1Department of Medicine, University of Melbourne, Austin Health, Repatriation Heidelberg Hospital, Heidelberg 3081, Victoria, Australia
2Vascular Division, Wynn Domain, Baker Heart Research Institute, Melbourne, Australia
3Heart Failure and Transplant Unit, St Vincent's Hospital, Darlinghurst, Sydney, New South Wales, Australia
Received 7 April 2004; revised 30 November 2004; accepted 2 December 2004; online publish-ahead-of-print 24 January 2005.
* Corresponding author. Tel: +61 3 9496 2159; fax: +61 3 9497 4554. E-mail address: l.burrell{at}unimelb.edu.au
See page 322 for the editorial comment on this article (doi:10.1093/eurheartj/ehi043)
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Abstract |
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Methods and results Rats were killed at days 1, 3, and 28 after MI, or treated for 4 weeks with the ACE inhibitor ramipril (1 mg/kg). Cardiac gene and protein expression of ACE and ACE2 were assessed by quantitative real-time reverse transcriptasepolymerase chain reaction and immunohistochemistry/activity assays/in vitro autoradiography, respectively. Both ACE (P=0.022) and ACE2 (P=0.015) mRNA increased in the border/infarct area compared with the viable area at day 3 after MI. By day 28, increases in ACE (P=0.005) and ACE2 (P=0.006) mRNA were also seen in the viable myocardium of MI rats compared with myocardium of control rats. ACE2 protein localized to macrophages, vascular endothelium, smooth muscle, and myocytes. Ramipril attenuated cardiac hypertrophy and inhibited cardiac ACE. In contrast, ramipril had no effect on cardiac ACE2 mRNA, which remained elevated in all areas of the MI rat heart. Immunoreactivity of both ACE and ACE2 increased in failing human hearts.
Conclusion The increase in ACE2 after MI suggests that it plays an important role in the negative modulation of the renin angiotensin system in the generation and degradation of angiotensin peptides after cardiac injury.
Key Words: Myocardium Infarction Injury Heart failure
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Introduction |
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The role of ACE2 remains to be determined, but its localization in the heart suggests an important role for cardiovascular function. Rats with hypertension have low ACE2 protein levels, whereas ACE2 knockout mice have severe impairment of cardiac contractility with upregulation of hypoxia-induced genes in the heart suggesting a link to myocardial ischaemia.7 Significant activation of the cardiac RAAS occurs after myocardial infarction (MI),810 but whether this extends to changes in ACE2 is unknown. To explore this possibility, we examined the temporal and regional expression of cardiac ACE2 and ACE mRNA and protein after MI, as well as the effect of treatment with the ACE inhibitor, ramipril. We also examined ACE2 expression in explanted failing hearts from heart transplant recipients.
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Methods |
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Surgical production of MI
Left ventricular (LV) free wall MI was induced in SpragueDawley rats (150200 g) by ligation of the left coronary artery.1113 Rats were allocated to sham operation in a random manner. These rats (control) underwent the same surgical procedure as the infarct rats but the suture was not tied. Only rats that survived 24 h post-operatively were included in Studies 1 and 2. In rats operated on to produce an infarct 80% were alive at 24 h and were then randomized to different groups. Rats with an infarct size of <20% were excluded from analysis.
Study 1: temporal and spatial expression of ACE and ACE2 following MI
Control and MI rats that survived 24 h post-operatively were randomized into groups and killed on day 1, 3, or 28. In all hearts, the LV/interventricular septum was dissected and weighed; a thin transverse slice was removed from the midline and fixed in 10% buffered formalin and paraffin embedded. Sections were stained both with Masson's trichrome for assessment of infarct size and with haematoxylin and eosin. The mean epicardial and endocardial scar circumference was compared with total LV circumference to calculate total infarct size.11 The remaining LV pieces were frozen at 80°C for in vitro autoradiography (IVA) and quantitative real-time reverse transcriptasepolymerase chain reaction (QRTPCR), which were performed on a randomly selected subset of hearts.
Study 2: effect of ACE inhibition on ACE and ACE2 after MI in the rat
MI rats were randomized to oral ramipril (1 mg/kg) or vehicle for 28 days. Sham-operated rats (control) were also studied and received only vehicle. All rats were killed on day 28. The LV was dissected, weighed, and infarct size determined. The remaining LV pieces were frozen for assessment of ACE and ACE2 mRNA by QRTPCR, and ACE and ACE2 by IVA and activity assay, respectively.
Study 3: immunolocalization of ACE2 in human heart
The LVs of explanted failing hearts from five male patients with ischaemic heart disease (IHD) who had undergone cardiac transplantation were assessed. Subjects were aged 49±2 years and had severe coronary atheroma with 86±4% stenosis of the left anterior descending coronary artery and 80±8% stenosis of the right coronary artery. All were on standard anti-heart failure medication including ACE inhibitors, beta-blockers, and diuretics. Control LV tissue (n=3) was obtained at post-mortem from male patients (age 49, 72, and 74 years) with no cardiac history and no pathological abnormalities of the heart. Tissues were fixed in 10% buffered formalin and paraffin embedded for immunohistochemical localization of ACE and ACE2.
Extraction of total RNA, synthesis of cDNA, and QRTPCR
Rat MI hearts were divided into two parts: viable myocardium and infarct/border zone, and RNA was isolated from both areas. For control rats, the whole LV was used. Total RNA was isolated using the RNeasy kit method (Qiagen). cDNA was synthesized with a reverse transcriptase reaction using standard techniques (Superscript II kit, Life Technologies, Gaithersburg, MD, USA) as previously described.14,15 QRTPCR is a fully quantitative method for the determination of amounts of mRNA. Briefly, gene-specific 5'-oligonucleotide corresponding to rat ACE (5'-CACCGGCAAGGTCTGCTT), ACE 3'-oligonucleotide primer (5'-CTTGGCATAGTTTCGTGAGGAA), and ACE probe (FAM5'-CAACAAGACTGCCACCTGCTGGTCC-TAMRA); for ACE2 gene-specific 5'-oligonucleotide corresponding to (5'-ACCCTTCTTACATCAGCCCTACTG), an ACE2 3'-oligonucleotide primer (5'-TGTCCAAAACCTACCCCACATAT), and ACE2 probe (FAM5'-ATGCCTCCCTGCTCATTTGCTTGGT-TAMRA) were designed using the software program Primer Express (PE Applied Biosystems, Foster City, CA, USA). QRTPCR was carried out using a multiplex method with 18S VIC as the endogenous control.14 A relative expression method was applied in this study using the control group as the calibrator. The calculations for fold induction are extrapolated as follows. The cycle number (Ct) for 18S was subtracted from the Ct of the gene of interest (in this case ACE or ACE2), resulting in the difference in Ct, that is, DCt. The average DCt was then calculated for the calibrator group and this value was subtracted from every DCt value in all groups, resulting in a DDCt value for all samples. The DDCt value was then entered into the equation 2(expDDCt), which resulted in a fold induction value. All groups were compared with the calibrator group (which has a value of 1).
Cardiac ACE activity
In Study 2, rat cardiac ACE (n=5 per group) was assessed by IVA on LV sections (20 µm) using the specific radioligand 125I-MK351A (Ki=30 pmol/L).12 Quantification of four sections from each rat was performed using a microcomputer-imaging device (Imaging Research, Ontario, Canada). In MI hearts, the viable myocardium, infarct, and border zone were quantified separately.13 The border zone is the area of high cellular infiltrate at the border zone of the scar tissue of the infarct. The optical density of the autoradiographs was calibrated in terms of radioactivity density (d.p.m./mm2).
Cardiac ACE2 activity
In Study 2, rat MI hearts (n=58 per group) were divided into viable myocardium and infarct/border zone, and membranes prepared from each area. The whole LV was used for control rats (n=8). Tissue was homogenized in ice-cold Tris-buffered saline (TBS: 25 mM TrisHCl, 125 mM NaCl, pH 7.4), and homogenates pelleted by ultracentrifugation at 100 000g for 60 min at 4°C, resuspended in fresh TBS, and rehomogenized. Following a second ultracentrifugation step, the final membrane pellet was resuspended in 0.5 mL TBS, aliquoted, and frozen at 70°C. Prior to ACE2 activity assay, an aliquot of each sample was thawed on ice, diluted 10-fold in ACE2 assay buffer (100 mM Tris, 1 M NaCl, pH 6.5), and briefly sonicated (Branson Cell Disruptor B-30) in order to disperse the membranes. Protein was determined using a Bio-Rad DC Protein Assay kit.
Quenched fluorescent substrate assay of ACE2 activity
Cardiac membrane preparations (100 µL diluted sample) were incubated in triplicate with an ACE2-specific quenched fluorescent substrate (QFS), (7-methoxycoumarin-4-yl)-acetyl-Ala-Pro-Lys (2,4-dintirophenyl) (Auspep, Parkville, Victoria, Australia) as previously published.5 Assays were performed with 50 µM QFS in a final volume of 200 µL ACE2 assay buffer (100 mM Tris, 1 M NaCl, pH 6.5). The final concentration of dimethylsulfoxide (used to solubilize QFS) was 0.7%. Reactions proceeded at 37°C for 60 min within a thermostatted fMax fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA), prior to reading the liberated fluorescence (ex=320 nm,
em=420 nm). As the QFS can be cleaved by prolyl endopeptidase, a specific inhibitor of this enzyme, Z-Pro-prolinal (1 µM), was included in all wells.16 To further confirm the specificity of the assay, the measurement was repeated in the presence of the ACE2 inhibitor C16.17 The specific activity of the preparations was expressed as units of fluorescence per milligram of membrane protein per hour.
ACE and ACE2 immunohistochemistry
Immunohistochemical staining for ACE2 (polyclonal antibody from Millennium Pharmaceuticals Inc., Cambridge, MA, USA) and ACE (human polyclonal ACE antibody18) were performed in rat heart (ACE2) and human heart (ACE and ACE2) as outlined subsequently. Both antibodies were used at a dilution of 1 : 500. The specificity of the ACE2 antibody has been shown in heart and kidney3 and that of ACE antibody in human vessels.18 Immunohistochemistry for macrophages was carried out as previously described.13
Immunohistochemistry was carried out on 4 µm sections prepared from paraffin embedded LV of human hearts as well as MI and control rat hearts. Sections were dewaxed and hydrated, then endogenous peroxidase activity quenched for 20 min incubation in 3% H2O2 in distilled water. The primary antibodies were applied for 1 h at room temperature. The Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) followed by diaminobenzidine (Sigma, St Louis, MO, USA) were used to visualize antibody binding. Slides were counterstained with haematoxylin and cover-slipped. Negative control slides were incubated with normal goat serum; the primary antibody was excluded.
Statistics
Data are presented as mean±SEM. The sample size was chosen on the basis that six to eight rats are sufficient to show infarct sizes of >20% compared with 0% in sham-operated rats. Significant differences were obtained when P<0.05, and all P-values were calculated from two-tailed tests of statistical significance. Data were analysed where appropriate using ANOVA and the Fisher least significant difference test for multiple comparisons. In addition, the myocardium of control rats was compared with the viable myocardium of MI rats using unpaired t-tests. Within MI hearts, the different areas of the heart (viable, infarct/border) were compared using paired t-tests. In Study 2, in MI rats the effect of ramipril was compared with vehicle in the different regions of the heart using unpaired t-tests. For QRTPCR, values for the controls were arbitrarily standardized to 1 by taking the average of the results of all control hearts, and the data for MI rats expressed relative to this value.
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Results |
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Study 1: temporal and spatial expression of ACE and ACE2 mRNA
Of 69 rats operated on to produce an MI, 81% (n=56) were alive at 24 h and were randomized to control day 1 (n=9), post-MI day 1 (n=10), control day 3 (n=9), post-MI day 3 (n=10), control day 28 (n=10), and post-MI day 28 (n=9). Four rats died during the course of Study 1 and were excluded from further analysis, as were rats with an MI of <20%. Hence, results are reported on 18 rats (post-MI day 1, n=8; post-MI day 3, n=4; post-MI day 28, n=6) and 27 control rats (n=9 per time point). Infarct size could not be determined at day 1 post-MI but was 49±4% at day 3 and 38±4% at day 28. Total heart mass and LV mass increased in MI compared with control rats at all time points (P<0.001, data not shown).
ACE and ACE2 mRNA expression
Figure 1 demonstrates the relative quantification of cardiac ACE and ACE2 mRNA, which was assessed in a subset of rat hearts and increased after MI in a temporal and spatial manner. There were nine control rats at each time point and four to eight MI rats at each time point. At day 3, ACE mRNA was elevated in the border/infarct zone [6.7±1.3 arbitrary units (AU)] compared with the MI-viable area (2.7±1.1 AU) (P=0.022), and also increased in the MI-viable myocardium by day 28 (2.4±0.3 AU) compared with control rats (1.08±0.2 AU) (P=0.005). Similar changes were noted in the temporal and spatial expression of ACE2 mRNA after MI. Significant increases in ACE2 expression were seen in the border/infarct zone of MI rats at day 3 (4.38±0.7 AU) compared with the MI-viable area (1.8±0.3 AU) (P=0.015), and these changes persisted at day 28 (3.0±0.5 AU). In addition, at day 28, ACE2 mRNA was elevated three-fold in the MI-viable myocardium (2.87±0.4 AU) compared with control myocardium (1.17±0.2 AU) (P=0.006).
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ACE and ACE2 mRNA expression
ACE and ACE2 mRNA expression was assessed in a subset of rat hearts (n=46 per group), and Figure 3 shows the quantification of mRNA expression. At day 28 post-MI, both ACE (P=0.005) and ACE2 (P=0.041) mRNA increased in the viable myocardium compared with control myocardium, confirming the results of Study 1. There were further increases in ACE mRNA in the border/infarct zone of MI rats compared with MI-viable myocardium (P=0.049). In MI rats, ACE inhibition decreased ACE mRNA in the viable myocardium (P=0.014) and the border/infarct zone (P=0.003) compared with the vehicle treatment. In contrast, ramipril had no effect on cardiac ACE2 mRNA, which remained elevated in both the viable and the border/infarct zone of the MI rat heart.
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Discussion |
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Components of the RAAS, such as ACE and Ang II are activated after MI8,1922 and the finding that ACE2 increases in a similar spatial and temporal manner to ACE supports a regulated response to injury and mechanical stress. ACE2 is also highly expressed by infiltrating mononuclear cells, suggesting that it may participate in the initial inflammatory response to injury. As ACE2 immunoreactivity was also observed in endothelial cells and myocytes some time after MI in both rat and humans, ACE2 may also be involved in the late phase of post-MI changes when injury and inflammation have abated but mechanical load remains high and the non-infarcted myocardium is undergoing a complex series of molecular and cellular events that lead to changes in the shape and function of the myocardium.23
The benefits of ACE inhibition to improve cardiac function and morbidity and mortality after MI24 are due in part to reduction in cardiac ACE and Ang II levels.20,25,26 Indeed, the present study confirmed that ACE inhibition was associated with reduced LV mass. Cardiac ACE was inhibited by ramipril but ACE2, as assessed by a range of techniques including specific assessment of ACE2 catalytic activity, was unchanged, which is consistent with the in vitro data, which have previously demonstrated that ACE inhibitors do not inhibit ACE2 activity.4
To date, there have been no studies on ACE2 in the context of MI. Increased cardiac ACE2 after MI may act as a counter-regulatory mechanism to limit the adverse effects of an elevated cardiac Ang II by increasing levels of the vasodilator Ang 1-7. It is possible that the relative balance of vasoconstrictor and vasodilatory angiotensin peptides is important in the modulation of both haemodynamic and trophic effects of these peptides within the heart. Support for this idea comes from studies in ACE2 knockout mice,7 which have severely impaired cardiac contractility in the setting of elevated Ang II levels and can be rescued by simultaneous genetic ablation of ACE. Furthermore, studies in the MI rat have shown that the development of heart failure is associated with increased cardiac Ang 1-7 immunoreactivity27 and that infusion of Ang 1-7 attenuates the development of heart failure after MI.28 It is possible that the relative balance of vasoconstrictor and vasodilator angiotensin peptides is important in the modulation of both haemodynamic and trophic effects of these peptides in the context of MI. Finally, the localization of ACE2 in rat and human heart to endothelial cells of intra-myocardial blood vessels and smooth muscle cells also supports a role for ACE2 in the control of local vasodilation.
It has been reported previously that ACE2 protein expression was similar in non-failing human hearts and the failing heart from one patient with idiopathic cardiomyopathy.3 However, a recent report examined 14 subjects with idiopathic cardiomyopathy and showed an increase in functional cardiac ACE2 activity as assessed by the ex vivo formation of Ang 1-7.29 The results of the present study extend the findings of increased cardiac ACE2 activity to the context of ischaemic heart disease.
The finding that hypoxia-induced gene expression is upregulated in ACE2-deficient mice may provide an explanation for changes in ACE2 in ischaemic myocardium. Although there have been no other studies on the expression of ACE2 after tissue injury, ACE2 expression is reduced in experimental hypertension7 and diabetes,14 while expression of collectrin, a protein with significant homology to ACE2, is upregulated in a model of progressive renal injury.30
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Perspectives |
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Supplementary material |
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Acknowledgements |
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References |
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