Nature volume 417, pages822–828 (2002)Cite this article
Abstract
Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and upregulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.
Main
Cardiovascular disease will be the greatest health care burden of the twenty-first century1. A major risk factor for heart disease is high blood pressure2. Hypertension is a multifactorial quantitative trait controlled by both genetic and environmental factors3. While much is known about environmental factors that can contribute to high blood pressure, such as diet and physical activity, less is known about the genetic factors that are responsible for predisposition to cardiovascular disease. Despite the identification of several putative genetic quantitative trait loci (QTL) associated with hypertension in animal models4, none of these loci have been translated into genes5. Thus, the molecular and genetic mechanisms underlying hypertension and other cardiovascular diseases remain obscure.
One important regulator of blood pressure homeostasis is the renin–angiotensin system (RAS). The protease renin cleaves angiotensinogen into the inactive decameric peptide angiotensin I (AngI). Angiotensin-converting enzyme (ACE) then catalyses the cleavage of the AngI into the active octomer angiotensin II (AngII), which can contribute to hypertension by promoting vascular smooth muscle vasoconstriction and renal tubule sodium reabsorption6,7. ACE mutant mice display spontaneous hypotension, partial male infertility, and kidney malformations8,9. In humans, an ACE polymorphism has been associated with determinants of renal and cardiovascular function10, and pharmacological inhibition of ACE and AngII receptors are effective in lowering blood pressure11 and preventing kidney disease12. In addition, inhibition of ACE and AngII receptors has beneficial effects in heart failure13.
Recently, a homologue of ACE, termed ACE2, has been identified; it is predominantly expressed in the vascular endothelial cells of the kidney and heart14,15. Unlike ACE, ACE2 functions as a carboxypeptidase, cleaving a single residue from AngI, generating Ang1-9 (refs 14, 15), and a single residue from AngII to generate Ang1-7 (ref. 14). These in vitro biochemical data suggest that ACE2 may modulate the RAS and thus affect blood pressure regulation. In addition, it has been shown that ACE2 can cleave other peptide substrates14,15. Two different ACE2 homologues have been identified in flies16,17. Nevertheless, the in vivo role of ACE2 in the cardiovascular system and the RAS is not known.
ACE2 maps to a QTL on the X chromosome in hypertensive rats
Hypertension and most cardiovascular diseases are multifactorial in nature and disease pathogenesis is influenced by multiple genetic susceptibility loci3. In various recombinant rat models, several QTL for hypertension have been identified. ace2 maps to the X chromosome in human14 and a QTL has been mapped to the X chromosome in several rat models of hypertension with no candidate gene ascribed to it as yet18,19,20. Angiotensin receptor II, which also maps to the X chromosome, has previously been excluded from this candidate interval21.
We speculated that ace2 could be a candidate gene for this QTL. Radiation hybrid mapping showed that the rat ace2 gene maps on the X chromosome with significant logarithm-of-odds (LOD) scores to markers DXRat9, DXWox14, DXWox15 and DXRat42, placing ace2 between DXRat9 and DXRat42 (Fig. 1a). Comparative mapping showed that the ace2 map position overlaps with a QTL interval for hypertension identified in Sabra salt-sensitive rats found between markers DXMgh12 and DXRat8 (SS-X) (ref. 18). Moreover, the chromosomal ace2 region maps to the BP3 QTL interval defined in stroke-prone spontaneously hypertensive rats (SHRSP) rats19, and the hypertensive BB.Xs QTL identified on the X chromosome of spontaneous hypertensive rats (SHR) by congenic analysis20 (Fig. 1a). Thus, ace2 maps to a QTL on the rat X chromosome that has been identified in three separate models of spontaneous and diet-induced hypertension.
Figure 1: Association of ACE2 and hypertension in the rat.
a, Results of radiation hybrid mapping of rat ace2, compared to the mapping of a QTL identified in Sabra salt-sensitive animals (SS-X), SHRSP (BP3), and SHR rats (BB.Xs). Polymorphic marker names are indicated to the left of the ideogram. LOD scores and theta values for markers linked to ace2 are shown. cR, centiradians. b, Northern blot analysis of ace2 mRNA from kidneys of Sabra SBH/y and SBN/y rats. c, Western blot analysis of ACE2 protein levels from kidneys of Sabra SBH/y and their control SBN/y rats, as well as SHR and SHRSP and their control WKY rats. Systolic blood pressure in mm Hg for the respective Sabra rats is indicated. Bars show mean values ± s.e.m. Asterisk, P < 0.05; double asterisk, P < 0.01 (n = 4, for all groups).
Downregulation of ACE2 expression in hypertensive rats
Because the kidney is a major site of blood pressure regulation22, we determined ace2 expression levels in the kidneys of these three hypertensive rat strains. We initially measured ace2 mRNA levels in the kidneys of salt-sensitive Sabra hypertensive (SBH/y) rats and control salt-resistant Sabra normotensive (SBN/y) rats. Salt loading (with deoxycorticosterone acetate (DOCA) salt) had no effect on ace2 mRNA expression in normotensive SBN/y rats. In SBH/y rats, salt loading and the development of hypertension were associated with a significant reduction in ace2 mRNA expression compared to normotensive SBN/y rats (Fig. 1b). ace2 mRNA was also lower in SBH/y rats fed a regular diet (‘normal chow’) when compared to SBN/y control rats fed a similar diet. This latter finding suggests that downregulation of ACE2 may be independent of blood pressure. However, the possibility that ACE2 expression is controlled by blood pressure cannot be excluded.
To measure ACE2 protein levels, we generated ACE2 (amino acids 206–225 of mouse ACE2) specific rabbit antiserum, which cross-reacts with both rat and human ACE2 (not shown). In line with the decreased ACE2 mRNA expression, ACE2 protein expression was markedly reduced in SBH/y animals that were fed a normal diet (Fig. 1c). Increase in blood pressure of SBH/y rats following a 4-week diet of DOCA salt correlated with a further decrease in ACE2 protein expression (Fig. 1c). Salt loading did not trigger increased blood pressure23, nor did it alter ACE2 expression in salt-resistant SBN/y control rats (Fig. 1c). ACE2 protein levels were also significantly decreased in the kidneys of SHRSP and SHR rats as compared to their WKY controls (Fig. 1c). Moreover, the levels of ACE2 mRNA were markedly reduced in SHRSP and SHR rats (not shown). Cloning and sequencing of the coding region of ace2 in the hypertensive rat strains did not reveal any sequence changes, indicating that reduced ACE2 expression probably results from polymorphisms that control ace2 gene expression. The map position and reduced expression of ACE2 in three different rat strains indicate that ace2 is a strong candidate gene for this hypertensive QTL on the X chromosome.
Generation of ACE2 knockout mice
To validate the candidacy of ace2 as a QTL and to test whether ACE2 has indeed an essential role in cardiovascular physiology and the pathogenesis of cardiovascular disease, we disrupted the ace2 gene in mouse by homologous recombination (see Supplementary Information). The null mutation of ace2 was verified by the absence of ace2 mRNA transcripts and protein by northern (not shown) and western blot analyses (Fig. 2a). ace mRNA expression in the kidneys and hearts was not altered in ace2 mutant mice (Fig. 2b). ACE2 null mice were born at the expected mendelian frequency, appeared healthy, and did not display any gross detectable alterations in all organs analysed. Moreover, in contrast to ace-/- male mice that display significantly reduced fertility, both male and female ace2 null mice are fertile.
Figure 2: ACE2-deficient mice.
a, Western blot analysis of ACE2 protein expression in the kidneys of ace2+/y ace-/y mice. b, Polymerase chain reaction with reverse transcription of RNA (RT–PCR) analysis of ACE mRNA expression in the heart and kidneys of ace2+/y and ace2-/y mice. c, Blood pressure measurements in 3-month-old ace2+/y (n = 8) and ace2-/y (n = 8) mice in the absence (left panels) or presence of the ACE blocker captopril. Mean values ± s.e.m. are shown (double asterisk; P < 0.01). d, The top panels show haematoxylin and eosin stained sections of hearts isolated from 6-month-old ace2+/y and ace2-/y mice. We note the enlarged left and right ventricles in ace2-/y mice. However, the overall heart size is comparable between both genotypes and there is no evidence for cardiac hypertrophy macroscopically or in isolated cardiomyocytes). The bottom panels show an absence of interstitial fibrosis in ace2-/y mice. As shown by PSR staining (see Methods).
Normal blood pressure in ace2 mutant mice
It has been previously shown that ace mutant mice display reduced blood pressure and kidney pathology8,9. Therefore, we first tested whether loss of ACE2 expression affects blood pressure homeostasis and/or kidney development or function. Loss of ACE2 did not result in alteration of blood pressure in 3-month-old ace2-/y male (Fig. 2c) or ace2-/- female mice (not shown) as compared to their control littermates. Because it was possible that ACE could compensate for the loss of ACE2, we treated ace2-deficient mice with captopril, which blocks ACE but not ACE2 function14,15. However, in vivo inhibition of ACE with captopril reduced the blood pressure of ace-/y male mice to a similar extent as was observed in captopril-treated wild-type littermates (Fig. 2c). Thus, even in a scenario of ACE inhibition, loss of ACE2 has no apparent direct effect on blood pressure homeostasis. In addition, no alterations in kidney ultrastructure or function or anaemia could be detected (not shown).
Loss of ACE2 impairs heart function
Pharmacological inhibition of ACE or AngII receptors suggested a role for the RAS in the regulation of heart function and cardiac hypertrophy13. However, neither ace8,9 nor angiotensinogen24 null mice develop any overt heart disease. ACE2 is highly expressed in the vasculature of the heart, so we analysed hearts of ace2-deficient mice. Hearts of ace2 mutant mice display a slight wall thinning of the left ventricle and increased chamber dimensions (Fig. 2d). Thinning of the anterior left ventricular wall and increase in the left ventricle end diastolic dimension in ace2-deficient hearts can be also seen by echocardiography (Table 1). These structural changes are primarily observed in 6-month-old male mice. However, heart weights and heart to body weight ratios were comparable between age-matched 3-month-old (not shown) and 6-month-old ace2-/y and ace2+/y mice (see Supplementary Information). Echocardiography also showed that the left ventriclar-mass and its ratio to body weight were normal (Table 1). We also failed to observe structural and biochemical changes characteristic of dilated cardiomyopathy as there was no indication of interstitial cardiac fibrosis (Fig. 2d) nor prototypical changes in ANF, BNP, α-MHC, β-MHC and skeletal muscle actin gene expression (not shown). In addition, individual cardiomyocytes of ACE2 null mice exhibited no evidence of hypertrophy and we did not observe any evidence of altered cardiomyocyte apoptosis in ACE2 null mice as detected by TdT-dependent dUTP-biotin nick end labelling (TUNEL) staining (not shown). Thus, despite mild dilation of hearts in 6-month-old ACE2 null mice, there was no evidence of cardiac hypertrophy or dilated cardiomyopathy.
Table 1 Heart functions of ace2 null mice
Assessment of cardiac function by echocardiography revealed that all ace2-/y male and ace2-/- female mice exhibit severe reduction in cardiac contractility as determined by decreased left ventricle fractional shortening, and decreased velocity of circumferential fibre shortening (Table 1 and Fig. 3a, b). The decrease in function was found to be more severe in 6-month-old male than in age-matched female mice. In addition, 3-month-old male mice had a less pronounced phenotype than older animals (Table 1 and Fig. 3b), suggesting a progression in the phenotype (Table 1). Consistent with the decreased cardiac contractility, 6-month-old male ace2-/y mice exhibited reduced blood pressure (Fig. 3c), a feature not found in age-matched ace2-/- females and 3-month-old males. This suggests that the reduction in blood pressure may be the result of severe cardiac dysfunction and not a direct effect of loss of ACE2 on systemic blood pressure. To confirm the echocardigraphic defects in cardiac function, invasive haemodynamic measurements were performed in ace2 null mice. These invasive haemodynamic measurements showed that both the maximum and minimum rates of change of left ventricular pressure, (dP/dt)max and (dP/dt)min, were markedly reduced in the ace2 mutant mice (Table 1), indicating severe impairment of contractile heart function. Loss of ACE2 also resulted in a significant decrease in aortic and ventricular pressures consistent with the observed reductions in cardiac contractility (data not shown). These results show that ACE2 is an important regulator of heart function in vivo.
Figure 3: Loss of ACE2 results in severe contractile dysfunction.
a, Echocardiographic measurements of contracting hearts in a 6-month-old ace2+/y mouse and two ace2-/y mice. Arrows indicate the distance between systolic contraction (LVESD) and diastolic relaxation (LVEDD). b, Per cent fractional shortening and velocity of circumferential fibre shortening (circumferences s-1) in 6-month-old ace2+/y (n = 8) and ace2-/y (n = 8) mice and 6-month-old ace2+/- (n = 5) and ace2-/- (n = 5) female mice. c, Tail-cuff blood pressure measurements in 6-month-old male ace2+/y (n = 8) and ace2-/y (n = 8) mice and 6-month-old female ace2+/- (n = 5) and ace2-/- (n = 5) mice. Mean values ± s.e.m. are shown. Asterisk, P < 0.05 and double asterisk, P < 0.01.
Upregulation of hypoxia-inducible genes in ace2 null mice
The severe contractile dysfunction and mild dilation in the absence of hypertrophy or cardiac fibrosis in ace2 null mice resembles cardiac stunning/hibernation in human and animal models25,26. Cardiac stunning and hibernation are adaptive responses to chronic hypoxia such as in coronary artery disease or following bypass surgery27. Because ACE2 is highly expressed in vascular endothelial cells but contractility is controlled by cardiomyocytes, we speculated that loss of ACE2 could result in cardiac hypoxia. We therefore analysed changes in the expression levels of hypoxia-inducible genes such as BNIP3 (ref. 28) and PAI-1 (ref. 29) by northern blotting. In the hearts of all ace2 null mice analysed, mRNA expression of BNIP3 and PAI-1 was markedly upregulated compared to their wild-type littermates (Fig. 4a and b). The magnitude of increased expression of these markers of hypoxia is similar to that previously observed in other hypoxic models such as in myocyte-specific vascular endothelial growth factor mutant mice30. Thus, loss of ACE2 results in the induction of a hypoxia-regulated gene expression profile.
Figure 4: Upregulation of hypoxia markers and increased angiotensin II levels in the absence of ACE2.
a, b, Northern blot analysis of BNIP3 and PAI-1 mRNA expression levels, two hypoxia-inducible genes in 6-month-old ace2+/y and ace2-/y male mice. a, Individual northern blot data; b, relative levels of BNIP3 and PAI-1 mRNA levels normalized to the gapdh control. Double asterisk, P < 0.01 (n = 5). c, Angiotensin I (AngI), and Angiotensin II (AngII) peptide levels in the heart, kidneys and plasma of male ace2+/y (n = 8) and ace2-/y (n = 8) littermate mice. Peptide levels were determined by radioimmunoassays. Mean peptide levels ± s.e.m. are shown. Double asterisk, P < 0.01.
Increased angiotensin II levels in ace2 null mice
Because ACE2 functions as a carboxypeptidase, cleaving a single residue from AngI to generate Ang1-9 (refs 14, 15) and a single residue from AngII to generate Ang1-7 (ref. 14), we hypothesized that ACE2 may regulate the RAS by competing with ACE for the substrate AngI and/or cleaving and inactivating AngII. If correct, loss of ACE2 should increase AngII levels in vivo. Using radioimmunoassays, AngII levels were indeed found to be significantly increased in the kidneys, hearts and plasma of ace2 mutant mice (Fig. 4c). In addition, an increase in AngI was observed in the heart and kidney of ace2 mutant mice (Fig. 4c), consistent with AngI being a substrate of ACE2 action in vivo. No differences in ACE mRNA levels were found in the hearts and kidneys of ace2 mutant mice compared to controls, indicating that the increased AngII tissue levels were not due to increased ACE expression (Fig. 2b). These data show that ACE2 functions as a regulator of the RAS modulating endogenous levels of AngI and AngII.
Ablation of ACE expression rescues ace2-deficient mice
To determine whether genetic ablation of ACE in combination with disruption of ACE2 has any effect on the heart phenotype in ace2 mutant mice, we generated ace/ace2 double mutant mice. These double mutant mice were born at the expected mendelian ratio and appear healthy. Blood pressure (Fig. 5a) and kidney defects (not shown) in the ace/ace2 double null mice were similar to that of ace single mutant mice. Fertility of the ace/ace2 double mutant mice was not addressed. Thus, loss of both ACE and ACE2 does not cause any apparent disease in addition to that seen in ace single mutant mice.
Figure 5: ACE/ACE2 double mutant mice do not develop cardiac dysfunction.
a, Blood pressure measurements in 6-month-old ace2+/y (n = 8), ace2-/y (n = 8), ace-/- (n = 8), and ace-/-/ace2-/y double mutant (n = 6) male mice. b, Per cent fractional shortening and velocity of circumferential fibre shortening in 6-month-old male ace2+/y (n = 8), ace2-/y (n = 8), ace-/- (n = 8) and ace-/-/ace2-/y double mutant (n = 6) littermates. c, Echocardiographic measurements of contracting hearts in 6-month-old male ace2+/y, ace2-/y, ace-/- and ace-/-/ace2-/y double mutant littermate mice. Mean values ± s.e.m. are shown. Asterisk, P < 0.05 and double asterisk, P < 0.01.
The heart function of ACE knockout mice has not been previously reported, to our knowledge, so we first analysed the heart parameters in these mice. In ace-/- mice, hearts are histologically normal (not shown) and no defect in heart function could be detected at 6 months of age (Fig. 5b, c). However, ablation of ACE expression on an ace2 mutant background completely abolished the cardiac dysfunction phenotype of ace2 single knockout mice (Fig. 5b, c). Using echocardiography, all heart functions of 6-month-old, age-matched ace/ace2 double mutant mice were comparable to that of their ace single mutant and wild-type littermates (Fig. 5, Table 1 and Supplementary Information). Restoration of heart functions occurred in both male and female ace/ace2 double mutant mice. Importantly, there was also no difference in blood pressure between ace and ace/ace2 knockout mice (Fig. 5a), further implying that the reduced blood pressure in older male ace2 mice is due to the dramatic decrease in heart function.
Heart defects in ACER mutant flies
ACE2 and ACE have critical roles in heart function in mice, so we analysed whether the role of ACE2 in the heart is evolutionarily conserved. Flies have two ACE2 homologues called ANCE16 and ACER17, both of which have a domain structure similar to ACE2 (see Supplementary Information). Both fly homologues are expressed in developing heart cells of the Drosophila embryo16,17. To determine what role these ACE homologues may have in heart development, we studied mutant flies that carry a P-element insertion in the Acer locus (P679) (ref. 31). Mutation of ACER results in early embryonic lethality (not shown). We, therefore, examined heart tube morphogenesis using two early markers (the even-skipped (Eve) and Tinman (Tin) proteins) for heart progenitor cells in Drosophila embryos.
Expression of Eve is the earliest available marker for heart progenitor cells32,33 and in wild-type stage-11 embryos, clusters of three Eve-positive heart progenitor cells form the earliest heart tube anlage (Fig. 6a). By contrast, ACER mutant flies display reduced numbers and disorganization of Eve positive progenitor cells (Fig. 6b). To study further whether ACER mutant mice have a defect in mesoderm that specifies heart progenitor cells, we used Tinman as a marker. Tinman provides the dorsal mesoderm with the competence to specify heart progenitors34, a role conserved in tinman-related homeobox genes such as murine Nkx2.5 that is essential for heart development35. Similar to alterations in Eve expression, loss of ACER results in reduced numbers and disorganization of the Tinman positive dorsal mesoderm (Fig. 6c, d). Thus, ACER mutant flies have defective heart morphogenesis and ACER may have a role in the specification of heart progenitors in Drosophila embryos.
Figure 6: Expression of heart progenitor markers in Drosophila ACER mutant embryos.
a, b, Expression of the heart progenitor cell marker even-skipped (Eve) in stage-11 wild-type and ACER mutant fly embryos. Bottom panels show close-ups of the Eve-positive heart progenitor cells. Arrows in a show clusters of three Eve-positive heart progenitor cells in wild-type embryos. In ACER mutant embryos, there is disorganization and reduction of Eve-positive cells (arrows). c, d, Expression of Tinman (Tin), a specification marker for heart progenitor cells, in stage-12 embroys. In ACER mutant embryos, the numbers of Tin-expressing cells is markedly reduced in the dorsal mesoderm as compared to wild-type Drosophila embryos.
Discussion
Here we have shown that ace2 maps to a QTL associated with hypertension in three rat models of high blood pressure. ACE2 levels are reduced in all of these hypertensive rat strains. In mice, genetic inactivation of ACE2 using homologous recombination results in increased AngII peptide levels, upregulation of hypoxia genes in the heart and severe cardiac dysfunction. Ablation of ACE expression on an ace2-deficient background completely abolished the heart-failure phenotype of ace2 single knockout mice. Our results in Drosophila show that disruption of the fly ACE2 homologue, ACER17, results in a severe and lethal defect of heart morphogenesis. Our fly data also indicate that ACER has a role in the specification of heart progenitor cells. Thus, fly ACER and mammalian ACE/ACE2 may have divergent roles in heart progenitor cell specification versus the control of heart function.
Most cardiovascular diseases are multifactorial quantitative traits controlled by both genetic and environmental factors3. One major factor for cardiovascular disease is the RAS. The map position and reduced expression of ACE2 in three different rat strains indicate that ace2 is a strong candidate gene for a hypertensive QTL on the X chromosome. However, loss of ACE2 in mice has no apparent effect on blood pressure even when ACE is inhibited. From previous studies in many systems, it has become clear that the interplay of several QTLs determine the phenotype. Currently it is thought that there are numerous genetic polymorphisms that serve to regulate blood pressure in humans and rodents3. Thus, altering one of the genes in the ‘wrong’ genetic mouse background would not be sufficient in itself to alter blood pressure. Only when a sufficient number of control mechanisms are altered in concert would high blood pressure ensue. But despite the lack of blood pressure changes in the ACE2 mutant mice, there is an increase in AngII levels. These data clearly indicate that ACE2 controls the levels of AngII in vivo, supporting the candidacy of ace2 as a QTL that controls the RAS. Thus, the absence of increases in blood pressure in ACE2 mutant mice does not exclude ace2 as a candidate gene for the QTL on the X chromosome. It will be interesting to determine whether single nucleotide polymorphisms in the human ACE2 locus correlate with changes in blood pressure.
Unexpectedly, loss of ACE2 in mice results in profound contractile dysfunction. The complete rescue of the heart phenotype in ACE/ACE2 double mutant mice indicates that ACE expression has a causative role in the onset of the heart dysfunction. Both ACE and ACE2 have been shown to be able to cleave AngI in vitro6,7,14,15. Whereas ACE generates AngII from AngI, ACE2 can counteract the function of ACE by cleaving AngII or by competing with ACE for the substrate AngI. Thus, our mouse data suggest that increased AngII mediates the heart phenotype. Nevertheless, our data does not exclude the possibility that other metabolites6,7,14,15 of ACE2 and ACE contribute to the observed phenotypes. It will be important to assess directly the role of AngII in the heart defects of ACE2 mutant mice by pharmacological or genetic inhibition of AngII receptors, and determine if any other ACE2-modulating peptides are altered in these mice. We note that in human patients, inhibition of ACE or AngII receptors can improve the outcome of heart failure13. The contribution of ACE2 in human heart failure needs to be determined.
The heart phenotype found in ACE2 mutant mice is similar to that seen in cardiac stunning/hibernation such as in humans with coronary artery disease and cases of bypass surgery25,26,27. In humans and in animal models of cardiac stunning/hibernation, chronic hypoxic conditions lead to compensatory changes in myocyte metabolism27, upregulation of hypoxia-induced genes30, and severe contractile dysfunction25,26,27,30. Because ACE2 is expressed in the vascular endothelium and not in cardiac myocytes15, local increases in AngII may lead to vasoconstriction, resulting in hypoperfusion and hypoxia in the myocardium. It has been established that AngII can induce oxidative stress in endothelial cells36,37, so the increase in AngII could also result in dysfunction of the vascular endothelium of the heart via the induction of oxidative stress.
Methods
Cloning of mouse and rat ACE2 and chromosomal QTL mapping
Murine ace2 was cloned from a proprietary EST database. Using a mouse ace2 probe, we then screened a rat kidney cDNA (Invitrogen) to obtain a full-length rat cDNA as determined by DNA sequencing (see Supplementary Information). For chromosomal mapping, a rat ace2 cDNA specific probe was used to screen a rat PAC library (RPCI-31, Research Genetics), identifying two positive clones (6M6 and 125K9). The end sequences of these clones were determined and rat-specific primers were designed (mc2L: 5′-TCAATTTACTGCTGAGGGGG-3′; and mc2R: 5′-GAGGGATAACCCAGTGCAAA-3′) to determine the chromosomal map position of ACE2 in rat by screening a radiation hybrid panel (RH07.5, Research Genetics). SHR and control Wistar Kyoto (WKY) rats were obtained from Harlan and maintained at the animal facilities of the Ontario Cancer Institute in accordance with institutional guidelines. Salt-resistant and salt-sensitive Sabra rats were bred and maintained at the animal facility of the Ben-Gurion University, Barzilai Medical Center, Israel. DOCA salt treatment was as described previously18.
Expression analysis
Total RNA was prepared from rat kidneys using tri-reagent. 20 µg of RNA was resolved on a 0.8% formamide gel, blotted to nylon membrane (Amersham) and probed with a partial rat ACE2 cDNA clone (9-1). The β-actin probe and multiple-tissue northern blots were purchased from Clontech. For western analysis, kidneys were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, 20 mM EDTA, and 1% triton-X100) supplemented with “Complete” protease inhibitor cocktail (Roche) and 1 mM Na3VO4. 100 µg of protein was resolved by SDS–polyacrylamide gel electrophoresis (PAGE) on 8% tris-glycine gels. ACE2 immuno-serum was obtained from rabbits immunized with a mouse-specific ACE2 peptide: DYEAEGADGYNYNRNQLIED. The serum was affinity-purified with the immunizing peptide using sulpho-link kit (Pierce). A commercially available β-actin antibody was used as loading control (Santa Cruz).
Generation of ACE2 mutant mice
A targeting vector (559 base pair short arm and 8.1 kilobase long arm) was constructed using the pKO Scrambler NTKV-1907 vector (Stratagene). A portion of the ace2 genomic DNA containing nucleotides +1069 to +1299 was replaced with the neomycin resistance cassette in the anti-sense orientation. The targeting construct was electroporated into E14K embryonic stem cells, and screening for positive homologous recombinant embryonic stem cell clones was performed by Southern blotting of EcoRI-digested genomic DNA hybridized to 5′ and 3′ flanking probes. Two independent ace-/y embryonic stem cell lines were injected into C57BL/6-derived blastocysts to generate chimaeric mice, which were backcrossed to C57BL/6 mice. Two embryonic stem cell lines gave independent germline transmission. Histology of all tissues, apoptopsis assays, blood serology and kidney morphometries were as described38. Complete ACE mutant mice have been previously described8 and were obtained from Jackson Laboratories. Mice were maintained at the animal facilities of the Ontario Cancer Institute in accordance with institutional guidelines.
Heart parameter measurements
For heart morphometry, hearts were perfused with 10% buffered formalin at 60 mm Hg (1 mm Hg = 133.3 Pa) and subsequently embedded in paraffin. Myocardial interstitial fibrosis was determined by quantitative morphometry using the colour-subtractive computer-assisted image analysis using Image Processing Tool Kit version 2.5 coupled with Photoshop 6.0 software. Picro-Sirius-red (PSR)-stained sections were used to calculate interstitial fibrosis as the ratio of the areas with positive PSR staining compared to the entire visual field. Echocardiographic assessments were performed as described39. Mice were anaesthetized with isoflurane/oxygen (1.25%/98.75%) and examined by transthoracic echocardiography using a Acuson Sequoia C256 equipped with a 15-MHz linear transducer. Haemodynamics measurements were performed as described40. Tail-cuff blood pressure measurement were taken using a Visitech BP-2000 Blood Pressure Analysis System manufactured by Visitech Systems (Apex). For captopril treatment, drinking water was supplemented with 400 mg l-1 captopril (Sigma) for two weeks before blood pressure measurement.
Plasma and tissue angiotensins
For plasma measurements blood was collected into chilled Vacutainer blood collection tubes (Becton Dickinson) containing a mixture of peptidase inhibitors (25 mM ethylenediaminetetraacetic acid (EDTA), 0.44 mM o-phenanthroline (PHEN), 1 mM 4-chloromercuribenzoic acid (PCMB), 0.12 mM pepstatin A and 3 µM acetyl-His-Pro-Phe-Val-Statine-Leu-Phe, a renin inhibitor. Frozen hearts and kidneys (stored at -80 °C) were homogenized on ice in 80% ethanol/0.1 M HCl containing the peptidase inhibitors described above including phenylmethylsulphonyl fluoride (PMSF, 100 µM). A sample of the homogenate was taken to determine total protein content, using the Bradford protein assay with bovine serum albumin as a standard (BioRad Protein Assay Reagent, BioRad Laboratories). Peptide extraction was performed as previously described41. Radioimmunoassay analysis of angiotensin peptide content in the extracts from plasma, heart and kidney tissue was performed as previously described42. The limits of detection for the AngII, and AngI radio immunoassays were 0.5 fmol per tube, and 5 fmol per tube, respectively.
Drosophila studies
The P-element allele of ACER, P679 (ref. 31), was used to generate homozygous ACER mutant embryos. Whole embryo staining with Eve and Tin antibodies was as described33.
References
Acknowledgements
We thank D. Ganten for supplying us with tissue from SHRSP rats. Eve and Tin antibodies were a gift from M. Frasch. We acknowledge the Samuel Lunenfeld Research Institute's CMHD Mouse Physiology Facility for their technical screening services. This study was supported by Amgen and by grants from the Israel Science Foundation and the German–Israeli Foundation for Scientific Research and Development to C.Y. and Y.Y. J.M.P. holds a Canadian Research Chair in Cell Biology. M.A.C. is supported in part by a Canadian Institutes of Health Research fellowship.
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Correspondence to Josef M. Penninger.
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Crackower, M., Sarao, R., Oudit, G. et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417, 822–828 (2002). https://doi-org-ssl.openlink.inha.ac.kr:8443/10.1038/nature00786
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