Angiotensin-Converting Enzyme 2 Suppresses Pathological Hypertrophy, Myocardial Fibrosis, and Cardiac Dysfunction
Background— Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)–mediated signaling and its adverse effects on the cardiovascular system.
Methods and Results— Ang II infusion (1.5 mg · kg−1 · d−1) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2−/y) mice compared with wild-type (WT) mice. Daily treatment of Ang II–infused wild-type mice with recombinant human ACE2 (rhACE2; 2 mg · kg−1 · d−1 IP) blunted the hypertrophic response and expression of hypertrophy markers and reduced Ang II–induced superoxide production. Ang II–mediated myocardial fibrosis and expression of procollagen type Iα1, procollagen type IIIα1, transforming growth factor-β1, and fibronectin were also suppressed by rhACE2. Ang II–induced diastolic dysfunction was inhibited by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1-7 levels. rhACE2 treatment inhibited Ang II–mediated activation of protein kinase C-α and protein kinase C-β1 protein levels and phosphorylation of the extracellular signal-regulated 1/2, Janus kinase 2, and signal transducer and activator of transcription 3 signaling pathways in wild-type mice. A subpressor dose of Ang II (0.15 mg · kg−1 · d−1) resulted in a milder phenotype that was strikingly attenuated by rhACE2 (2 mg · kg−1 · d−1 IP). In adult ventricular cardiomyocytes and cardiofibroblasts, Ang II–mediated superoxide generation, collagen production, and extracellular signal-regulated 1/2 signaling were inhibited by rhACE2 in an Ang 1-7–dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wild-type mice.
Conclusions— Elevated Ang II induced hypertension, myocardial hypertrophy, fibrosis, and diastolic dysfunction, which were exacerbated by ACE2 deficiency, whereas rhACE2 attenuated Ang II– and pressure-overload–induced adverse myocardial remodeling. Hence, ACE2 is an important negative regulator of Ang II–induced heart disease and suppresses adverse myocardial remodeling.
Received November 4, 2009; accepted June 14, 2010.
Activation of the renin-angiotensin system (RAS) and the subsequent generation of angiotensin (Ang) II are important mediators of myocardial fibrosis, pathological hypertrophy, and heart failure.1–3 Pathological hypertrophy and increased myocardial interstitial fibrosis contribute to increased ventricular wall stiffness, thereby impairing cardiac diastolic function, and represent an important risk factor for heart failure in experimental models and patients.4–6 Drugs that target Ang II and the Ang II type 1 receptor (AT1) are widely used for the treatment of cardiovascular diseases such as hypertension, myocardial infarction, and heart failure.7 Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing a range of peptide substrates, including Ang I, Ang II, des-Arg9-bradykinin, apelin-13, and opioids.8–11 Ang 1-7, one of the major enzymatic products of ACE2, has been shown to reduce Ang II–induced cardiac hypertrophy and remodeling and pressure-overload–induced heart failure.12,13 ACE2-deficient mice can develop impaired cardiac function with adverse ventricular remodeling, enhanced oxidative stress, and inflammatory cytokine expression.14,15
Clinical Perspective on p 728
In the present study, we directly assessed the hypothesis that ACE2 is a negative regulator of Ang II–mediated heart disease. Ang II–mediated oxidative stress, cardiac hypertrophy, and fibrosis are exacerbated in ACE2-deficient mice, which results in worsening of diastolic dysfunction. Recombinant human ACE2 (rhACE2) attenuated Ang II–mediated hypertension, ventricular hypertrophy, and fibrosis and improved diastolic dysfunction, with a marked reversal of Ang II–induced activation of pathological signaling pathways. In response to pressure overload, rhACE2 partially attenuated the adverse myocardial remodeling. These beneficial effects of rhACE2 correlated with the reduction in Ang II and increase in Ang 1-7 levels. We conclude that ACE2 plays a pivotal role in the inhibition of myocardial hypertrophy, fibrosis, and cardiac dysfunction, and rhACE2 represents a novel therapeutic strategy for cardiovascular disorders.
Please see the online-only Data Supplement for additional details.
Experimental Animals and Protocols
Mutant mice were backcrossed into a pure C57BL/6 background for >10 generations as described previously.15,16 Ten-week-old male ACE2 knockout (ACE2KO) mice (Ace2−/y) and their littermate wild-type (WT; Ace2+/y) mice were used. An osmotic minipump (model 1002, Alza Corp, Palo Alto, Calif) was implanted subcutaneously at the dorsum of the neck to infuse a pressor dose of Ang II (1.5 mg · kg−1 · d−1), a subpressor dose of Ang II (0.15 mg · kg−1 · d−1), or saline (vehicle) for 14 days. Ang II–infused WT mice were then treated with placebo or rhACE2 (2 mg · kg−1 · d−1 IP). All experiments were performed in accordance with institutional guidelines and the standards of the Canadian Council on Animal Care.
The aortic banding model was used to generate pressure-overload–induced heart failure in 8- to 9-week-old male C57/BL6 WT mice, as we have described previously.17 After 6 weeks of pressure overload, banded WT mice were randomized to receive rhACE2 (2 mg · kg−1 · d−1 IP) or placebo for 3 weeks.
Tail-Cuff Systolic Blood Pressure
Systolic blood pressure of each mouse was measured by the tail-cuff method with an IITC blood pressure monitoring system (IITC Life Science, Woodland Hills, Calif).
Echocardiography and Tissue Doppler Imaging
Transthoracic echocardiography was performed and analyzed in a blinded manner as described previously with a Vevo 770 high-resolution imaging system equipped with a 30-MHz transducer (RMV-707B; VisualSonics, Toronto, Canada).18
Trichrome and picrosirius red staining and visualization and analysis of collagen volume fraction were performed as described previously.17
TaqMan Real-Time Polymerase Chain Reaction and Western Blot Analysis
RNA expression levels of various genes and Western blot analyses were determined as described previously.15–17 The primers and probes for mRNA expression analysis by TaqMan (Applied Biosystems, Foster City, Calif) real-time polymerase chain reaction are listed in online-only Data Supplement Table I; 18S ribosomal RNA was used as the endogenous control. Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif), Cell Signaling Inc (Danvers, Mass), and R&D Systems (Minneapolis, Minn).15–17
Generation and Characterization of rhACE2
The extracellular domain of human ACE2 (amino acid residues 1 to 740, molecular weight 101 kDa)19 was expressed recombinantly in Chinese hamster ovary (CHO) cells as described previously (online-only Data Supplement).20
Angiotensin Peptide Measurement
Plasma Ang II and Ang 1-7 and myocardial Ang II levels were measured by radioimmunoassay in the Hypertension and Vascular Research Center core laboratory at Wake Forest University School of Medicine as described previously.14,16
Invasive Pressure-Volume Measurements
Invasive pressure-volume measurements were made in nonintubated and anesthetized mice (1% isoflurane mixed with 100% O2) with the SPR-869 microtip catheter and the Millar pressure-volume system MPVS-400 (Millar Instruments, Houston, Tex) as described previously21 at 3 to 4 hours after removal of the osmotic pumps. In aortic-banded mice, only invasive pressure measurements were obtained.
Isolation and Culture of Adult Cardiomyocytes and Cardiofibroblasts
Adult murine left ventricular (LV) cardiomyocytes and cardiofibroblasts were isolated and cultured as described previously.22
Dihydroethidium Fluorescence and Lucigenin-Enhanced Chemiluminescence
Dihydroethidium, an oxidative fluorescent dye, was used to measure superoxide (O2−) levels in cultured cardiomyocytes and heart tissues from ACE2KO and WT mice as described previously.16 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in cultured cardiomyocytes and hearts of mice was quantified by lucigenin-enhanced chemiluminescence as described previously.15,16
Results are shown as mean±SEM. Statistical analysis was performed with SPSS 11.5 software either by ANOVA followed by multiple-comparison testing (Student-Neuman-Keuls test) or the Fisher exact test as appropriate. P<0.05 was considered statistically significant.
Ang II–Induced Myocardial Hypertrophy and Fibrosis, Diastolic Dysfunction, and Oxidative Stress Are Exacerbated by the Loss of ACE2
To determine the regulatory role of ACE2 in Ang II–induced heart disease, we subjected ACE2KO (Ace2−/y) and WT (Ace2+/y) mice to a 14-day period of Ang II infusion (1.5 mg · kg−1 · d−1). Echocardiographic M-mode images (Figure 1A) and 2-dimensional short-axis midventricular views (Figure 1B) revealed normal systolic function with no change in fractional shortening, ejection fraction, or velocity of circumferential shortening between WT and ACE2KO mice (online-only Data Supplement Table II). Notably, there was greater concentric remodeling in the ACE2KO mice in response to chronic stimulation by Ang II, with a greater increase in ventricular wall thickness (Figures 1A and 1B; online-only Data Supplement Table II). Concentric remodeling of the LV resulted in diastolic dysfunction as assessed by transmitral Doppler filling and tissue Doppler imaging. In Ang II–treated WT mice, the transmitral A wave was increased (with a reduced E/A ratio) with reduced E′ (and increased E/E′ ratio; Figures 1C and 1D; online-only Data Supplement Table II). LV isovolumetric relaxation time was increased significantly, whereas the E′/A′ showed greater reduction, primarily due to marked elevation of the A′ wave in Ang II–treated ACE2KO mice compared with WT mice (Figures 1C and 1D; online-only Data Supplement Table II).
Interestingly, myocardial ACE2 protein level in WT mice was reduced by 60% in response to Ang II (Figure 1E), which may have facilitated Ang II–induced myocardial injury. Gravimetric analysis showed that Ang II infusion resulted in a greater increase in the heart weight–tibia length ratio and LV weight–tibia length ratio in ACE2KO than in WT mice (Figure 1F). Real-time polymerase chain reaction analysis revealed that loss of ACE2 augmented Ang II–induced mRNA expression of molecular markers of pathological hypertrophy, including brain natriuretic peptide (Figure 1G) and α-skeletal actin (Figure 1H), without having a differential effect on β-myosin heavy chain mRNA expression (Figure 1I) or protein levels (Figure 1J). Ang II–mediated NADPH oxidase activation and superoxide generation are pivotal mechanisms of Ang II–mediated injury in the cardiovascular system.3,23,24 Consistent with the exacerbation of Ang II pathological effects in ACE2KO mice, Ang II–induced superoxide production (Figures 2A and 2B) and NADPH oxidase activation (Figure 2B) were greater in ACE2KO mice than in WT mice, likely driven by the greater elevation in myocardial Ang II levels in ACE2KO mice (Figure 2C). Ang II is a well-known activator of increased tissue fibrosis via its profibrotic effects.1–3 Ang II resulted in greater mRNA expression of procollagen type Iα1 (Figure 2D), procollagen type IIIα1 (Figure 2E), fibronectin (Figure 2F), and transforming growth factor-β 1 (TGF-β1; Figure 2G) in ACE2KO mice than in WT mice. These molecular footprints of increased fibrosis were confirmed by histological analyses (Figures 2H and 2I) that showed greater interstitial and perivascular myocardial fibrosis in ACE2KO mice. The collagen volume fraction showed a greater increase in ACE2KO mice (15.9±2.1%; n=8) than in WT mice (9.1±1.8%; n=8; P<0.05) in response to Ang II. Collectively, these data show that loss of ACE2 increases myocardial Ang II levels and worsens concentric remodeling, which results in greater deterioration of diastolic dysfunction in response to exogenous Ang II.
Treatment With rhACE2 Attenuates Ang II–Mediated Cardiac Hypertrophy and Fibrosis and Pathological Signaling
The purity, molecular weight, and identity of rhACE2 were confirmed by high-performance liquid chromatography, SDS-PAGE, and Western blot analyses (online-only Data Supplement Figure I). Human ACE2 shares 65% homology with rodent ACE2 with a conserved catalytic site (online-only Data Supplement Figure II),14,19 and rhACE2 (1 μg/mL) produced marked and similar elevations in plasma ACE2 activity in murine and human plasma (online-only Data Supplement Figures IIIA and IIIB). Plasma ACE2 activity was markedly increased at 1 and 2 weeks after initiation of rhACE2 at 2 mg · kg−1 · d−1 IP in WT mice (online-only Data Supplement Figure IIIC). WT mice showed a marked increase in LV wall thickness (online-only Data Supplement Table III), as illustrated by M-mode imaging (Figure 3A) and a short-axis view (Figure 3B), without deterioration in systolic function (Figure 3A; online-only Data Supplement Table III), in response to Ang II. The primary functional abnormality in these mice was diastolic dysfunction characterized by increased A-wave amplitude (with resultant reduction in the E/A ratio) and reduced E′ (with resultant reduction in the E′/A′ ratio; Figures 3C and 3D; online-only Data Supplement Table III). The use of rhACE2 inhibited Ang II–induced hypertrophy on the basis of LV wall thickness (Figures 3A and 3B; online-only Data Supplement Table III), with reversal of diastolic dysfunction (Figures 3C and 3D; online-only Data Supplement Table III). Invasive pressure-volume hemodynamic assessment confirmed Ang II–induced diastolic dysfunction characterized by elevated LV end-diastolic pressure, increased slope of the end-diastolic pressure-volume relationship, and an increased LV relaxation time constant, which were all reduced by treatment with rhACE2 (online-only Data Supplement Figure IV).
Morphometric analyses further confirmed the marked antihypertrophic effects of rhACE2 against Ang II–induced hypertrophy (Figure 3E), with reduced expression of hypertrophy markers (Figures 3F through 3H). Normalization of increased β-myosin heavy chain mRNA expression was confirmed by Western blot analysis (Figure 3I). Both matrix metalloproteinase-2 and ADAM12 (a disintegrin and metalloprotease 12), which have been linked to agonist-mediated hypertrophy, were upregulated by Ang II and suppressed by rhACE2 (Figures 3J and 3K), consistent with the effects on myocardial hypertrophy. Hence, rhACE2 reduced the Ang II–induced hypertrophy associated with improvement in diastolic dysfunction to levels comparable to those in the vehicle-treated group. WT mice that received rhACE2 showed reduced Ang II–induced mRNA expression of the fibrosis-associated genes procollagen type Iα1 (Figure 4A), procollagen type IIIα1 (Figure 4B), fibronectin (Figure 4C), and TGF-β1 (Figure 4D). Picrosirius red (Figure 4E) and trichrome (Figure 4F) staining showed that Ang II–triggered interstitial and perivascular fibrosis was suppressed by rhACE2 treatment. Collagen volume fraction was reduced from 11.6±1.8% in WT mice treated with Ang II and placebo (n=8) to 3.2±1.3% in WT mice treated with Ang II and rhACE2 (n=8; P<0.05). Given the critical role of Ang II and ACE2 in kidney disease,20,25,26 we also examined the impact of rhACE2 on Ang II–induced renal fibrosis. Ang II resulted in a marked increase in the expression of procollagen type Iα, procollagen type IIIα, α-smooth muscle actin, and TGF-β1, as well as in collagen I and III protein levels (online-only Data Supplement Figure V), whereas picrosirius red staining showed increased tubulointerstitial fibrosis in response to Ang II (online-only Data Supplement Figure V). Treatment with rhACE2 largely attenuated these markers of increased tubulointerstitial fibrosis (online-only Data Supplement Figure V). Clearly, rhACE2 can suppress the hypertrophic and profibrotic effects of Ang II, thereby reducing Ang II–induced diastolic dysfunction.
Consistent with the induction of hypertrophy and fibrosis, Ang II infusion caused activation and elevated phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK 1/2; Figure 4G), Janus kinase 2 (JAK2; Figure 4H), and signal transducer and activator of transcription 3 (STAT3; Figure 4I) without altering p38 mitogen-activated protein kinase (Figure 4J) in WT mice. Interestingly, rhACE2 inhibited the increased phosphorylation of extracellular signal-regulated (ERK) 1/2 (Figure 4G) while reducing the phosphorylation of JAK2 and STAT3 by 50% to 60% (Figures 4H and 4I). Western blot analyses also revealed increased protein expression of protein kinase C (PKC)-α (Figure 4K) and PKC-β1 (Figure 4L) in response to Ang II, which was suppressed strikingly by the administration of rhACE2. These observations confirm that the potent antihypertrophic and antifibrotic effects mediated by rhACE2 are mediated, at least in part, via suppression of Ang II–induced ERK1/2, JAK2-STAT3, and PKC signaling pathways.
Treatment With rhACE2 Reverses Ang II–Induced Oxidative Stress and Modulates Angiotensin Peptide Levels
The structural, functional, and biochemical rescue provided by rhACE2 could be linked to a reduction in Ang II or increased Ang 1-7 signaling. To test this hypothesis, we measured plasma and myocardial Ang II levels, as well as levels of the product of ACE2 metabolism of Ang II, plasma Ang 1-7. Ang II infusion led to a predictable increase in plasma and myocardial Ang II levels, both of which were both reduced by 60% with rhACE2 treatment (Figures 5A and 5B). In WT mice that received Ang II, plasma Ang 1-7 levels increased and were further elevated by rhACE2, which provides definitive evidence that ACE2 is an important enzyme that degrades Ang II into Ang 1-7 in vivo (Figure 5C). Infusion of Ang II for 14 days resulted in a 64±5-mm Hg increase in peak systolic blood pressure over vehicle-infused WT mice (Figure 5D), whereas rhACE2 treatment partially reduced the pressor effect of Ang II without affecting basal systolic blood pressure in vehicle-treated mice (Figure 5D). Exposure to elevated Ang II resulted in a predictable and significant increase in superoxide levels in the heart in vivo (Figures 5E through 5J). Recombinant human ACE2 largely prevented this increase in superoxide production (Figure 5K) via suppression of Ang II–induced activation of the NADPH oxidase system (Figure 5L). The superoxide scavenger polyethylene glycol–superoxide dismutase (500 U/mL; Figure 5K) and the NADPH oxidase inhibitor diphenyleneiodonium (10 μmol/L; Figure 5L), were used to confirm these measurements. These results showed that rhACE2 can reverse pathological signaling and superoxide production in association with reduced Ang II or increased Ang 1-7 levels.
Treatment With rhACE2 Prevents Subpressor Ang II–Mediated and Pressure-Overload–Induced Pathological Remodeling
A low dose of Ang II (0.15 mg · kg−1 · d−1 for 2 weeks) did not change systolic blood pressure (online-only Data Supplement Figure VI). Echocardiographic 2-dimensional short-axis midventricular views (Figure 6A) and M-mode images (Figure 6B) revealed normal systolic function with no change in fractional shortening, ejection fraction, or velocity of circumferential shortening but with a mild but measurable increase in LV wall thickness in response to the subpressor dose of Ang II (Figures 6A, 6B, and 6E; online-only Data Supplement Table IV). The mild concentric remodeling of the LV resulted in diastolic dysfunction as assessed by transmitral Doppler filling (Figure 6C) and tissue Doppler imaging (Figure 6D), with a reduced E/A ratio (Figure 6F) and increased E′/A′ ratio (Figure 6H; online-only Data Supplement Table IV). Morphometric assessment (Figure 6I) and analysis of the expression of the hypertrophy genes α-SA (α-skeletal actin; Figure 6J), BNP (brain natriuretic peptide; Figure 6K), and β-MHC (β-myosin heavy chain; Figure 6L) showed a measurable increase in response to Ang II, whereas the Ang II–induced mRNA expression of the fibrosis-associated genes procollagen type Iα1 (Figure 6M), procollagen type IIIα1 (Figure 6N), fibronectin (Figure 6O), and TGF-β1 (Figure 6P) resulted in a mild degree of myocardial fibrosis (Figure 6Q). Treatment with rhACE2 attenuated the low-dose Ang II–induced myocardial hypertrophy, diastolic dysfunction, and myocardial fibrosis (Figures 6A through 6Q). These results show that rhACE2 can directly suppress Ang II–mediated pathological hypertrophy and diastolic dysfunction independent of systemic hemodynamic effects.
In the clinically relevant pressure-overload model of heart failure, there was an early and marked downregulation of myocardial ACE2 protein (Figure 7A), which implies that loss of ACE2 can facilitate adverse myocardial remodeling. We next evaluated the effects of rhACE2 on the pathological hypertrophy and functional response to pressure overload. After 6 weeks of pressure overload, WT mice were randomized to receive placebo or rhACE2 (2 mg · kg−1 · d−1 IP) for 3 weeks. Treatment with rhACE2 for 3 weeks partially reduced the hypertrophy (Figure 7B) and resulted in a partial improvement in fractional shortening (Figure 7C) and prevention of ventricular dilation (Figure 7D). Invasive hemodynamic assessment confirmed partial rescue of functional deterioration, with a reduction in the elevated LV end-diastolic pressure (Figure 7E) and partial restoration of myocardial performance as measured by +dP/dt (Figure 7F) and −dP/dt (Figure 7G). Molecular markers of pathological hypertrophy, α-skeletal actin (Figure 7H), brain natriuretic peptide (Figure 7I), and β-myosin heavy chain (Figure 7J), and expression of procollagen type Iα1 (Figure 7K) and procollagen type IIIα1 (Figure 7L) all showed a near normalization that resulted in a reduction in myocardial fibrosis (Figure 7M) in response to rhACE2. These results clearly demonstrate that rhACE2 can provide therapeutic benefits against pathological myocardial remodeling in a clinically relevant model of heart failure.
ACE2 Is a Negative Regulator of Oxidative Stress and Collagen Production in Cultured Adult Cardiomyocytes and Cardiofibroblasts
The in vivo action of Ang II can result in a wide array of effects, including changes in blood pressure and hemodynamic load. To evaluate a more direct effect of rhACE2 on Ang II signaling, we examined the impact of rhACE2 on Ang II effects in these cultured cells. We first confirmed the presence of ACE2 in cultured adult murine cardiomyocytes and cardiofibroblasts (Figure 8A). Dihydroethidium fluorescence imaging showed a marked increase in superoxide production in response to Ang II (100 nmol/L) in cardiomyocytes that was blocked by rhACE2 in a dose-dependent manner and by biological superoxide degradation with polyethylene glycol–superoxide dismutase (Figures 8A through 8D). NADPH oxidase activity showed a similar increase with Ang II that was blocked by increasing doses of rhACE2 and by the chemical inhibitor of NADPH oxidase, diphenyleneiodonium (Figure 8C). Preincubation with the Mas receptor peptide antagonist D-Ala7-Ang 1-727 largely prevented the suppression of Ang II–mediated superoxide generation (Figures 8B and 8E) and NADPH oxidase activation (Figure 8F), which confirmed that enhanced Ang 1-7 signaling is a key mediator of rhACE2 action. Expression analyses showed that Ang II–induced expression of procollagen type Iα (Figure 8G), procollagen type IIIα (Figure 8H), fibronectin (Figure 8I), TGF-β1 (Figure 8J), and α-smooth muscle actin (Figure 8K) was reduced strikingly by rhACE2. The increased phosphorylation of ERK1/2 in cardiomyocytes and cardiofibroblasts in response to Ang II was prevented in part by rhACE2, which was reversed by cotreatment with D-Ala7-Ang 1-7 (Figure 8L). These in vitro results proved that rhACE2 can mediate direct effects on adult cardiomyocytes and cardiofibroblasts, which are mediated in part by the promotion of Ang 1-7 signaling.
ACE2 is the first known homolog of human ACE and functions as a pleiotropic monocarboxypeptidase responsible for the degradation of a range of peptides with a high catalytic efficiency.8–10 In the present study, we clearly define the critical role of ACE2 in mediating conversion of Ang II to Ang 1-7 and as a negative regulator of Ang II–induced heart disease. The downregulation of ACE2 by Ang II and aortic banding is likely a key player in the adverse remodeling that is characteristic of agonist-mediated and pressure-overload–induced heart disease. Ang II, a major bioactive effector of the RAS, is aberrantly activated in heart disease, eliciting a variety of biological actions spanning the diverse roles of the RAS in cardiovascular homeostasis and disease.1–3,7 ACE2 has emerged as an important determinant of heart disease,16,28 and increasing serum ACE2 activity correlates with worsening LV function and poor clinical outcomes in patients with heart failure.29
The present data show that in a setting of elevated Ang II levels, absence of ACE2 results in greater pathological ventricular hypertrophy and fibrosis, which results in worsening diastolic dysfunction. In contrast, treatment with rhACE2 prevented Ang II–induced hypertrophy and myocardial fibrosis. Mechanistically, we linked these changes to a greater elevation in myocardial Ang II levels, which led to enhanced NADPH oxidase activation and superoxide generation, which were important mediators of pathological hypertrophy and fibrosis in the ACE2KO hearts, and to a corresponding reduction in response to rhACE2.30 The development of myocardial fibrosis and pathological hypertrophy results in diastolic dysfunction and diastolic heart failure due to increased myocardial stiffness.5 Importantly, Ang II–induced diastolic dysfunction was completely ameliorated by rhACE2, in association with marked reduction of hypertrophy and myocardial fibrosis. In addition, using a subpressor dose of Ang II and the pressure-overload model of heart failure, we showed that rhACE2 can have direct cardioprotective actions independent of systemic hemodynamic effects. The present findings are consistent with the ability of increased ACE2 to blunt the high blood pressure and myocardial damage seen in rat models.31,32 The protective effects of rhACE2 against pressure-overload–induced heart failure are consistent with the exacerbation of pressure-overload heart failure in ACE2KO mice28 and support a key role of the RAS in the cardiac response to pressure overload.33
Ang II activates a plethora of signaling cascades, including those of the mitogen-activated protein kinase, PKC, and JAK2-STAT3 signaling pathways, which results in myocardial hypertrophy and increased fibrosis.1,3,34,35 Ang II activation of PKC signaling and enhancement of nuclear accumulation of STAT family members are associated with cardiac fibrosis and hypertrophy,36,37 whereas inhibition of these pathways attenuates collagen deposition and myocardial fibrosis, hypertrophy, and diastolic dysfunction.38,39 Ang II–AT1-mediated activation of ERK1/2 plays a key role in the downregulation of ACE2 expression, which leads to an amplification of Ang II–mediated effects.40 The abolishment of ERK1/2, JAK2-STAT3, and PKC signaling by rhACE2 is responsible, at least in part, for attenuation of Ang II–induced myocardial hypertrophy and fibrosis and improvement of diastolic dysfunction.
rhACE2 reduced the plasma and myocardial Ang II levels and increased plasma Ang 1-7 levels. This critical switch in the biochemical milieu had a pivotal role in minimizing Ang II–induced cardiac and vascular pathology. Ang 1-7 can suppress Ang II–mediated myocardial hypertrophy and fibrosis independent of blood pressure.12,13 Importantly, rhACE2 did not alter baseline plasma Ang II, Ang 1-7, or blood pressure in WT mice, which suggests that substrate availability is a limiting factor in ACE2 enzymatic activity.41 We propose that ACE2 functions as a negative regulator of the RAS predominantly in the setting of increased Ang II, as in heart disease. ACE2 is expressed and functional in cardiomyocytes and cardiofibrobasts and acts as a local negative regulator of the RAS independent of a blood pressure–lowering effect. Cardiac fibroblasts express AT1 receptors and Ang 1-7 receptors,42 and Ang II is known to induce production of collagen and TGF-β1,1,42 both of which were largely inhibited by rhACE2.
In summary, in the setting of elevated Ang II, loss of ACE2 increases Ang II–induced myocardial hypertrophy and fibrosis and increases oxidative stress, which results in worsening diastolic dysfunction. In contrast, rhACE2 prevents Ang II–induced hypertrophy and fibrosis in part because of enhanced Ang 1-7 signaling. In the pressure-overload model of heart failure, rhACE2 partially prevented the functional deterioration of cardiac function and the degree of pathological remodeling. ACE2 can act as a protective mechanism in the heart to limit the pathological effects of an activated systemic and/or local RAS.
Dr Oudit is a Clinician-Investigator Scholar of the Alberta Heritage Foundation for Medical Research and a Distinguished Clinician Scientist of the Heart and Stroke Foundation of Canada and Canadian Institutes of Health Research, and Dr Kassiri is a New Investigator of the Heart and Stroke Foundation of Canada. Dr Zhong was also supported by the National Natural Science Foundation of China. We acknowledge technical assistance from the Cardiovascular Research Centre core facilities at the University of Alberta and support from an Alberta HEART Interdisciplinary Team Grant.
Sources of Funding
We acknowledge financial support from the Canadian Institute for Health Research (Dr Oudit, grant No. 86602; Dr Kassiri, grant No. 84279), the Alberta Heritage Foundation for Medical Research (Drs Oudit and Zhong), and EuGeneHeart (EU 6th Framework Programs), the Austrian National Bank, and the Institute of Molecular Biotechnology, Austria (Dr Penninger).
Dr Shuster is Chief Operating Officer of Apeiron Biologics and owns stock in the company; Dr Loibner is Chief Executive Officer of Apeiron Biologics and owns stock in the company; and Dr Penninger is the founder of Apeiron Biologics and owns stock in the company. The remaining authors report no conflicts.
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Activation of the tissue and systemic renin-angiotensin system and the generation of angiotensin II play a key role in cardiovascular diseases. Angiotensin-converting enzyme 2 (ACE2) is the first known homolog of human ACE and functions as a pleiotropic monocarboxypeptidase. In the present study, we showed that ACE2 negatively regulates the pathophysiological effects of a pressor and subpressor dose of angiotensin II on myocardial structure and function. Whereas loss of ACE2 increases angiotensin II levels, increased ACE2 action by the use of recombinant human ACE2 lowered angiotensin II and increased angiotensin 1-7 levels in vivo, which provides definitive evidence for a key role of ACE2 in the metabolism of angiotensin II. These changes in peptide levels were associated with a plethora of molecular and cellular alterations, including inhibition of superoxide production and reduced activation of various key signaling pathways. The resultant phenotypic changes, characterized by increased myocardial and renal fibrosis, pathological hypertrophy, and diastolic dysfunction, were inhibited by ACE2. The beneficial effects of recombinant human ACE2 were also demonstrated in the clinically relevant model of pressure-overload–induced heart failure. In response to exogenous angiotensin II and pressure overload, ACE2 levels were decreased, thereby perpetuating the pathological effects of angiotensin II. The direct effects of angiotensin II on adult ventricular cardiomyocytes and cardiac fibroblasts were suppressed by recombinant human ACE2 in an angiotensin 1-7–dependent manner. Recombinant human ACE2 can provide a novel therapeutic approach for patients with cardiovascular disease.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.110.955369/DC1.