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Cardioprotective Effects Mediated by Angiotensin II Type 1 Receptor Blockade and Enhancing Angiotensin 1-7 in Experimental Heart Failure in Angiotensin-Converting Enzyme 2–Null Mice

Originally publishedhttps://doi.org/10.1161/HYPERTENSIONAHA.112.191650Hypertension. 2012;59:1195–1203

Abstract

Loss of angiotensin (Ang)-converting enzyme 2 (ACE2) and inability to metabolize Ang II to Ang 1-7 perpetuate the actions of Ang II after biomechanical stress and exacerbate early adverse myocardial remodeling. Ang receptor blockers are known to antagonize the effect of Ang II by blocking Ang II type 1 receptor (AT1R) and also by upregulating the ACE2 expression. We directly compare the benefits of AT1R blockade versus enhancing Ang 1-7 action in pressure-overload–induced heart failure in ACE2 knockout mice. AT1R blockade and Ang 1-7 both resulted in marked recovery of systolic dysfunction in pressure-overloaded ACE2-null mice. Similarly, both therapies attenuated the increase in NADPH oxidase activation by downregulating the expression of Nox2 and p47phox subunits and also by limiting the p47phox phosphorylation. Biomechanical stress-induced increase in protein kinase C-α expression and phosphorylation of extracellular signal–regulated kinase 1/2, signal transducer and activator of transcription 3, Akt, and glycogen synthase kinase 3β were normalized by irbesartan and Ang 1-7. Ang receptor blocker and Ang 1-7 effectively reduced matrix metalloproteinase 2 activation and matrix metalloproteinase 9 levels. Ang II–mediated cellular effects in cultured adult cardiomyocytes and cardiofibrolasts isolated from pressure-overloaded ACE2-null hearts were inhibited to similar degree by AT1R blockade and stimulation with Ang 1-7. Thus, treatment with the AT1R blocker irbesartan and Ang 1-7 prevented the cardiac hypertrophy and improved cardiac remodeling in pressure-overloaded ACE2-null mice by suppressing NADPH oxidase and normalizing pathological signaling pathways.

Introduction

Several lines of experimental and clinical evidence implicate a key role for the renin-angiotensin system in the pathophysiology of a number of cardiovascular diseases, such as myocardial infarction, hypertension, and heart failure.1,2 Angiotensin II (Ang II), acting via the Ang II type 1 receptor (AT1R) and Ang II type 2 receptor, modulates production of reactive oxygen species (ROS), impairing myocardial contractility and extracellular matrix remodeling, thereby negatively impacting on heart function.3 Angiotensin-converting enzyme 2 (ACE2), a homologue of angiotensin-converting enzyme, is a monocarboxypeptidase that metabolizes Ang II to yield angiotensin 1-7 (Ang 1-7) and lowers the Ang II/Ang 1-7 ratio.49 Ang II receptor blockers that selectively antagonize the AT1R became a valid alternative approach to interfere with the renin-angiotensin system axis and also upregulates ACE2, resulting in the generation of Ang 1-7.10,11 Ang 1-7 acts on the Mas receptor and plays an important role in counteracting the actions of Ang II.1218

Ang II–mediated oxidative stress, cardiac hypertrophy, contractile dysfunction, and fibrosis are exacerbated in ACE2-deficient mice,5,7 whereas recombinant human ACE2 is able to attenuate these responses and improve cardiac function, with a marked reversal of Ang II–mediated signaling.6 Many of the cardiac pathological effects of renin-angiotensin system activation and Ang II appear to be mediated through ROS, produced by a specific NADPH oxidase-dependent pathway in an AT1R-dependent manner.19,20 Also involved are a battery of prohypertrophic signaling pathways with downstream activation of matrix metalloproteinase (MMPs).3,6 We hypothesized that the cardiac phenotype in ACE2-null mice can be rescued by switching off the Ang II-AT1R axis and/or switching on the Ang 1-7/Mas receptor axis as an integral dual counterregulatory system.

Methods

Detailed methods are available in the online-only Data Supplement.

Experimental Animals and Protocols

Ace2/y mutant mice that were backcrossed into the C57BL/6 background for ≥8 generations were used in the present study.5,6,21 All of the experiments were performed in accordance with institutional guidelines, the Canadian Council on Animal Care, and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Pressure-Overload

Young (8- to 9-week–old) male Ace2/y (ACE2KO) mice were subjected to pressure-overload as described previously.7,22 ACE2KO mice were treated with the AT1R blocker irbesartan (50 mg/kg per day)21 or the antioxidant and NADPH oxidase inhibitor apocynin (240 mg/kg per day)23 in the drinking water, and in parallel experiments, ACE2KO mice were implanted with miniosmotic pumps to deliver Ang 1-7 (24 μg/kg per hour; Bachem).7,14

Echocardiographic and Hemodynamic Measurements

Transthoracic echocardiography and hemodynamic measurements were performed in anesthetized mice as described previously.6

Isolation and Culture of Adult Cardiomyocytes and Cardiofibroblasts

Adult murine left ventricular (LV) cardiomyocytes and cardiofibroblasts from ACE2KO-2 Weeks AB mice were isolated and cultured as described previously.6 Irbesartan (1 μmol/L) or Ang 1-7 (Bachem; 100 nmol/L) was added to the cardiomyocytes and cardiofibroblasts for 20 minutes before exposure of Ang II (Sigma; 100 nmol/L).

Superoxide Assay and Dihydroethidium Fluorescence

The chemiluminescence lucigenin assay to measure NADPH oxidase activity and dihydroethidium fluorescence were performed as described previously.6,7

Taqman Real-Time PCR, Western Blot Analysis, and Gelatin Zymography

Taqman real-time PCR, Western blot analysis, and gelatin zymography were carried out as described previously.6,7 Assessment of phosphorylated p47phox was measured by immunoprecipitation, followed by Western blot, as described previously.7

Plasma and Myocardial Ang 1-7 Levels

Myocardial and plasma Ang 1-7 levels were measured at the Hypertension Core Laboratory (Wake Forest University, Winston-Salem, NC), as described previously.6,7

Histological Analysis and Immunofluorescence

LV fibrosis and cardiomyocyte hypertrophy were measured by Masson trichrome and Picrosirius red staining, as described previously.6 Isolated cardiofibroblasts were double-stained for α-smooth muscle actin (SMA) and vimentin, along with nuclear-staining with 4′,6-diamidino-2-phenylindole (Invitrogen) and visualized and imaged using fluorescence microscopy.

Statistical Analysis

All of the data are shown as mean±SEM. All of the statistical analyses were performed using SPSS software (Chicago, IL; version 10.1). The effects of genotype, irbesartan, and Ang 1-7 were evaluated using ANOVA followed by the Student-Neuman-Keuls test for multiple comparison testing, and comparison between 2 groups were made using the Student t test.

Results

AT1R Blockade and Ang 1-7 Supplementation Reversed the Adverse Myocardial Remodeling in Pressure-Overload ACE2-Deficient Hearts

Pressure-overload resulted in a hypertrophic response at 2 weeks, as assessed by morphometry, LV weight normalized to tibial length, and expression of hypertrophic markers compared with atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain in ACE2KO hearts (Figure 1). Pressure-overload resulted in increased LV wall thickness and LV end-diastolic dimension with lowered fractional shortening in ACE2KO hearts (Table S1, available in the online-only Data Supplement). The functional deterioration was confirmed by invasive hemodynamic measurement showing elevated LV end-diastolic pressure and reduced ±dP/dtmax (Table S1). Myocardial Ang II level is elevated and Ang 1-7 level is reduced in ACE2KO hearts subjected to pressure-overload.7 We tested the hypothesis that blocking AT1R using irbesartan or supplementing Ang 1-7 would prevent the deleterious effects of Ang II actions in vivo and improve cardiac function. Both AT1R blockade and Ang 1-7 supplementation resulted in less cardiac hypertrophy with marked improvement in heart function in ACE2KO mice receiving irbesartan or Ang 1-7 in response to 2 weeks of pressure-overload (Figure 1 and Table S1). Although treatment with irbesartan and Ang 1-7 resulted in a significant lowering of systolic blood pressure, the proximal aortic pressure was equivalent in all of the experimental groups (Figure S1). We confirmed that systemic delivery of Ang 1-7 elevated plasma Ang 1-7 levels with a mild elevation seen in response to AT1R blockade without changes in myocardial Ang 1-7 levels (Figure S2). These results show that both AT1R blockade and Ang 1-7 supplementation have equivalent protective effects against adverse myocardial remodeling in an ACE2-null environment.

Figure 1.

Figure 1. Angiotensin (Ang) II type 1 receptor (AT1R) blockade and Ang 1-7 therapy reduce cardiac hypertrophy, based on morphometry and expression analysis of ANF and BNP in ACE2KO mice subjected to pressure-overload. Both treatments prevented an increase in left ventricular dilation and hypertrophy based on Masson trichrome-stained short axis views (A), LVW normalized to tibial length (B), cardiac ANF (C), BNP (D), and β-MHC (E) mRNA expression seen in ACE2KO mice at 2 weeks postbanding. n=8 from sham-operated; n=10 for all other groups. Sham indicates sham-operated; AB, aortic banding; IRB, irbesartan; Ang 1-7, angiotensin 1-7; LVW, left ventricular weight; TL, tibial length; ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; β-MHC, β-myosin heavy chain; R.E., relative expression. #P<0.05 vs all other groups.

Suppression of NADPH Oxidase, Pathological Signaling, and MMP Axis by AT1R Blockade and Enhanced Ang 1-7 Signaling

NADPH oxidase activation and enhanced oxidative stress are common features of the pathological effects of Ang II.3,24,25 The antioxidant and NADPH oxidase inhibitor apocynin suppressed the increased myocardial NADPH oxidase activity and superoxide production in pressure-overloaded ACE2KO hearts (Figure 2A and 2B), resulting in suppression of brain natriuretic peptide expression and improvement in heart function (Figure 2C through 2E). AT1R blockade and Ang 1-7 supplementation effectively mitigated NADPH oxidase activation and ROS production measured by dihydroethidium fluorescence (Figure 2F and 2G), which correlated with reduction in the expression of NADPH oxidase subunits p47phox and NOX2 (gp91phox; Figure 2H and 2I). Furthermore, immunoprecipitation followed by Western blotting indicated that there is a significant increase in phosphorylated-p47phox levels with increased gp91phox protein levels in pressure-overloaded ACE2KO hearts, which were effectively blocked by treatment with irbesartan and Ang 1-7 (Figure 2J and 2K).

Figure 2.

Figure 2. Treatment with apocynin, irbesartan, and angiotensin 1-7 inhibits NADPH oxidase and superoxide generation and reverses the adverse remodeling in angiotensin-converting enzyme 2 knockout (ACE2KO) mice subjected to pressure-overload. Apocynin inhibited NADPH oxidase activity (A) and reactive oxygen species formation assessed by dihydroethidium (DHE) fluorescence (B), resulting in normalized brain natriuretic peptide (BNP) expression (C) and recovery of systolic dysfunction (D and E). Ang II type 1 receptor (AT1R) blockade and Ang 1-7 supplementation inhibit NADPH oxidase activity (F), reactive oxygen species production (G), mRNA expression of p47phox (H) and NOX2 (gp91phox; I), serine phosphorylation of p47phox determined by immunoprecipitation (J). and expression of gp91phox protein (K) in ACE2KO hearts subjected to pressure-overload. In J, the 2 lanes on the right include a lane without antibody, followed by the last lane with the isotypic control antibody where we detected the IgG heavy chain but not p47phox. n=4 from each group. FS indicates fractional shortening; LVEDD, left ventricular end diastolic dimension; A.U., arbitrary units; R.E., relative expression; gp91 tat, gp91phox ds tat peptide; scr tat, scrambled gp91phox ds tat peptide. #P<0.05 vs all other groups.

Ang II–mediated activation of AT1R leads to a cascade of molecular events, including activation of the extracellular signal–regulated kinase (ERK) 1/2, signal transducer and activator of transcription 3 (STAT3), and phosphatidylinositol 3-kinase signaling pathways.3,6,7 Both irbesartan and Ang 1-7 completely blocked the activation of ERK1/2 (Figure 3A) and STAT3 (Figure 3B), suggesting that AT1R blockade or Mas receptor activation was capable of suppressing pathological signaling. We then examined the role of irbesartan and Ang 1-7 in regulating the activity of the phosphatidylinositol 3-kinase/Akt pathway. During cardiac hypertrophy, glycogen synthase kinase 3β (serine 9) is phosphorylated by Akt, leading to suppression of its kinase activity.26 We found that pressure-overloaded ACE2KO hearts exhibit increased phosphorylation of Akt and glycogen synthase kinase 3β, which was blocked by irbesartan and Ang 1-7 (Figure 3C and 3D). We hypothesized that there might be a common upstream target that regulates the activity of these pathways. Indeed, protein kinase C (PKC)-α is capable of regulating the activity of ERK1/2 and Akt pathways in response to Ang II,27,28 whereas Ang II–induced activation of NADPH oxidase involves PKC-α–induced phosphorylation of p47phox and its translocation to the membrane.3 Pressure-overload in ACE2KO hearts increased PKC-α expression by 2-fold compared with sham control, and this effect was prevented by irbesartan and Ang 1-7 (Figure 3E).

Figure 3.

Figure 3. Suppression of pathological signaling in pressure-overloaded angiotensin (Ang)-converting enzyme 2 knockout (ACE2KO) hearts by irbesartan and Ang 1-7. Western blot analysis showing increased phosphorylation of ERK 1/2 (A), STAT3 (B), Akt (C), glycogen synthase kinase (GSK-3β; D), and increased expression of PKC-α (E) in ACE2KO AB vs sham controls and attenuation of activation of these signaling pathways by Ang II type 1 receptor (AT1R) blockade and Ang 1-7 supplementation. Distinct lines refer to the sham-operated, placebo, irbesartan (IRB), and Ang 1-7 groups, respectively. n=4 from each group. R.E. indicates relative expression; ERK 1/2, extracellular signal-regulated kinase 1/2; STAT3, signal transducer and activator of transcription 3; GSK-3β, glycogen synthase kinase 3β; PKC-α, protein kinase C-α. #P<0.05 vs all other groups.

Enhanced formation of ROS is linked to the activation of MMPs and degradation of key components of the extracellular matrix.29,30 Gelatin zymography showed greater activation of the MMP2 system with higher levels of the active MMP2, in addition to increase MMP9 levels. Both pro- and active-MMP2 and -MMP9 levels returned to normal levels as in he sham control, on exposure to irbesartan and Ang 1-7 (Figure 4A through 4C). These results show that AT1R blockade and Mas receptor activation suppress MMP expression. Picrosirius red staining revealed marked hypertrophy and disorganization of the extracellular matrix, which was prevented by treatment with irbesartan and Ang 1-7 (Figure 4D and 4E), suggesting both cardiomyocyte and extracellular matrix–dependent effects.

Figure 4.

Figure 4. Suppression of matrix metalloprotease (MMP) activation by irbesartan (IRB) and angiotensin (Ang) 1-7 with normalization of the disorganized myocardial extracellular matrix in pressure-overloaded angiotensin-converting enzyme 2 knockout (ACE2KO) myocardium. Gelatin zymography showing greater cleavage and activation of MMP2 (A), which is quantified and shown (B) with greater pro-MMP9 levels (C) in the ACE2KO myocardium after pressure-overload. Picrosirius red staining showing normal extracellular matrix in ACE2KO hearts at baseline, whereas pressure-overload led to a disorganized extracellular matrix and cardiomyocyte hypertrophy (D). These changes were prevented by treatment with IRB and Ang 1-7 (D) with myocyte cross-sectional area shown quantitatively (E). n=4 from each group. #P<0.05 vs all other groups. B, □, pro-MMP2; ▩, MMP2.

Cellular Effects of AT1R Blockade and Ang 1-7 Supplementation in Cardiomyocytes and Cardiofibroblasts

To gain further insight into the beneficial effects of AT1R blockade and Ang 1-7 effects, we isolated and cultured adult cardiomyocytes and cardiofibroblasts from pressure-overloaded ACE2KO hearts. Acute stimulation of cardiomyocytes with Ang II (100 nmol/L) resulted in a marked increase in NADPH oxidase activity and superoxide formation with increased phosphorylation of ERK1/2 (Figure 5A through 5C). In cardiofibroblasts, Ang II stimulation increased the expression of α-SMA (Figure 5D) with activation of the ERK1/2 signaling pathway (Figure 5E), resulting in increased accumulation of α-SMA, a well-accepted marker of activated fibroblasts (Figure 5F). In both cell types, irbesartan and Ang 1-7 were equally efficacious at preventing the Ang II–mediated changes (Figure 5). These results demonstrate that the adverse remodeling in pressure-overloaded ACE2-deficient hearts is mediated by a combination of pathological effects of Ang II on cardiomyocytes and cardiofibroblasts that can be effectively blocked by either AT1R antagonism or treatment with Ang 1-7.

Figure 5.

Figure 5. Irbesartan (IRB) and angiotensin (Ang) 1-7 suppresses oxidant stress, signaling, and remodeling induced by Ang II in ACE2-null cardiomyocytes and cardiofibroblasts isolated from pressure-overloaded hearts. Ang II increases oxidant stress, as evident by increased NADPH oxidase activity (A) and dihydroethidium (DHE) fluorescence (B), along with phosphorylation of ERK 1/2 (C) in isolated cardiomyocytes, which were lowered by IRB and Ang 1-7 pretreatment. In cardiofibroblasts, Ang II exposure stimulated mRNA expression of α-SMA (D) with increased phosphorylation of ERK1/2 (E), resulting in increased immunostaining for α-SMA (F), which was prevented by pretreatment with IRB and Ang 1-7 (D through F). n=4 from each group. A.U. indicates arbitrary unit; R.E., relative expression; ERK 1/2, extracellular signal-regulated kinase 1/2; α-SMA, α-smooth muscle actin. #P<0.05 vs all other groups.

Discussion

Genetic and functional loss of ACE2 is associated with an age-dependent cardiomyopathy,4,5 adverse myocardial remodeling in response to myocardial infarction,21,31 and pressure-overload7,32 and worsens Ang II–induced cardiac dysfunction.6 The enhanced susceptibility to heart disease in relation to a loss of ACE2 correlates with elevated Ang II and lowered Ang 1-7 levels in the heart.6,7,21 Recombinant human ACE2 attenuates Ang II–induced diastolic dysfunction by lowering the Ang II:Ang 1-7 ratio.6 Collectively, these results shown that ACE2 plays a key role in metabolizing Ang II into Ang 1-7 in vivo. The Ang II/AT1R axis is a well-known trigger of heart disease,3,33,34 whereas Ang 1-7/Mas is cardioprotective and antagonizes Ang II effects.1218 Thus, it is conceivable that blocking the action of Ang II at the AT1R or enhancing the endogenous levels of Ang 1-7 would result in a reduction of cardiac hypertrophy and improve heart function. In this study, we showed that blocking Ang II/AT1R or enhancing Ang 1-7/Mas action resulted in a marked reduction in the pathological effects in the ACE2KO hearts, illustrating a marked degree of redundancy between these counterregulatory pathways. Treatment with AT1R blocker and Ang 1-7 supplementation increased plasma Ang 1-7 levels, which is consistent with previous studies showing that a systemic increase in Ang 1-7 levels and/or increased Mas receptor expression are cardioprotective.13,14,16 We also showed these effects in isolated cardiomyocytes and cardiofibroblasts, showing a clear cellular basis for the phenotypic rescue in ACE2-deficient hearts independent of blood pressure.

Treatment with irbesartan and Ang 1-7 blocked NADPH oxidase activation, thereby attenuating superoxide formation. This was associated with reduced expression of p47phox and gp91phox and inhibition of p47phox phosphorylation, resulting in reduced NADPH oxidase activation and superoxide formation. Apocynin, an antioxidant and NADPH oxidase inhibitor,23 ameliorates the pressure-overload–induced dysfunction in ACE2-null mice, confirming a key role of the NADPH oxidase pathway. Functional interactions exist between Mas and AT1R in the heart and vasculature, wherein the complex formation between Mas and AT1R is inhibitory to AT1R function.16,17,35 Pressure-overload in ACE2KO hearts activated PKC/Akt and ERK1/2 pathways. Interestingly, inhibition of PKC activation observed with AT1R blockade and Ang 1-7 supplementation paralleled changes in NADPH oxidase activity and blunted the hypertrophic response with attenuated activation of both ERK1/2 and Akt. The cardioprotection of AT1R blocker and Ang 1-7 is mediated by interruption of PKC-α–dependent NADPH oxidase activation, resulting in lowered activation of ERK1/2, Akt, glycogen synthase kinase 3β, and attenuation of cardiac hypertrophy. Biomechanical stress and Ang-II–induced generation of ROS are known to activate MMP2 in a p47phox-dependent manner.29,36 Reductions in pro- and active-MMP2 and -MMP9 levels by AT1R blockade and Ang 1-7 represent important mediators of the protective effects of these therapies on heart function.

Treatment with an AT1R blocker after coronary artery ligation demonstrated an increase in cardiac ACE2 mRNA levels and activity, suggesting that AT1R blocker can partially increase the ACE2 mRNA and formation of Ang 1-7.10 In our model, we demonstrated that AT1R blockade can afford cardioprotection independent of ACE2. Upregulation of the Mas receptor may also contribute toward the cardioprotective effects mediated by AT1R blocker, whereas also facilitating the action of Ang 1-7 in a positive feedback manner.16,17 Ang 1-7 attenuates the development of heart failure postmyocardial infarction14,15 and in response to pressure-overload.12,13 Ang 1-7 also antagonizes the Ang II–induced myocardial hypertrophy and fibrosis.18 Enhancing Ang 1-7 action using the Mas agonist AVE0991 reduces postmyocardial infarction remodeling and cardiac dysfunction.37 The beneficial effects of Ang 1-7 are not limited to cardiomyocytes and include important effects on cardiofibroblasts, such as antifibrotic and antihypertrophic effects associated with reduced ERK1/2 activity.38,39 These data further suggest that hypertrophy may result from increased action of Ang II and/or loss of Ang 1-7 effects, and supplementation of Ang 1-7 may prove to be effective therapy for pressure-overload–induced heart failure, where the ACE2 system is known to be downregulated.6

Perspectives

The present study demonstrates that AT1R blocker and Ang 1-7 treatment provide comparable cardioprotection in pressure-overload–induced hypertrophy and heart failure in ACE2-null mice. Importantly, the doses of irbesartan and Ang 1-7 tested achieved similar improvement in the structural and functional phenotypes. Our study unveiled a marked degree of redundancy in the Ang II/AT1R and Ang 1-7/Mas receptor counterregulatory pathways in the absence of ACE2. Irbesartan is known to upregulate ACE2 mRNA and Mas receptor expression, which may contribute to the cardioprotection in several experimental models of cardiovascular disease. The Ang 1-7/Mas receptor axis plays a key role in cardioprotection against pressure-overload–induced hypertrophy and heart failure, where the ACE2 system is known to be downregulated. This suggests a possible role for Ang 1-7 in human heart failure, and Ang 1-7 can prove to be a potential therapeutic agent. However, redundancy of AT1R blocker and Ang 1-7 treatment in a wild-type background is unknown and is an important consideration for the translational aspect of Ang 1-7 treatment.

Acknowledgments

G.Y.O. is a clinician-investigator of the Alberta Innovates-Health Solutions and the Distinguish Clinician Scientist of the Heart and Stroke Foundation of Canada and Canadian Institutes of Health Research.

Sources of Funding

We acknowledge the financial support from the Canadian Institute for Health Research (grant 86602 to G.Y.O.) and Alberta Innovates-Health Solutions (to G.Y.O., S.B., and J.Z.).

Disclosures

None.

Footnotes

The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.112.191650/-/DC1.

Correspondence to Gavin Y. Oudit,
Division of Cardiology, Department of Medicine, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
. E-mail

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