Heart Failure - Circulationcirc.ahajournals.org/content/122/7/717.full.pdf · heart failure in experimental models and patients.4–6 ... (Santa Cruz, Calif), Cell Signaling Inc (Danvers,

Heart Failure

Angiotensin-Converting Enzyme 2 Suppresses PathologicalHypertrophy, Myocardial Fibrosis, and Cardiac Dysfunction

JiuChang Zhong, MD; Ratnadeep Basu, MD; Danny Guo, BSc; Fung L. Chow, MSc;Simon Byrns, BSc; Manfred Schuster, PhD; Hans Loibner, PhD; Xiu-hua Wang, PhD;

Josef M. Penninger, MD; Zamaneh Kassiri, PhD; Gavin Y. Oudit, MD, PhD

Background—Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizingseveral peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)–mediatedsignaling 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 pathologicalhypertrophy in ACE2 knockout (Ace2�/y) mice compared with wild-type (WT) mice. Daily treatment of Ang II–infusedwild-type mice with recombinant human ACE2 (rhACE2; 2 mg � kg�1 � d�1 IP) blunted the hypertrophic response andexpression of hypertrophy markers and reduced Ang II–induced superoxide production. Ang II–mediated myocardial fibrosisand expression of procollagen type I�1, procollagen type III�1, transforming growth factor-�1, and fibronectin were alsosuppressed by rhACE2. Ang II–induced diastolic dysfunction was inhibited by rhACE2 in association with reduced plasmaand myocardial Ang II and increased plasma Ang 1-7 levels. rhACE2 treatment inhibited Ang II–mediated activation ofprotein kinase C-� and protein kinase C-�1 protein levels and phosphorylation of the extracellular signal-regulated 1/2, Januskinase 2, and signal transducer and activator of transcription 3 signaling pathways in wild-type mice. A subpressor dose ofAng 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, whichwere exacerbated by ACE2 deficiency, whereas rhACE2 attenuated Ang II– and pressure-overload–induced adversemyocardial remodeling. Hence, ACE2 is an important negative regulator of Ang II–induced heart disease and suppressesadverse myocardial remodeling. (Circulation. 2010;122:717-728.)

Key Words: angiotensin � signal transduction � hypertrophy � remodeling � diastole

Activation of the renin-angiotensin system (RAS) and thesubsequent generation of angiotensin (Ang) II are im-

portant mediators of myocardial fibrosis, pathological hyper-trophy, and heart failure.1–3 Pathological hypertrophy andincreased myocardial interstitial fibrosis contribute to in-creased ventricular wall stiffness, thereby impairing cardiacdiastolic function, and represent an important risk factor forheart failure in experimental models and patients.4–6 Drugsthat target Ang II and the Ang II type 1 receptor (AT1) arewidely used for the treatment of cardiovascular diseases suchas hypertension, myocardial infarction, and heart failure.7

Angiotensin-converting enzyme 2 (ACE2) is a pleiotropicmonocarboxypeptidase 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 themajor enzymatic products of ACE2, has been shown toreduce Ang II–induced cardiac hypertrophy and remodelingand pressure-overload–induced heart failure.12,13 ACE2-deficient mice can develop impaired cardiac function withadverse ventricular remodeling, enhanced oxidative stress,and inflammatory cytokine expression.14,15

Clinical Perspective on p 728

In the present study, we directly assessed the hypothesisthat ACE2 is a negative regulator of Ang II–mediated heartdisease. Ang II–mediated oxidative stress, cardiac hypertro-

Received November 4, 2009; accepted June 14, 2010.From the Division of Cardiology (J.C.Z., D.G., F.L.C., S.B., G.Y.O.), Department of Medicine (J.C.Z., R.B., D.G., F.L.C., S.B., Z.K., G.Y.O.)

Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Canada; Department of Physiology (R.B., X.-h.W., Z.K.), University of Alberta,Edmonton, Canada; Apeiron Biologics (M.S., H.L.), Vienna, Austria; and Institute of Molecular Biotechnology of the Austrian Academy of Sciences(J.M.P.), Vienna, Austria.

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.110.955369/DC1.Correspondence to Gavin Y. Oudit, MD, PhD, FRCP(C), Division of Cardiology, Department of Medicine, Mazankowski Alberta Heart Institute,

University of Alberta, Edmonton, Alberta, T6G 2S2, Canada. E-mail [email protected]© 2010 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.110.955369

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phy, and fibrosis are exacerbated in ACE2-deficient mice,which results in worsening of diastolic dysfunction. Recom-binant human ACE2 (rhACE2) attenuated Ang II–mediatedhypertension, ventricular hypertrophy, and fibrosis and im-proved diastolic dysfunction, with a marked reversal of AngII–induced activation of pathological signaling pathways. Inresponse to pressure overload, rhACE2 partially attenuatedthe adverse myocardial remodeling. These beneficial effectsof rhACE2 correlated with the reduction in Ang II andincrease in Ang 1-7 levels. We conclude that ACE2 plays apivotal role in the inhibition of myocardial hypertrophy,fibrosis, and cardiac dysfunction, and rhACE2 represents anovel therapeutic strategy for cardiovascular disorders.

MethodsPlease see the online-only Data Supplement for additional details.

Experimental Animals and ProtocolsMutant mice were backcrossed into a pure C57BL/6 background for�10 generations as described previously.15,16 Ten-week-old maleACE2 knockout (ACE2KO) mice (Ace2�/y) and their littermatewild-type (WT; Ace2�/y) mice were used. An osmotic minipump(model 1002, Alza Corp, Palo Alto, Calif) was implanted subcuta-neously 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 thentreated with placebo or rhACE2 (2 mg � kg�1 � d�1 IP). Allexperiments were performed in accordance with institutional guide-lines and the standards of the Canadian Council on Animal Care.

Aortic BandingThe aortic banding model was used to generate pressure-overload–induced heart failure in 8- to 9-week-old male C57/BL6 WT mice, as wehave described previously.17 After 6 weeks of pressure overload, bandedWT mice were randomized to receive rhACE2 (2 mg � kg�1 � d�1 IP)or placebo for 3 weeks.

Tail-Cuff Systolic Blood PressureSystolic blood pressure of each mouse was measured by the tail-cuffmethod with an IITC blood pressure monitoring system (IITC LifeScience, Woodland Hills, Calif).

Echocardiography and Tissue Doppler ImagingTransthoracic echocardiography was performed and analyzed in ablinded manner as described previously with a Vevo 770 high-resolution imaging system equipped with a 30-MHz transducer(RMV-707B; VisualSonics, Toronto, Canada).18

HistologyTrichrome and picrosirius red staining and visualization andanalysis of collagen volume fraction were performed as describedpreviously.17

TaqMan Real-Time Polymerase Chain Reactionand Western Blot AnalysisRNA expression levels of various genes and Western blot analyseswere determined as described previously.15–17 The primers andprobes for mRNA expression analysis by TaqMan (Applied Biosys-tems, Foster City, Calif) real-time polymerase chain reaction arelisted in online-only Data Supplement Table I; 18S ribosomal RNAwas used as the endogenous control. Antibodies were obtained fromSanta Cruz Biotechnology (Santa Cruz, Calif), Cell Signaling Inc(Danvers, Mass), and R&D Systems (Minneapolis, Minn).15–17

Generation and Characterization of rhACE2The extracellular domain of human ACE2 (amino acid residues 1 to740, molecular weight 101 kDa)19 was expressed recombinantly in

Chinese hamster ovary (CHO) cells as described previously (online-only Data Supplement).20

Angiotensin Peptide MeasurementPlasma Ang II and Ang 1-7 and myocardial Ang II levels weremeasured by radioimmunoassay in the Hypertension and VascularResearch Center core laboratory at Wake Forest University School ofMedicine as described previously.14,16

Invasive Pressure-Volume MeasurementsInvasive pressure-volume measurements were made in nonintubatedand anesthetized mice (1% isoflurane mixed with 100% O2) with theSPR-869 microtip catheter and the Millar pressure-volume systemMPVS-400 (Millar Instruments, Houston, Tex) as described previ-ously21 at 3 to 4 hours after removal of the osmotic pumps. Inaortic-banded mice, only invasive pressure measurements wereobtained.

Isolation and Culture of Adult Cardiomyocytesand CardiofibroblastsAdult murine left ventricular (LV) cardiomyocytes and cardiofibro-blasts were isolated and cultured as described previously.22

Dihydroethidium Fluorescence andLucigenin-Enhanced ChemiluminescenceDihydroethidium, an oxidative fluorescent dye, was used to measuresuperoxide (O2

�) levels in cultured cardiomyocytes and heart tissuesfrom ACE2KO and WT mice as described previously.16 Nicotin-amide adenine dinucleotide phosphate (NADPH) oxidase activity incultured cardiomyocytes and hearts of mice was quantified bylucigenin-enhanced chemiluminescence as described previously.15,16

Statistical AnalysisResults are shown as mean�SEM. Statistical analysis was per-formed with SPSS 11.5 software either by ANOVA followed bymultiple-comparison testing (Student-Neuman-Keuls test) or theFisher exact test as appropriate. P�0.05 was considered statisticallysignificant.

ResultsAng II–Induced Myocardial Hypertrophy andFibrosis, Diastolic Dysfunction, and OxidativeStress Are Exacerbated by the Loss of ACE2To 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 IIinfusion (1.5 mg � kg�1 � d�1). Echocardiographic M-modeimages (Figure 1A) and 2-dimensional short-axis midven-tricular views (Figure 1B) revealed normal systolic func-tion with no change in fractional shortening, ejectionfraction, or velocity of circumferential shortening betweenWT and ACE2KO mice (online-only Data SupplementTable II). Notably, there was greater concentric remodel-ing in the ACE2KO mice in response to chronic stimula-tion by Ang II, with a greater increase in ventricular wallthickness (Figures 1A and 1B; online-only Data Supple-ment Table II). Concentric remodeling of the LV resultedin diastolic dysfunction as assessed by transmitral Dopplerfilling and tissue Doppler imaging. In Ang II–treated WTmice, the transmitral A wave was increased (with areduced E/A ratio) with reduced E� (and increased E/E�ratio; Figures 1C and 1D; online-only Data SupplementTable II). LV isovolumetric relaxation time was increasedsignificantly, whereas the E�/A� showed greater reduction,

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primarily due to marked elevation of the A� wave in AngII–treated ACE2KO mice compared with WT mice (Fig-ures 1C and 1D; online-only Data Supplement Table II).

Interestingly, myocardial ACE2 protein level in WTmice was reduced by 60% in response to Ang II (Figure1E), which may have facilitated Ang II–induced myocar-dial injury. Gravimetric analysis showed that Ang IIinfusion resulted in a greater increase in the heart weight–tibia length ratio and LV weight–tibia length ratio inACE2KO than in WT mice (Figure 1F). Real-time poly-merase chain reaction analysis revealed that loss of ACE2augmented Ang II–induced mRNA expression of molecu-lar markers of pathological hypertrophy, including brainnatriuretic peptide (Figure 1G) and �-skeletal actin (Figure1H), without having a differential effect on �-myosinheavy chain mRNA expression (Figure 1I) or proteinlevels (Figure 1J). Ang II–mediated NADPH oxidaseactivation and superoxide generation are pivotal mecha-nisms of Ang II–mediated injury in the cardiovascularsystem.3,23,24 Consistent with the exacerbation of Ang IIpathological effects in ACE2KO mice, Ang II–induced

superoxide production (Figures 2A and 2B) and NADPHoxidase activation (Figure 2B) were greater in ACE2KOmice than in WT mice, likely driven by the greaterelevation in myocardial Ang II levels in ACE2KO mice(Figure 2C). Ang II is a well-known activator of increasedtissue fibrosis via its profibrotic effects.1–3 Ang II resultedin greater mRNA expression of procollagen type I�1(Figure 2D), procollagen type III�1 (Figure 2E), fibronec-tin (Figure 2F), and transforming growth factor-� 1 (TGF-�1; Figure 2G) in ACE2KO mice than in WT mice. Thesemolecular footprints of increased fibrosis were confirmedby histological analyses (Figures 2H and 2I) that showedgreater interstitial and perivascular myocardial fibrosis inACE2KO mice. The collagen volume fraction showed agreater increase in ACE2KO mice (15.9�2.1%; n�8) thanin WT mice (9.1�1.8%; n�8; P�0.05) in response to AngII. Collectively, these data show that loss of ACE2increases myocardial Ang II levels and worsens concentricremodeling, which results in greater deterioration of dia-stolic dysfunction in response to exogenous Ang II.

Figure 1. Loss of ACE2 increases patho-logical hypertrophy and diastolic dys-function in response to Ang II. A throughD, Echocardiographic assessment ofmice by M-mode (A), short-axis view (B)(The red arrows indicate the LV wallthickness), mitral Doppler (C), and tissueDoppler (D) in Ang II–infused WT andACE2KO mice showed greater hypertro-phy and worsening diastolic dysfunctionin ACE2KO mice. E, Western blot analy-sis of myocardial ACE2 protein showeda marked reduction in ACE2 protein lev-els in response to Ang II, with ACE2KOhearts serving as a negative control. Fthrough J, Morphometric assessment ofpathological hypertrophy based on heartweight (HW)/tibia length (TL) and LVweight (LVW)/TL ratios (F) and TaqManreal-time polymerase chain reactionanalysis of genetic markers of pathologi-cal hypertrophy, including brain natri-uretic peptide (BNP; G), �-skeletal actin(�-SA; H), and �-myosin heavy chain(�-MHC; I) mRNA expression, as well asWestern blot analysis of �-MHC proteinlevels (J), in Ang II–infused WT andACE2KO mice showed a greaterincrease in expression in ACE2KO mice,with equivalent changes in �-MHC. R.E.indicates relative expression. n�6 forWT�Vehicle and ACE2KO�Vehicle; n�8for WT�Ang II and ACE2KO�Ang II.*P�0.05 vs corresponding vehicle-treated group; #P�0.05 vs WT�Ang IIgroup.

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Treatment With rhACE2 Attenuates AngII–Mediated Cardiac Hypertrophy and Fibrosisand Pathological SignalingThe purity, molecular weight, and identity of rhACE2 wereconfirmed by high-performance liquid chromatography,SDS-PAGE, and Western blot analyses (online-only DataSupplement Figure I). Human ACE2 shares 65% homologywith rodent ACE2 with a conserved catalytic site (online-onlyData Supplement Figure II),14,19 and rhACE2 (1 �g/mL)produced marked and similar elevations in plasma ACE2activity in murine and human plasma (online-only DataSupplement Figures IIIA and IIIB). Plasma ACE2 activitywas markedly increased at 1 and 2 weeks after initiation ofrhACE2 at 2 mg � kg�1 � d�1 IP in WT mice (online-only DataSupplement Figure IIIC). WT mice showed a marked in-crease in LV wall thickness (online-only Data SupplementTable III), as illustrated by M-mode imaging (Figure 3A) anda short-axis view (Figure 3B), without deterioration in sys-tolic function (Figure 3A; online-only Data Supplement

Table III), in response to Ang II. The primary functionalabnormality in these mice was diastolic dysfunction charac-terized by increased A-wave amplitude (with resultant reduc-tion in the E/A ratio) and reduced E� (with resultant reductionin the E�/A� ratio; Figures 3C and 3D; online-only DataSupplement Table III). The use of rhACE2 inhibited AngII–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–induceddiastolic dysfunction characterized by elevated LV end-dia-stolic pressure, increased slope of the end-diastolic pressure-volume relationship, and an increased LV relaxation timeconstant, which were all reduced by treatment with rhACE2(online-only Data Supplement Figure IV).

Morphometric analyses further confirmed the marked an-tihypertrophic effects of rhACE2 against Ang II–inducedhypertrophy (Figure 3E), with reduced expression of hyper-

Figure 2. Increased NADPH oxidaseactivity and worsening myocardial fibro-sis in ACE2KO mice in response to AngII. A through C, Representative dihydro-ethidium (10 �mol/L) fluorescenceimages (A), relative fluorescence values(B), and NADPH oxidase activity quanti-fied by lucigenin-enhanced chemilumi-nescence assay (B) showed greatersuperoxide generation and NADPH oxi-dase activity due to a greater elevationin myocardial Ang II levels (C) inACE2KO mice in response to Ang II(n�8 for vehicle and n�16 for AngII–treated groups). D through G, TaqManreal-time polymerase chain reactionanalysis showed a greater elevation inmRNA expression of procollagen typeI�1 (D), procollagen type III�1 (E),fibronectin (F), and TGF-�1 (G) inresponse to Ang II in ACE2KO mice thanin WT mice. H through I, Representativeimages of picrosirius red (H) andtrichrome staining (I) showed a greaterincrease in interstitial and perivascularmyocardial fibrosis in response to Ang IIin ACE2KO mice than in WT mice. R.E.indicates relative expression. n�6 forWT�Vehicle and ACE2KO�Vehicle; n�8for WT�Ang II and ACE2KO�Ang II.*P�0.05 vs corresponding vehicle-treated group; #P�0.05 vs WT�Ang IIgroup.




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trophy markers (Figures 3F through 3H). Normalization ofincreased �-myosin heavy chain mRNA expression wasconfirmed by Western blot analysis (Figure 3I). Both matrixmetalloproteinase-2 and ADAM12 (a disintegrin and metal-loprotease 12), which have been linked to agonist-mediatedhypertrophy, were upregulated by Ang II and suppressed byrhACE2 (Figures 3J and 3K), consistent with the effects onmyocardial hypertrophy. Hence, rhACE2 reduced the AngII–induced hypertrophy associated with improvement in dia-stolic dysfunction to levels comparable to those in thevehicle-treated group. WT mice that received rhACE2showed reduced Ang II–induced mRNA expression of thefibrosis-associated genes procollagen type I�1 (Figure 4A),procollagen type III�1 (Figure 4B), fibronectin (Figure 4C),and TGF-�1 (Figure 4D). Picrosirius red (Figure 4E) andtrichrome (Figure 4F) staining showed that Ang II–triggeredinterstitial and perivascular fibrosis was suppressed byrhACE2 treatment. Collagen volume fraction was reducedfrom 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 andrhACE2 (n�8; P�0.05). Given the critical role of Ang II andACE2 in kidney disease,20,25,26 we also examined the impactof rhACE2 on Ang II–induced renal fibrosis. Ang II resulted

in a marked increase in the expression of procollagen type I�,procollagen type III�, �-smooth muscle actin, and TGF-�1,as well as in collagen I and III protein levels (online-onlyData Supplement Figure V), whereas picrosirius red stainingshowed increased tubulointerstitial fibrosis in response toAng II (online-only Data Supplement Figure V). Treatmentwith rhACE2 largely attenuated these markers of increasedtubulointerstitial fibrosis (online-only Data Supplement Fig-ure V). Clearly, rhACE2 can suppress the hypertrophic andprofibrotic 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 phosphoryla-tion levels of extracellular signal-regulated kinase 1/2 (ERK1/2; Figure 4G), Janus kinase 2 (JAK2; Figure 4H), andsignal transducer and activator of transcription 3 (STAT3;Figure 4I) without altering p38 mitogen-activated proteinkinase (Figure 4J) in WT mice. Interestingly, rhACE2 inhib-ited the increased phosphorylation of extracellular signal-regulated (ERK) 1/2 (Figure 4G) while reducing the phos-phorylation of JAK2 and STAT3 by 50% to 60% (Figures 4Hand 4I). Western blot analyses also revealed increased proteinexpression of protein kinase C (PKC)-� (Figure 4K) and

Figure 3. Prevention of Ang II–inducedcardiac hypertrophy in WT mice byrhACE2. A through D, Echocardiographicand Doppler assessment of Ang II–in-fused WT mice by M-mode (A), short-axis view (B) (The red arrows indicatethe LV wall thickness), mitral Doppler (C),and tissue Doppler (D) showed a markedattenuation of hypertrophy and restora-tion of diastolic function within normallimits in mice treated with rhACE2. E,Morphometric assessment of pathologi-cal hypertrophy showed a markedreduction in hypertrophy in WT miceinfused with Ang II in response torhACE2. F through H, TaqMan real-timepolymerase chain reaction analysis ofmRNA expression of genetic markers ofmyocardial hypertrophy, including brainnatriuretic peptide (F), �-skeletal actin(G), and �-myosin heavy chain mRNA (H)and protein levels (I), and mRNA levelsof genetic markers of adverse myocar-dial remodeling, including matrixmetalloproteinase-2 (MMP2; J) andADAM12 (K), showed that rhACE2 dras-tically reduced the Ang II–inducedexpression of these genes. n�6for WT�Vehicle�Placebo andWT�Vehicle�rhACE2; n�8 for WT�AngII�Placebo and WT�Ang II�rhACE2.ADAM12 indicates a disintegrin and met-alloprotease 12; other abbreviations asin Figure 1. *P�0.05 vs WT�Vehicle�Placebo group; #P�0.05 vs WT�AngII�Placebo group.




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PKC-�1 (Figure 4L) in response to Ang II, which wassuppressed strikingly by the administration of rhACE2. Theseobservations confirm that the potent antihypertrophic andantifibrotic effects mediated by rhACE2 are mediated, at leastin part, via suppression of Ang II–induced ERK1/2, JAK2-STAT3, and PKC signaling pathways.

Treatment With rhACE2 Reverses AngII–Induced Oxidative Stress and ModulatesAngiotensin Peptide LevelsThe structural, functional, and biochemical rescue providedby rhACE2 could be linked to a reduction in Ang II orincreased Ang 1-7 signaling. To test this hypothesis, wemeasured plasma and myocardial Ang II levels, as well aslevels of the product of ACE2 metabolism of Ang II, plasmaAng 1-7. Ang II infusion led to a predictable increase inplasma and myocardial Ang II levels, both of which wereboth reduced by 60% with rhACE2 treatment (Figures 5Aand 5B). In WT mice that received Ang II, plasma Ang 1-7levels increased and were further elevated by rhACE2, which

provides definitive evidence that ACE2 is an importantenzyme that degrades Ang II into Ang 1-7 in vivo (Figure5C). 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 treatmentpartially reduced the pressor effect of Ang II without affect-ing basal systolic blood pressure in vehicle-treated mice(Figure 5D). Exposure to elevated Ang II resulted in apredictable and significant increase in superoxide levels inthe heart in vivo (Figures 5E through 5J). Recombinanthuman ACE2 largely prevented this increase in superoxideproduction (Figure 5K) via suppression of Ang II–inducedactivation of the NADPH oxidase system (Figure 5L). Thesuperoxide scavenger polyethylene glycol–superoxide dis-mutase (500 U/mL; Figure 5K) and the NADPH oxidaseinhibitor diphenyleneiodonium (10 �mol/L; Figure 5L), wereused to confirm these measurements. These results showedthat rhACE2 can reverse pathological signaling and superox-ide production in association with reduced Ang II or in-creased Ang 1-7 levels.

Figure 4. Treatment with rhACE2 attenu-ates Ang II–mediated myocardial fibrosisand ERK1/2, JAK2-STAT3, and PKC sig-naling in WT mice. A through D, TaqManreal-time polymerase chain reactionanalysis revealed that treatment withrhACE2 reduced mRNA expression offibrosis-associated genes, including pro-collagen type I�1 (A), procollagen typeIII�1 (B), fibronectin (C), and TGF-�1 (D)in WT mice infused with Ang II. R.E. indi-cates relative expression. E through F,Determination of myocardial fibrosis bypicrosirius red (E) and Masson trichromestaining (F) showed a marked increase inresponse to Ang II that was suppressedby rhACE2 treatment. n�6 to 8. *P�0.05vs WT�Vehicle�Placebo group;#P�0.05 vs WT�Ang II�Placebo group.G through J, Representative Westernblot analysis with quantification shownbelow illustrating that Ang II–inducedphosphorylation (p) levels of ERK1/2 (G),JAK2 (H), STAT3 (I), and p38 mitogen-activated protein kinase (p38MAPK; J)was markedly suppressed by rhACE2. Kthrough L, Representative Western blotanalysis with quantification shown belowillustrating that the Ang II–inducedincreases in protein levels of PKC-� (K)and PKC-�1 (L) were drastically reducedby rhACE2. A.U. indicates arbitrary units;T, total unphosphorylated level. n�4.*P�0.05 vs WT�Vehicle�Placebogroup; #P�0.05 vs WT�Ang II� Pla-cebo group.




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Treatment With rhACE2 Prevents SubpressorAng II–Mediated and Pressure-Overload–InducedPathological RemodelingA low dose of Ang II (0.15 mg � kg�1 � d�1 for 2 weeks) didnot change systolic blood pressure (online-only Data Supple-ment Figure VI). Echocardiographic 2-dimensional short-axismidventricular views (Figure 6A) and M-mode images (Fig-ure 6B) revealed normal systolic function with no change infractional shortening, ejection fraction, or velocity of circum-ferential shortening but with a mild but measurable increasein LV wall thickness in response to the subpressor dose ofAng II (Figures 6A, 6B, and 6E; online-only Data Supple-ment Table IV). The mild concentric remodeling of the LVresulted in diastolic dysfunction as assessed by transmitralDoppler filling (Figure 6C) and tissue Doppler imaging(Figure 6D), with a reduced E/A ratio (Figure 6F) andincreased E�/A� ratio (Figure 6H; online-only Data Supple-ment Table IV). Morphometric assessment (Figure 6I) andanalysis of the expression of the hypertrophy genes �-SA(�-skeletal actin; Figure 6J), BNP (brain natriuretic peptide;Figure 6K), and �-MHC (�-myosin heavy chain; Figure 6L)showed a measurable increase in response to Ang II, whereasthe Ang II–induced mRNA expression of the fibrosis-associated genes procollagen type I�1 (Figure 6M), procol-

lagen type III�1 (Figure 6N), fibronectin (Figure 6O), andTGF-�1 (Figure 6P) resulted in a mild degree of myocardialfibrosis (Figure 6Q). Treatment with rhACE2 attenuated thelow-dose Ang II–induced myocardial hypertrophy, diastolicdysfunction, and myocardial fibrosis (Figures 6A through6Q). These results show that rhACE2 can directly suppressAng II–mediated pathological hypertrophy and diastolic dys-function independent of systemic hemodynamic effects.

In the clinically relevant pressure-overload model of heartfailure, there was an early and marked downregulation ofmyocardial ACE2 protein (Figure 7A), which implies thatloss of ACE2 can facilitate adverse myocardial remodeling.We next evaluated the effects of rhACE2 on the pathologicalhypertrophy and functional response to pressure overload.After 6 weeks of pressure overload, WT mice were random-ized to receive placebo or rhACE2 (2 mg � kg�1 � d�1 IP) for3 weeks. Treatment with rhACE2 for 3 weeks partiallyreduced the hypertrophy (Figure 7B) and resulted in a partialimprovement in fractional shortening (Figure 7C) and pre-vention of ventricular dilation (Figure 7D). Invasive hemo-dynamic assessment confirmed partial rescue of functionaldeterioration, with a reduction in the elevated LV end-dia-stolic pressure (Figure 7E) and partial restoration of myocar-dial performance as measured by �dP/dt (Figure 7F) and

Figure 5. Treatment with rhACE2reduced Ang II pressor response andplasma angiotensin peptide levels andsuppressed oxidative stress in WT mice.A through C, Plasma Ang II (A), myocar-dial Ang II (B), and plasma Ang 1-7 (C)levels in mice, with marked elevation inAng II levels and Ang 1-7 levels inresponse to Ang II infusion and a result-ant reduction in plasma and myocardialAng II levels and increased Ang 1-7 lev-els with treatment with rhACE2. n�8 forvehicle and n�14 for Ang II–treatedgroups. D, Systolic blood pressure levelswere measured noninvasively by the tail-cuff method at 0, 3, 7, 10, 12, and 14days in WT mice with Ang II pumps.n�6 for vehicle and n�9 for AngII–treated groups. E through L, Repre-sentative dihydroethidium (DHE) fluores-cence images (E through J), relative fluo-rescence values (K), and NADPHoxidase activity (L) quantified bylucigenin-enhanced chemiluminescenceassay showed Ang II–induced superox-ide generation and NADPH oxidase acti-vation were prevented by rhACE2. Pre-indicates before treatment; Post3d, 3days after treatment; Post7d, 7 daysafter treatment; Post10d, 10 days aftertreatment; Post12d, 12 days after treat-ment; Post14d, 14 days after treatment;PEG-SOD, polyethylene glycol–superox-ide dismutase (500 U/mL); A.U., arbitraryunits; and DPI, diphenyleneiodonium(10 �mol/L). *P�0.05 vs all other groups;#P�0.05 vs WT�Ang II�Placebo group.




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�dP/dt (Figure 7G). Molecular markers of pathologicalhypertrophy, �-skeletal actin (Figure 7H), brain natriureticpeptide (Figure 7I), and �-myosin heavy chain (Figure 7J),and expression of procollagen type I�1 (Figure 7K) andprocollagen type III�1 (Figure 7L) all showed a near normal-ization that resulted in a reduction in myocardial fibrosis(Figure 7M) in response to rhACE2. These results clearlydemonstrate that rhACE2 can provide therapeutic benefitsagainst pathological myocardial remodeling in a clinicallyrelevant model of heart failure.

ACE2 Is a Negative Regulator of Oxidative Stressand Collagen Production in Cultured AdultCardiomyocytes and CardiofibroblastsThe in vivo action of Ang II can result in a wide array ofeffects, including changes in blood pressure and hemodynam-ic load. To evaluate a more direct effect of rhACE2 on AngII signaling, we examined the impact of rhACE2 on Ang IIeffects in these cultured cells. We first confirmed the pres-

ence of ACE2 in cultured adult murine cardiomyocytes andcardiofibroblasts (Figure 8A). Dihydroethidium fluorescenceimaging showed a marked increase in superoxide productionin response to Ang II (100 nmol/L) in cardiomyocytes thatwas blocked by rhACE2 in a dose-dependent manner and bybiological superoxide degradation with polyethylene glycol–superoxide dismutase (Figures 8A through 8D). NADPHoxidase activity showed a similar increase with Ang II thatwas blocked by increasing doses of rhACE2 and by thechemical inhibitor of NADPH oxidase, diphenyleneiodonium(Figure 8C). Preincubation with the Mas receptor peptideantagonist D-Ala7-Ang 1-727 largely prevented the suppres-sion of Ang II–mediated superoxide generation (Figures 8Band 8E) and NADPH oxidase activation (Figure 8F), whichconfirmed that enhanced Ang 1-7 signaling is a key mediatorof rhACE2 action. Expression analyses showed that AngII–induced expression of procollagen type I� (Figure 8G),procollagen type III� (Figure 8H), fibronectin (Figure8I), TGF-�1 (Figure 8J), and �-smooth muscle actin (Figure

Figure 6. Treatment with rhACE2 pre-vents diastolic dysfunction, pathologicalhypertrophy, and myocardial fibrosis inresponse to a subpressor dose of Ang II.A through D, Echocardiographic assess-ment of mice by 2-dimensional short-axis view (A) (The red arrows indicate theLV wall thickness), M-mode (B), mitralDoppler flow (C), and tissue Dopplerimaging (D) showed a mild increase inventricular wall thickness and early evi-dence of diastolic dysfunction inresponse to Ang II, which were pre-vented by treatment with rhACE2. Ethrough H, Quantitative assessment ofLV posterior wall thickness (LVPWT; E)and diastolic dysfunction based on E/Aratio (F) with no change in E� (G) butwith a reduction in E�/A� ratio (H). Ithrough L, Increased pathological hyper-trophy based on morphometric assess-ment (I), �-skeletal actin (J), brain natri-uretic peptide (K), and �-myosin heavychain (L) in response to subpressor AngII was reduced by treatment withrhACE2. M through Q, Increased myo-cardial fibrosis based on expressionanalysis of profibrotic genes, procollagentype I�1 (M), procollagen type III�1 (N),fibronectin (O), and TGF-�1 (P) andpicrosirius red and Masson trichromestaining (Q) in response to Ang II wasnormalized in response to rhACE2. n�6for vehicle and n�9 for groups treatedwith low Ang II (0.15 mg � kg�1 � d�1 for14 days). Abbreviations as in Figure 1.*P�0.05 compared with WT�Vehicle�Placebo group; #P�0.05 compared withWT�Ang II�Placebo group.




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8K) was reduced strikingly by rhACE2. The increasedphosphorylation of ERK1/2 in cardiomyocytes and cardiofi-broblasts in response to Ang II was prevented in part byrhACE2, which was reversed by cotreatment with D-Ala7-Ang 1-7 (Figure 8L). These in vitro results proved thatrhACE2 can mediate direct effects on adult cardiomyocytesand cardiofibroblasts, which are mediated in part by thepromotion of Ang 1-7 signaling.

DiscussionACE2 is the first known homolog of human ACE andfunctions as a pleiotropic monocarboxypeptidase responsiblefor the degradation of a range of peptides with a high catalyticefficiency.8–10 In the present study, we clearly define thecritical role of ACE2 in mediating conversion of Ang II toAng 1-7 and as a negative regulator of Ang II–induced heartdisease. The downregulation of ACE2 by Ang II and aorticbanding is likely a key player in the adverse remodeling thatis characteristic of agonist-mediated and pressure-overload–induced heart disease. Ang II, a major bioactive effector ofthe RAS, is aberrantly activated in heart disease, eliciting avariety of biological actions spanning the diverse roles of theRAS in cardiovascular homeostasis and disease.1–3,7 ACE2has emerged as an important determinant of heart disease,16,28

and increasing serum ACE2 activity correlates with worsen-

ing LV function and poor clinical outcomes in patients withheart failure.29

The present data show that in a setting of elevated Ang IIlevels, absence of ACE2 results in greater pathologicalventricular hypertrophy and fibrosis, which results in wors-ening diastolic dysfunction. In contrast, treatment withrhACE2 prevented Ang II–induced hypertrophy and myocar-dial fibrosis. Mechanistically, we linked these changes to agreater elevation in myocardial Ang II levels, which led toenhanced NADPH oxidase activation and superoxide gener-ation, which were important mediators of pathological hyper-trophy and fibrosis in the ACE2KO hearts, and to a corre-sponding reduction in response to rhACE2.30 Thedevelopment of myocardial fibrosis and pathological hyper-trophy results in diastolic dysfunction and diastolic heartfailure due to increased myocardial stiffness.5 Importantly,Ang II–induced diastolic dysfunction was completely ame-liorated by rhACE2, in association with marked reduction ofhypertrophy and myocardial fibrosis. In addition, using asubpressor dose of Ang II and the pressure-overload model ofheart failure, we showed that rhACE2 can have directcardioprotective actions independent of systemic hemody-namic effects. The present findings are consistent with theability of increased ACE2 to blunt the high blood pressureand myocardial damage seen in rat models.31,32 The protec-

Figure 7. hrACE2 partially prevents func-tional deterioration while reversingpathological hypertrophy in response topressure overload. A, Western blot anal-ysis of myocardial ACE2 protein inresponse to aortic banding (AB) in WTmice showed an early and markeddownregulation in response to pressureoverload. B through G, Reduction of hy-pertrophy (B) with partial reversal offunctional deterioration based on echo-cardiographic assessment of fractionalshortening (FS; C), LV end-diastolic dila-tion (LVEDD; D), and hemodynamicassessment with LV end-diastolic pres-sure (LVEDP; E), �dP/dtmax (F), and�dP/dtmax (G). n�8 for WT�Shamgroups and n�14 for WT�AB groups.3wAB indicates 3 weeks of aortic band-ing; 9wAB, 9 weeks of aortic banding. Hthrough M, Prevention of the Ang II–me-diated upregulation of hypertrophy dis-ease markers �-skeletal actin (H), brainnatriuretic peptide (I), and �-myosinheavy chain (J) and the profibrotic genesprocollagen type I�1 (K) and procollagentype III�1 (L) in association with a reduc-tion in myocardial fibrosis as shown bypicrosirius red staining (M) in responseto rhACE2. n�6 for WT�Sham groupsand n�8 for WT�AB groups. 3wAB indi-cates 3 weeks of aortic banding; 9wAB,9 weeks of aortic banding; other abbre-viations as in Figure 1. *P�0.05 vsWT�Sham�Placebo group; #P�0.05 vsWT�AB�Placebo group.




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tive effects of rhACE2 against pressure-overload–inducedheart failure are consistent with the exacerbation of pressure-overload heart failure in ACE2KO mice28 and support a keyrole of the RAS in the cardiac response to pressureoverload.33

Ang II activates a plethora of signaling cascades, includingthose of the mitogen-activated protein kinase, PKC, andJAK2-STAT3 signaling pathways, which results in myocar-dial hypertrophy and increased fibrosis.1,3,34,35 Ang II activa-tion of PKC signaling and enhancement of nuclear accumu-lation of STAT family members are associated with cardiacfibrosis and hypertrophy,36,37 whereas inhibition of thesepathways attenuates collagen deposition and myocardial fi-brosis, hypertrophy, and diastolic dysfunction.38,39 Ang II–AT1-mediated activation of ERK1/2 plays a key role in thedownregulation of ACE2 expression, which leads to anamplification of Ang II–mediated effects.40 The abolishmentof ERK1/2, JAK2-STAT3, and PKC signaling by rhACE2 isresponsible, at least in part, for attenuation of Ang II–inducedmyocardial hypertrophy and fibrosis and improvement ofdiastolic dysfunction.

rhACE2 reduced the plasma and myocardial Ang II levelsand increased plasma Ang 1-7 levels. This critical switch in

the biochemical milieu had a pivotal role in minimizing AngII–induced cardiac and vascular pathology. Ang 1-7 cansuppress Ang II–mediated myocardial hypertrophy and fibro-sis independent of blood pressure.12,13 Importantly, rhACE2did not alter baseline plasma Ang II, Ang 1-7, or bloodpressure in WT mice, which suggests that substrate availabil-ity is a limiting factor in ACE2 enzymatic activity.41 Wepropose that ACE2 functions as a negative regulator of theRAS predominantly in the setting of increased Ang II, as inheart disease. ACE2 is expressed and functional in cardio-myocytes and cardiofibrobasts and acts as a local negativeregulator of the RAS independent of a blood pressure–lowering effect. Cardiac fibroblasts express AT1 receptorsand Ang 1-7 receptors,42 and Ang II is known to induceproduction of collagen and TGF-�1,1,42 both of which werelargely inhibited by rhACE2.

In summary, in the setting of elevated Ang II, loss of ACE2increases Ang II–induced myocardial hypertrophy and fibro-sis and increases oxidative stress, which results in worseningdiastolic dysfunction. In contrast, rhACE2 prevents AngII–induced hypertrophy and fibrosis in part because of en-hanced Ang 1-7 signaling. In the pressure-overload model ofheart failure, rhACE2 partially prevented the functional

Figure 8. rhACE2 prevents Ang II–in-duced oxidative stress, expression ofprofibrotic genes, and ERK1/2 signalingin cultured cardiomyocytes and cardiacfibroblasts. A, Western blot analysisshowed ACE2 protein expression in WT(and its absence in ACE2KO) cardiomyo-cytes and cardiofibroblasts. B through F,Relative dihydroethidium fluorescence (Band E) and NADPH oxidase activityquantified by lucigenin-enhanced chemi-luminescence (C and F) with representa-tive dihydroethidium fluorescenceimages (D) in cultured WT cardiomyo-cytes treated with Ang II (100 nmol/L) inthe absence and presence of an Ang 1-7antagonist (D-Ala7; 10 �mol/L) andrhACE2 (0.2 or 2 mg/mL). n�5. Gthrough K, TaqMan real-time polymerasechain reaction analysis revealed thattreatment with rhACE2 (2 mg/mL)reduced mRNA expression of procolla-gen type I�1 (G), procollagen type III�1(H), fibronectin (I), TGF-�1 (J), and�-smooth muscle actin (�-SMA; K) incultured WT cardiofibroblasts treatedwith Ang II (100 nmol/L). n�6. L, Phos-phorylation (p) levels of ERK1/2 in cul-tured WT adult cardiomyocytes and car-diofibroblasts treated with Ang II (100nmol/L) in the absence and presence ofrhACE2 (2 mg/mL) and Ang 1-7 antago-nist (D-Ala7; 10 �mol/L). n�5. *P�0.05vs vehicle-treated group; #P�0.05 vsAng II group; �P�0.05 vs Ang II �rhACE2group. A.U. indicates arbitrary units;PEGSOD, polyethylene glycol–superox-ide dismutase; DPI, diphenyleneiodo-nium; D-Ala7, D-Ala7-Ang 1-7; T, totalunphosphorylated level; and R.E., rela-tive expression.




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deterioration of cardiac function and the degree of patholog-ical remodeling. ACE2 can act as a protective mechanism inthe heart to limit the pathological effects of an activatedsystemic and/or local RAS.

AcknowledgmentsDr Oudit is a Clinician-Investigator Scholar of the Alberta HeritageFoundation for Medical Research and a Distinguished Clinician Scien-tist of the Heart and Stroke Foundation of Canada and CanadianInstitutes of Health Research, and Dr Kassiri is a New Investigator of theHeart and Stroke Foundation of Canada. Dr Zhong was also supportedby the National Natural Science Foundation of China. We acknowledgetechnical assistance from the Cardiovascular Research Centre corefacilities at the University of Alberta and support from an AlbertaHEART Interdisciplinary Team Grant.

Sources of FundingWe acknowledge financial support from the Canadian Institute forHealth Research (Dr Oudit, grant No. 86602; Dr Kassiri, grant No.84279), the Alberta Heritage Foundation for Medical Research (DrsOudit and Zhong), and EuGeneHeart (EU 6th Framework Programs),the Austrian National Bank, and the Institute of Molecular Biotech-nology, Austria (Dr Penninger).

DisclosuresDr Shuster is Chief Operating Officer of Apeiron Biologics and ownsstock in the company; Dr Loibner is Chief Executive Officer ofApeiron Biologics and owns stock in the company; and Dr Penningeris the founder of Apeiron Biologics and owns stock in the company.The remaining authors report no conflicts.

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CLINICAL PERSPECTIVEActivation of the tissue and systemic renin-angiotensin system and the generation of angiotensin II play a key role incardiovascular diseases. Angiotensin-converting enzyme 2 (ACE2) is the first known homolog of human ACE andfunctions as a pleiotropic monocarboxypeptidase. In the present study, we showed that ACE2 negatively regulates thepathophysiological effects of a pressor and subpressor dose of angiotensin II on myocardial structure and function. Whereasloss of ACE2 increases angiotensin II levels, increased ACE2 action by the use of recombinant human ACE2 loweredangiotensin II and increased angiotensin 1-7 levels in vivo, which provides definitive evidence for a key role of ACE2 inthe metabolism of angiotensin II. These changes in peptide levels were associated with a plethora of molecular and cellularalterations, including inhibition of superoxide production and reduced activation of various key signaling pathways. Theresultant phenotypic changes, characterized by increased myocardial and renal fibrosis, pathological hypertrophy, anddiastolic dysfunction, were inhibited by ACE2. The beneficial effects of recombinant human ACE2 were also demonstratedin the clinically relevant model of pressure-overload–induced heart failure. In response to exogenous angiotensin II andpressure overload, ACE2 levels were decreased, thereby perpetuating the pathological effects of angiotensin II. The directeffects of angiotensin II on adult ventricular cardiomyocytes and cardiac fibroblasts were suppressed by recombinanthuman ACE2 in an angiotensin 1-7–dependent manner. Recombinant human ACE2 can provide a novel therapeuticapproach for patients with cardiovascular disease.




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Hans Loibner, Xiu-hua Wang, Josef M. Penninger, Zamaneh Kassiri and Gavin Y. OuditJiuChang Zhong, Ratnadeep Basu, Danny Guo, Fung L. Chow, Simon Byrns, Manfred Schuster,

Fibrosis, and Cardiac DysfunctionAngiotensin-Converting Enzyme 2 Suppresses Pathological Hypertrophy, Myocardial

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Zhong et al. ACE2 Suppresses Pathological Myocardial Remodeling

SUPPLEMENTAL MATERIAL

Angiotensin-Converting Enzyme 2 Suppresses Pathological Hypertrophy,

Myocardial Fibrosis and Cardiac Dysfunction

by

JiuChang Zhong MD, Ratnadeep Basu MD, Danny Guo BSc, Fung L. Chow MSc, Simon Byrns BSc, Manfred Schuster PhD, Hans Loibner PhD, Xiu-hua Wang PhD, Josef M. Penninger MD,

Zamaneh Kassiri PhD and Gavin Y. Oudit MD, PhD

Supplemental Methods

Tail-Cuff Systolic Blood Pressure. For the measurement of tail-cuff systolic blood pressure (TC-SBP),

conscious mice were placed in the restrainers and their body temperature was maintained at ~ 34 °C by the

warming chamber. The IITC tail cuff sensor containing both the inflation cuff and the photoelectric sensor

was placed on the tail and attached to the restrainer. The cuff was inflated to a pressure of 200 mmHg and

then deflated slowly. Upon reappearance of pulse signals, TC-SBP data from the IITC amplifier was

recorded, analyzed and reported by the IITC software (IITC Life Science Blood Pressure System,

Woodland Hills, CA). The mice were trained on three occasions before actual recordings were made and

the corresponding TC-SBPs were averaged from three readings and used for the averaged comparisons.

Echocardiography and Tissue Doppler Imaging. Transthoracic echocardiography was performed and

analyzed in a blinded manner as described previously using a Vevo 770 high-resolution imaging system

equipped with a 30-MHz transducer (RMV-707B; VisualSonics, Toronto, Canada).1-3 Mice were

anesthetized with 0.75% isoflurane for the duration of the recordings. M-mode images were obtained for

measurements of LV wall thickness (LVWT), LV end-diastolic diameter (LVEDD) and LV end-systolic

diameter (LVESD) (measures of LV dilation). LV fractional shortening (FS) was calculated as FS


(%)=(LVEDD–LVESD)/LVEDD)x100 and LV ejection fraction (EF) (%)=(LVEDV-

LDESV)/LVEDVx100 as measures of systolic function. Diastolic function was assessed using pulsed-wave

Doppler imaging of the transmitral filling pattern with the early transmitral filling wave (E-wave) followed

by the late filling wave due to atrial contraction (A-wave). Isovolumetric relaxation time (IVRT) was

calculated as the time from closure of the aortic valve to initiation of the E-wave. The deceleration time

(DT) of the E-wave was determined by measuring the time needed for the down-slope of the peak of the E-

wave to reach the baseline while the rate of E-wave deceleration rate (EWDR) was calculated as the E-wave

divided by the DT. Tissue Doppler imaging (TDI) was made at the mitral valve annulus in the modified

four-chamber view at the base of the LV with the assessment of peak annular systolic (S’), early diastolic

(E’) and late diastolic (A’) peak annular velocities.4, 5 The TDI technique represents a novel and validated

technique to assess systolic and diastolic function with reduction in E’ and E’/A’ ratio and elevation in E/E’

being considered a valid marker of elevated LV filling pressure and diastolic dysfunction. 4, 5

Invasive Pressure-Volume Measurements. Invasive pressure-volume measurements were made in non-

intubated and anesthesized mice (1% isoflurane mixed with 100% O2) using the SPR-839 microtip catheter

and the Millar PV system (MPVS-400, Millar Instruments, Texas) as previously described.6 The catheter

was inserted into the LV via the right carotid artery in a spontaneously breathing closed chest anesthesized

mouse. Inferior vena cava occlusion and calibrations to convert the raw conductance signals to true volumes

were carried out as previously described.6 At the end of 14-days of vehicle or Ang II infusion (1.5 mg.kg-

1.d-1) in WT mice, osmotic pumps were removed for 2 hrs prior to invasive hemodynamic measurements.

Histology. For heart morphometry, hearts were arrested with 1M KCl, perfuse-fixed with buffered 10%

formalin, and embedded in paraffin. Trichrome and Picro-sirius red (PSR) staining and visualization were

carried out as previously described.7, 8 Trichrome-stained sections were used for assessment of overall tissue

architecture and interstitial and perivascular fibrosis. PSR staining of kidney sections were used to assess

for tubulointerstitial fibrosis.

Generation and Characterization of Human Recombinant ACE2. The extracellular domain of human

ACE2 (amino acid residues 1-740, MW=101 kDa)9 was expressed recombinantly in CHO cells under serum


free conditions in a chemically defined medium. The expression product was purified to homogeneity by

applying a capture step on a DEAE Sepharose® anion exchanger resin. The eluted fractions containing the

expression product were submitted to a polishing step on a Superdex® 200 gel filtration column. The

expression product was compared to the commercially available ACE2 standard 933-ZN (R&D Systems).

Chemical and immunological properties of both products were almost identical while rhACE2 showed a

93% enzymatic activity with Mca-APK-(Dnp)-OH substrate in comparison to rhACE2 standard 933-ZN

(R&D Systems). The enzymatic turnover of rhACE2 with Ang II substrate was 5.2±0.1 µmol.mg-1.min-1

and the elimination half-life of rhACE2 was 10.4 hrs in Rhesus monkeys. The purity of the expression

product was 99.99% as measured by HPLC.

HPLC methods, Gel Chromatography and Western Blot Analysis for rhACE2. The expression product

was purified to homogeneity by applying a capture step on a DEAE Sepharose® anion exchanger resin. The

eluted fractions containing the expression product were submitted to a polishing step on a Superdex® 200

gel filtration column. The expression product was analyzed by SEC-HPLC using a Zorbax GF250 column

at a flow rate of 1 ml/min (Supplemental Fig. 2A). Running buffer was 220 mM Na2HPO4 supplemented

with 10% CH3CN, pH:6.8, sample dilution was 1:25 and injected volume was 50µl. ACE2 eluted as a sharp

single peak at 8.4 minutes. This retention time correlates with a molecular mass of nearly 297 kDa in

comparison to GF-Standard (Biorad). The calculated molecular weight of the glycosylated protein

corresponds to 85 kDa. Integration of the signal monitored at 214 nm indicates a purity >99%, the signal

monitored at 280 nm showed a purity >99%.

Recombinant human ACE2 was analyzed by reducing SDS-PAGE (NOVEX, Tris-Glycine Gel 4-

12%) (Supplemental Fig. 2B). 300 ng of the final purified product (Lane B) is compared to 300 ng ACE2

standard (Lane A, 933-ZN, R&D Systems). Recombinant ACE2 appeared as single protein band of a

molecular weight between 120 and 100 kDa while ACE2 standard was double banded. Band intensities of

both products were similar. Purity of rhACE2 was >98%. Identity of protein bands visualized in SDS-

PAGE was confirmed immunologically by Western blot analysis (Supplemental Fig. 2C) using the murine

monoclonal ACE2 specific antibody MAB933 (R&D Systems) as the detection antibody. The expression

product showed a distinct band in the range between 120 and 100 kDa.


Plasma ACE2 Activity. Human recombinant ACE2 (1 µg/ml) preincubated with 2 µM DX600 was added

to normal murine plasma and plasma obtained from healthy volunteers and plasma ACE2 activity was

measured as previously described.10 Fluorogenic substrate concentration was 100 µM (final) and reaction

volume (final) 100µl (50 µl fluorogenic peptide solution, 50 µl enzyme in plasma with or without DX600 or

buffer). Plasma ACE2 activity obtained from mice treated daily with 2 mg/kg i.p. of rhACE2 was also

measured at 1 week and 2 weeks. Plasma was collected at 3-4 hours following the last dose of hrACE2.

Isolation and Culture of Adult Cardiomyocytes and Cardiofibroblasts. Adult murine left ventricular

cardiomyocytes and cardiofibroblasts were isolated and cultured as previously described.11 Briefly, 11-week

old mice were injected with 0.05 ml of 1000USP/ml heparin for 15 min and then anesthetised using 2%

isoflurane (1 L/min oxygen flow rate) provided through a nose cone. After opening the chest cavity, the

heart was quickly excised and perfused using a Langendorff system within 45 s. Following 3-min perfusion,

the heart was then digested with 2.4 mg/mL collagenase type 2 (Worthington) for 7-8 min. After sufficient

digestion, the ventricles were removed, dissociated using forceps and transfer pipettes, and resuspended in

stopping buffer (10% FBS perfusion buffer). The isolated cardiomyocytes were then exposed to increasing

calcium concentrations (100 µM, 400 µM, and 900 µM) for 15 min each before being plated onto laminin

coated culture dishes in plating buffer (Eagle’s MEM with 10% FBS, Sigma) and placed at 37 ℃ in a sterile

2% CO2 incubator. The discarded stopping buffer was set aside for cardiofibroblasts collection. One hour

after plating, the plating buffer was gently aspirated and replaced with culture buffer (serum free Eagle’s

MEM with 0.1% BSA) and then placed into the incubator for 18 h before treatment. The discarded stopping

buffer is centrifuged at 20 g for 3 min and the resulting supernatant was collected in a 15 mL conical tube.

This was then centrifuged at 1500 rpm for 5 min and the pellet was collected and washed in 10% FBS

DMEM (GIBCO). The solution is once again centrifuged at 1500 rpm for 5 min and the pellet was collected

and plated onto a 10 cm culture dish in 10% FBS DMEM. The cardiofibroblasts were then passaged 2 times

and put into 24 h serum free DMEM prior to treatment. Ang 1-7 antagonist D-Ala7-Ang 1-7 (A779,

Bachem Co.; 10 μM) and rhACE2 (0.2 mg/mL or 2 mg/mL) were added to the cardiomyocytes and

cardiofibroblasts for 2 h and 30 min prior to 30-min exposure of Ang II (Sigma; 100 nM), respectively. The


treated cells were used for dihydroethidium (DHE) staining or collected for TaqMan real-time PCR,

Western bloting analysis or lucigenin-enhanced chemiluminescence assay.

TaqMan Real-time PCR. RNA expression levels of various genes were determined by TaqMan real-time

PCR as previously described.7, 12 Total RNA was extracted from flash-frozen tissue or cardiofibroblasts

using TRIzol, and cDNA was synthesized from 1 μg RNA by using a random hexamer. For each gene, a

standard curve was generated using known concentrations of cDNA (0.625, 1.25, 2.5, 5, 10 and 20 µg) as a

function of cycle threshold (CT). Expression analysis of the reported genes was performed by TaqMan real-

time-PCR using ABI 7900 Sequence Detection System. The SDS2.2 software (integral to ABI7900 real-

time machine) fits the CT values for the experimental samples and generates values for cDNA levels. All

samples were run in triplicates in 384 well plates. 18S rRNA was used as an endogenous control. The

primers and probes for mRNA expression analysis by Taqman realtime PCR are listed in Supplementary

Table 1 (see below).

Dihydroethidium Fluorescence. Dihydroethidium (DHE), an oxidative fluorescent dye, was used to

measure superoxide (O2–) levels in cultured cardiomyocytes and heart tissues from ACE2KO and WT mice

as previously described.12 Briefly, the cultured and treated cardiomyocytes were washed with clear media

(Eagle’s MEM) and then incubated at 37 °C for 30 min with DHE (20 μM) in clear media. For heart

samples, 20 μm fresh frozen tissue sections were washed with hanks balanced salt solution (HBSS) with

magnesium and calcium and then incubated at 37 °C for 30 min with DHE (20 μM) in HBSS. For a

separated experiment, heart tissue sections from mice with Ang II pumps or Ang II-treated cardiomyocytes

were incubated with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (500U/mL) at 37 °C

for 30 min prior to 30-min exposure of DHE (20 μM). In addition, one cell plate or one tissue slide was kept

without DHE for blank control. The cell plates or tissue slides were wrapped with foil to minimize them

exposure to light. Fluorescent images were observed with an Olympus Fluoview laser-scanning confocal

microscope mounted on an Olympus microscope selected with CY3 (red) channel.

Lucigenin-Enhanced Chemiluminescence. The activities of nicotinamide adenine dinucleotide phosphate

(NADPH) oxidase in cultured cardiomyocytes and hearts of mice were quantified by lucigenin-enhanced


chemiluminescence as previously described.8, 12 Briefly, the cardiomyocytes and heart homogenates (200 μg

total proteins) were collected in 100 μl of phosphate buffer solution (PBS) mixture with phosphatase

inhibitor and protease inhibitor and then centrifuged at 1000 g for 10 min. The supernatants were then

collected and added NADPH (1mM) and lucigenin (50 μM) for NADPH oxidase activities assay with FB-

12 luminometer in the presence or absence of diphenylene iodonium (DPI, 10 μM), a selective inhibitor of

flavin-containing enzymes including NADPH oxidase. Data were calculated as the change in the rate of

luminescence per minute per milligram of cardiomyocytes or tissue.

Western Blot Analysis. The phosphorylated and/or total proteins from cultured cells and heart tissues of

mice were measured by Western blot analysis as previously described.12, 13 To gain insight of the regulatory

mechanisms of ACE2, we investigated a number of intracellular signaling pathways known to be associated

with fibrotic and hypertrophic remodeling. Protein was extracted in 25 mM Tris, 62.5 mM NaCl, 1.25 mM

PMSF, 62.5 mM Glycerol-2-phosphate, 12.5 mM sodium pyrophosphate, 125 μM NaF, 6.25 μg/ml

leupeptine, 312.5 μM sodium orthovanadate, 12.5% glycerol, pH 7.4, supplemented with 5% sodium

dodecyl sulphate (SDS) and 1% Triton X-100. After quantification using the BCA Protein Array Kit

(Pierce, Rockford, IL, USA), protein samples were separated by 8%~12% SDS-polyacrylamide gel

electrophoresis and then transferred to nitrocellulose membrane (Millipore). The membrane was blocked

with 5% milk in Tris-Buffered Saline Tween-20 (TBST) for 2 h and then incubated overnight at 4 ℃ with

primary antibody against ACE2 (90 KD), PKCα (80 kDa), PKCβ1 (79 kDa), α-tubulin (55 kDa) and total

and phosphorylated ERK1 (44 kDa), ERK2 (42 kDa), p38-MAPK (38 kDa), JAK2 (130 kDa) and STAT3

(80 kDa) (Santa Cruz and Cell Signaling Inc.). The primary antibody was then removed, and membrane

washed thoroughly with TBST. The membrane was then incubated with goat anti-rabbit IgG coupled to

horseradish peroxidase (HRP) at a 1:3000 dilution in TBST for l h at room temperature, then rinsed

thoroughly with TBST as before. Aim proteins were detected by ECL using X-O-Mat X-ray film and

analyzed by the ImageJ software (U.S. National Institutes of Health, Bethesda, MD).


Supplemental Tables

Supplemental Table 1. Primers and probes sequences for TaqMan real-time PCR analysis*.

Genes Primers /Probes Sequences (Primer: 5'-3'; Probe: 5'-FAM- -TAMRA-3')

BNP Forward primer Reverse primer Probe

CTGCTGGAGCTGATAAGAGA TGCCCAAAGCAGCTTGAGAT CTCAAGGCAGCACCCTCCGGG

ß-MHC Forward primer Reverse primer Probe:

GTGCCAAGGGCCTGAATGAG GCAAAGGCTCCAGGTCTGA ATCTTGTGCTACCCAGCTCTAA

α-SA Forward Primer Reverse Primer Probe

CAGCCGGCGCCTGTT CCACAGGGCTTTGTTTGAAAA TTGACGTGTACATAGATTGACTCGTTTTACCTCATTTTG

Pro- collagen I-α1

Forward primer Reverse primer Probe

CTTCACCTACAGCACCCTTGTG TGACTGTCTTGCCCCAAGTTC CTGCACGAGTCACACC

Pro-collagen III-α1


TGTCCTTTGCGATGACATAATCTG AATGGGATCTCTGGGTTGGG ATGAGGAGCCACTAGACT

TGFβ1


CCTGCAAGACCATCGACATG ACAGGATCTGGCCACGGAT CTGGTGAAACGGAAGCGCATCGAA

MMP-2 Forward Primer: Reverse Primer: Probe:

AACTACGATGATGACCGGAAGTG TGGCATGGCCGAACTCA TCTGTCCTGACCAAGGATATAGCCTATTCCTCG

ADAM-12 Forward Primer: Reverse Primer: Probe:

GAAACCCCCATCCTTGACAA TTCCACCAGAAGAACCAGTCTTTAA AGTCCACCATCAACTTTCTGTCACCAAAGC

* Primer/probe mix for Fibronectin (product #: Mm01256742_m1) and α-Smooth muscle actin (αSMA) (product #: Mm00725412_S1) were purchased from Applied Biosystems Inc. BNP, brain natriuretic peptide; βMHC, β-myosin heavy chain; αSA, α-skeletal actin; TGFβ1, transforming growth factor-beta 1.


Supplemental Table 2. Echocardiographic assessment of cardiac function in WT and ACE2KO mice infused with a pressor dose of Ang II (1.5 mg.kg-1.d-1).

WT+Vehicle WT+Ang II ACE2KO+

Vehicle ACE2KO+

Ang II n 9 12 9 12

HR (bpm) 497±13 505±19 481±21 494±15

E-wave (mm/s) 743±27 722±29 743±35 545±31*,†

A-wave (mm/s) 434±16 539±18* 438±23 431±19

E/A Ratio 1.71±0.1 1.33±0.14* 1.7±0.14 1.27±0.15*

IVRT (ms) 13.5±0.8 14.5±1.2 12.1±1.3 22.4±1.7*,†

DT (ms) 27.9±1.6 25.2±1.8 28.7±1.5 30.5±1.9

EWDR (mm/s2) 26.6±2.4 28.7±3.1 25.9±2.4 17.8±2.8

E′ (mm/s) 25.6±2.4 15.7±2.5* 28.3±3.1 16.1±1.6*

E/E′ Ratio 29±3.1 46±3.4* 26.3±2.7 31.8±4.2

A′ (mm/s) 16.1±1.3 19.3±2.3 18.1±1.4 26.2±2.4*

E′ /A′ Ratio 1.5±0.09 0.85±0.07* 1.56±0.11 0.62±0.06*,†

LVEDD (mm) 3.73±0.09 3.42±0.1 3.72±0.09 3.51±0.12

LVESD (mm) 2.45±0.07 2.23±0.07 2.48±0.07 2.24±0.08

LVFS (%) 34.3±1.9 34.8±2.9 33.3±2.3 36.2±2.2

LVEF (%) 63.3±3 64.9±3.4 62.7±2.6 66.4±2.9

VCFc (circ/s) 6.49±0.19 6.56±0.26 6.52±0.21 6.74±0.34

LVPWT (mm) 0.67±0.05 0.94±0.08* 0.69±0.06 1.13±0.06*,†

HR, heart rate; E-wave, peak early transmitral inflow mitral E velocity; A-wave, transmitral inflow velocity due to atrial contraction; IVRT, isovolumetric relaxation time; DT, deceleration time; EWDR, E-wave deceleration rate (E-wave/DT); E′, early diastolic tissue Doppler velocity; LVEDD, left ventricular (LV) end diastolic diameter; LVESD, LV end systolic diameter; LVFS, LV fractional shortening; LVEF, LV ejection fraction; VCFc, Velocity of circumferential shortening corrected for heart rate; LVPWT, LV posterior wall thickness. Results are presented as mean±S.E.M. *p<0.05 compared with corresponding vehicle group; †p<0.05 compared with WT+Ang II group.


Supplemental Table 3. Echocardiographic assessment of cardiac function in WT mice infused with a pressor dose of Ang II (1.5 mg.kg-1.d-1) and in response to treatment with rhACE2 (2 mg.kg-1.d-1).

WT+Vehicle+ Placebo

WT+Vehicle+ rhACE2

WT+Ang II+ Placebo

WT+Ang II+rhACE2

n 9 9 12 12

HR (bpm) 493±12 508±14 502±17 496±15

E-wave (mm/s) 749±18 758±15 718±23 742±18

A-wave (mm/s) 431±15 430±21 543±16* 442±19

E/A Ratio 1.74±0.11 1.76±0.14 1.31±0.12* 1.69±0.14

IVRT (ms) 13.7±0.6 13.2±0.9 14.8±1.1 14.8±1.1

DT (ms) 28.3±1.4 26.8±1.7 25±1.7 27.9±1.3

EWDR (mm/s2) 26.4±2.1 25.3±2.5 28.7±3.2 25.9±2.9

E′ (mm/s) 24.1±2.1 25.8±2.8 15.9±2.2* 27.3±3.1

E/E′ Ratio 31.1±3.3 29.4±3.1 45.2±3.2* 27.2±2.5

A′ (mm/s) 16.4±1.1 17.1±2.4 18.4±1.9 20.7±2.3

E′/A′ Ratio 1.47±0.07 1.51±0.1 0.86±0.08* 1.32±0.11

LVEDD (mm) 3.76±0.08 3.77±0.09 3.43±0.1 3.72±0.12

LVESD (mm) 2.49±0.06 2.51±0.05 2.29±0.08 2.5±0.06

LVFS (%) 33.8±1.9 33.4±2.1 33.2±2.9 32.8±3.1

LVEF (%) 62.3±3 61.7±2.5 63.9±3.4 62.1±3.1

VCFc (circ/s) 6.45±0.17 6.36±0.14 6.61±0.23 6.42±0.19

LVPWT (mm) 0.68±0.05 0.67±0.06 0.95±0.08* 0.72±0.08

see supplemental Table 2 for abbreviations. Results are presented as mean±S.E.M. *p<0.05 compared with all other groups.


Supplemental Table 4. Echocardiographic assessment of cardiac function in WT mice infused with a subpressor dose of Ang II (0.15 mg.kg-1.d-1) and in response to treatment with rhACE2 (2 mg.kg-1.d-1).

WT+Vehicle+

Placebo WT+Vehicle+

rhACE2 WT+Ang II+

Placebo WT+Ang II+

rhACE2 N 9 9 10 10

HR (bpm) 481±13 476±15 473±17 484±15

E-wave (mm/s) 744±26 738±30 714±17 740±28

A-wave (mm/s) 426±17 422±16 463±23 428±22

E/A Ratio 1.75±0.13 1.75±0.11 1.54±0.08* 1.73±0.07

IVRT (ms) 13.7±0.8 14.1±0.9 13.4±1 13.2±0.21

DT (ms) 27.9±1.4 26.8±1.6 26.6±1.4 25.1 ±0.52

EWDR (mm/s2) 26.7±2.1 27.5±1.8 27±1.9 28.3±1.5

E′ (mm/s) 23.6±1.9 24.9±2.3 22.1±0.8 26.8±1.8

E/E′ Ratio 31.5±3.2 29.6±3.2 32.6±1.3 27.6 ±1.3

A′ (mm/s) 16.1±1.2 17.3±1.7 24.4±1.9 18.9±2

E′ /A′ Ratio 1.47±0.09 1.44±0.08 0.9±0.05* 1.38±0.07

LVEDD (mm) 3.72±0.09 3.71±0.1 3.67±0.12 3.73±0.08

LVESD (mm) 2.47±0.06 2.41±0.07 2.42±0.1 2.48±0.09

LVFS (%) 33.6±2.1 35±2.9 32.4±1.3 33.5±1.4

LVEF (%) 63.8±3.2 64.9±3.4 62.4±2.1 63.1±1.9

VCFc (circ/s) 6.42±0.21 6.53±0.23 6.37±0.27 6.2±0.18

LVPWT (mm) 0.67±0.05 0.66±0.05 0.74±0.03* 0.65±0.03

see supplemental Table 2 for abbreviations. Results are presented as mean±S.E.M. *p<0.05 compared with all other groups.

Minutes0 2 4 6 8 10 12 14 16 18 20

mA

U

0

50

100

150

200

250

300

0

50

100

150

200

250

300

8.38

Minutes

0 2 4 6 8 10 12 14 16 18 20

mA

U

0

10

20

30

0

10

20

30

8.38

12.1

5

120100

90

kDa

807060

50

a b

A

B C

Zhong et al. Supplemental Figure 1

human ACE2 (1) --------------------------------------------------MSSSSWLLLSLVAVTAAHSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNY mouse ACE2 (1) --------------------------------------------------MSSSSWLLLSLVAVTTAQSLTEENAKTFLNNFNQEAEDLSYQSSLASWNY rat ACE2 (1) --------------------------------------------------MSSSCWLLLSLVAVATAQSLIEEKAESFLNKFNQEAEDLSYQSSLASWNY human ACE2 (51) NTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDN-PQECLLLEPGLN mouse ACE2 (51) NTNITEENAQKMSEAAAKWSAFYEEQSKTAQSFSLQEIQTPIIKRQLQALQQSGSSALSADKNKQLNTILNTMSTIYSTGKVCNPKN-PQECLLLEPGLD rat ACE2 (51) NTNITEENAQKMNEAAAKWSAFYEEQSKIAQNFSLQEIQNATIKRQLKALQQSGSSALSPDKNKQLNTILNTMSTIYSTGKVCNSMN-PQECFLLEPGLD human ACE2 (150) EIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLM mouse ACE2 (150) EIMATSTDYNSRLWAWEGWRAEVGKQLRPLYEEYVVLKNEMARANNYNDYGDYWRGDYEAEGADGYNYNRNQLIEDVERTFAEIKPLYEHLHAYVRRKLM rat ACE2 (150) EIMATSTDYNRRLWAWEGWRAEVGKQLRPLYEEYVVLKNEMARANNYEDYGDYWRGDYEAEGVEGYNYNRNQLIEDVENTFKEIKPLYEQLHAYVRTKLM human ACE2 (250) NAY-PSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTA mouse ACE2 (250) DTY-PSYISPTGCLPAHLLGDMWGRFWTNLYPLTVPFAQKPNIDVTDAMMNQGWDAERIFQEAEKFFVSVGLPHMTQGFWANSMLTEPADGRKVVCHPTA rat ACE2 (250) EVY-PSYISPTGCLPAHLLGDMWGRFWTNLYPLTTPFLQKPNIDVTDAMVNQSWDAERIFKEAEKFFVSVGLPQMTPGFWTNSMLTEPGDDRKVVCHPTA human ACE2 (349) WDLGKG-DFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIV mouse ACE2 (349) WDLGHG-DFRIKMCTKVTMDNFLTAHHEMGHIQYDMAYARQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLPSDFQEDSETEINFLLKQALTIV rat ACE2 (349) WDLGHG-DFRIKMCTKVTMDNFLTAHHEMGHIQYDMAYAKQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLPSNFQEDNETEINFLLKQALTIV human ACE2 (448) GTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGP------LHK mouse ACE2 (448) GTLPFTYMLEKWRWMVFRGEIPKEQWMKKWWEMKREIVGVVEPLPHDETYCDPASLFHVSNDYSFIRYYTRTIYQFQFQEALCQAAKYNGS------LHK rat ACE2 (448) GTLPFTYMLEKWRWMVFQDKIPREQWTKKWWEMKREIVGVVEPLPHDETYCDPASLFHVSNDYSFIRYYTRTIYQFQFQEALCQAAKHDGP------LHK human ACE2 (542) CDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMY mouse ACE2 (542) CDISNSTEAGQKLLKMLSLGNSEPWTKALENVVGARNMDVKPLLNYFQPLFDWLKEQNRNSFVGWNTEWSPYADQSIKVRISLKSALGANAYEWTNNEMF rat ACE2 (542) CDISNSTEAGQKLLNMLSLGNSGPWTLALENVVGSRNMDVKPLLNYFQPLFVWLKEQNRNSTVGWSTDWSPYADQSIKVRISLKSALGKNAYEWTDNEMY human ACE2 (642) LFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSI mouse ACE2 (642) LFRSSVAYAMRKYFSIIKNQTVPFLEEDVRVSDLKPRVSFYFFVTSPQNVSDVIPRSEVEDAIRMSRGRINDVFGLNDNSLEFLGIHPTLEPPYQPPVTI rat ACE2 (642) LFRSSVAYAMREYFSREKNQTVPFGEADVWVSDLKPRVSFNFFVTSPKNVSDIIPRSEVEEAIRMSRGRINDIFGLNDNSLEFLGIYPTLKPPYEPPVTI human ACE2 (742) WLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF mouse ACE2 (742) WLIIFGVVMALVVVGIIILIVTGIKGRKKKNETKREENPYDSMDIGKGESNAGFQNSDDAQTSF rat ACE2 (742) WLIIFGVVMGTVVVGIVILIVTGIKGRKKKNETKREENPYDSMDIGKGESNAGFQNSDDAQTSF




Parameter Units WT+Vehicle+Placebo

WT+Vehicle+rhACE2

WT+Ang II+Placebo

WT+Ang II+rhACE2

N 5 5 8 8

SYSTOLIC

+dP/dt,max mmHg/s 10213±452 10092±544 99867±402 10623±622

dP/dt/EDV mmHg/s/uL 368±82 376±68 397±74 361±57

EF % 58.9±3.5 56.8±2.9 62.5±4.1 61.8±5.1

CO mL/min 13.6±1.9 14.5±2.4 13.5±2.6 12.8±2.1

PRSW mmHg 69.5±6.8 74.5±6 65.7±4.9 72.3±8.9

ESPVR mmHg/uL 12.1±2.1 10.4±1.7 14.5±2.9 13.1±2.9

DIASTOLIC

LVEDP mmHg 7.63±1.31 8.12±1.24 12.9±2.1* 7.94 ±1.86

‐dP/dt,min mmHg/s 94560±375 90081±411 91445±370 96441±393

EDPVR(Stiffness)

mmHg/uL 0.34±0.03 0.29±0.04 0.56±0.06* 0.37±0.05

Tau (Glanz) ms 7.16±1.21 7.03±1.38 11.3±1.9* 8.04±1.45




Supplemental Figure Legends Supplemental Figure 1. Biochemical characterization of recombinant human ACE2. (A) SEC-HPLC chromatogram of rhACE2 analyzed on a Zorbax GF250 column. The effluent was monitored online at 214 (left) and 280 nm (right). (B) SDS-PAGE analysis of recombinant human ACE2, 15 µl of 20 µg/ml samples were loaded: (a) ACE2 standard 933-ZN ACE2 and (b) final purified expression product. (C) Western Blot analysis of the same samples: protein bands were identified by chemiluminescence. Supplemental Figure 2. Protein sequence alignment of human, mouse and rat ACE2. Blackened shading indicates amino acid identity and gray shading indicates degree of amino acid similarity. Based on published sequence by Crackower MA et al. Nature 2002 and Tipnis SR et al. J Biol Chem 2000. Supplemental Figure 3. Plasma ACE2 activity. (A-B) Fluorescence-based assay of plasma ACE2 activity in mouse and human plasma in response to 1 ug/ml of rhACE2 and its suppressibility by the specific ACE2 inhibitor, DX600 (2 µM) (A) which is quantified and shown in B. (C) Plasma ACE2 activity in mice injected with placebo or rhACE2 (2 mg.kg-

1.d-1) for 1 week and 2 weeks and sampled at 3-4 hrs following the last injection. n=6 for each group. *p<0.05 compared with corresponding control group; #p<0.05 compared with the APN01 group. APN01=rhACE2. Supplemental Figure 4. Invasive pressure-volume measurements using a closed-chest approach in mice treated with high dose Ang II (1.5 mg.kg-1.d-1 x 2 weeks) and rhACE2 (2 mg.kg-1.d-1; i.p.) showing Ang II-induced diastolic dysfunction with preserved systolic function which was prevented by treatment with rhACE2.*p<0.05 compared with all other groups. +dP/dtmax, peak rate of LV pressure rise; CO=Cardiac output; EF=Ejection fraction; PRSW=Preload recruited stroke work; ESPVR=End-systolic pressure-volume relationship; EDPVR=End-diastolic pressure- volume relationship, LVEDP=LV end-diastolic pressure; -dP/dtmin, peak rate of LV pressure decline and Tau (G), relaxation time constant calculated by Glantz method. Supplemental Figure 5. Suppression of Ang II (1.5 mg.kg-1.d-1) induced renal fibrosis by treatment with rhACE2. (A-D) Realtime Taqman PCR analysis showing marked increase in the expression of pro-collagen I-α1 (A), pro-collagen III-α1 (B), α-smooth muscle actin (C) and transforming growth factor-beta 1 (D) in response to Ang II which was suppressed by rhACE2. (E-G) Western blot analysis of collagen I (E) and collagen III (F) and Picrosirius red staining of kidney sections (G) showing increased collagen formation and tubulointerstitial fibrosis in response to Ang II which was completely suppressed by rhACE2. n=6 for WT+Vehicle+Placebo, WT+ Vehicle+rhACE2; n=8 for WT+Ang II+Placebo, WT+Ang II+rhACE2. *p<0.05 compared with WT+Vehicle+Placebo group; #p<0.05 compared with WT+Ang II+Placebo group. Supplemental Figure 6. Tail-cuff systolic blood pressure in wildtype mice treated with subpressor dose of Ang II (0.15 mg.kg-1.d-1 x 2 weeks) (n=6) or placebo (n=6) showing no significant elevation in systolic blood pressure over the two week period.


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