. Inhibition of TGF-β1induced-collagen production and the activation of Smad signal pathway by the CSD peptide in HAFsThe CSD peptide inhibited the TGF-β1-induced activation of Smad2/3and pronouncedly reduced the TGF-β1-induced increase in production of collagens by78%. By comparison, the Scr peptide (5μM), as a negative control, did not alter the effects of TGF-β1.The CSD peptide (5μM) attenuated TGF-β1induced-decrease of MMPs, whereas the Scr peptide did not significantly affect the changes of MMPs induced by TGF-β1.Conculsion:Our data suggested that Cav-1is an important regulator of AF and a promising therapeutic target for AF. However, this in vitro study merely lays the groundwork for future studies on the role of Cav-1in AF. Additional studies are warranted to confirm the results.