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Human Molecular Genetics, 2004, Vol. 13, No. 14 1441-1450
DOI: 10.1093/hmg/ddh147
Human Molecular Genetics, Vol. 13, No. 14 © Oxford University Press 2004; all rights reserved

Over-expression of angiotensin converting enzyme-1 augments cardiac hypertrophy in transgenic rats

Xiao-Li Tian1, Yigal Martin Pinto1, Olivier Costerousse1, Wolfgang M. Franz2, Andrea Lippoldt3, Sigrid Hoffmann3, Thomas Unger4 and Martin Paul1,*

1Department of Clinical Pharmacology, Benjamin Franklin Medical Center, Free University of Berlin, Berlin 12200, Germany, 2Department of Internal Medicine, University of Lübeck, Lübeck D-23538, Germany, 3Hypertension Research, Max-Delbrück Center (MDC) for Molecular Medicine, Berlin 13092, Germany and 4Department of Pharmacology, University of Kiel, Kiel 24105, Germany

Received March 5, 2004; Revised April 13, 2004; Accepted April 22, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Increased cardiac angiotensin converting enzyme-1 (ACE1) is found in individuals who carry a deletion in intron 16 of ACE1 gene or in individuals who suffer from cardiac disorders, such as hypertrophy. However, whether a single increase in ACE1 expression leads to spontaneous cardiac defects remains unknown. To determine if the increased cardiac ACE1 actively plays a role or is merely the consequence of pathological changes in the process of cardiac hypertrophy, we generated a transgenic rat model with selective over-expression of human ACE1 in the cardiac ventricles. The left ventricular ACE1 activity is elevated about 50-fold in transgenic rats. Angiotensin-1 perfusion of isolated hearts demonstrated a significant decrease in coronary artery flow compared with non-transgenic littermates, suggesting that the transgenic ACE1 is functional. Neither cardiac hypertrophy nor other morphological abnormalities were observed in transgenic rats under standard living conditions. It was found, however, after induction of hypertension by suprarenal aortic banding, that the degree of cardiac hypertrophy in transgenic rats was significantly higher than that of banded control rats. The expressions of both ANF and collagen III, molecular markers of cardiac hypertrophy, were also increased in banded transgenic rats compared with banded control. Our results suggest that increased cardiac ACE1 does not trigger but augments cardiac hypertrophy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the renin–angiotensin system, renin cleaves angiotensinogen into angiotensin 1 (ANG I), which is further catalyzed into angiotensin II (ANG II), an active form, by angiotensin converting enzyme-1 (ACE1). ANG II acts as a potent vasopressor, mitogen, neurotransmitter and aldosterone-stimulating factor, therefore, playing an important role in vascular tone, cell metabolism and growth as well as electrolyte balance through its receptors (AT1 and AT2) (1,2). ACE1 is a predominant enzyme in the generation of ANG II in blood and tissues in vivo (3). Although ACE1 degrades bradykinin, a vasodilator, into an inactive form (4), ANG II is its major functional product (5–7). Existing data suggest that the cardiac ACE1 is up-regulated in cardiac disorders such as ventricular hypertrophy (8), myocardial infarction (9) and congestive heart failure (10). In animal models, ACE1 inhibitors were able to prevent or reverse the cardiac hypertrophy caused by either pressure- (11) or volume-overload (12), to increase the myocardial capillary length density (13,14), to prevent the fibrosis in hypertrophied hearts (15) as well as to limit or reduce the infarct size of the heart (16,17). In clinics, ACE1 inhibitors are used widely owing to their beneficial effects on the treatment of these cardiac diseases (18–21). These strongly suggest that ACE1 is involved in the pathogenesis of these cardiac disorders. However, direct and functional evidences in support of this concept are lacking. Previously, ACE1 insertion/deletion (I/D) genetic variances in intron 16 of the human ACE1 gene were identified (22). D/D alleles occur in a considerable human population and cause an increase in ACE1 activities of both plasma and cardiac tissue (23). It is expected that this finding would provide direct evidence for the hypothesis that the increased ACE1 is deleterious to the heart if an association could be established between the D allele and cardiac disorders as well as hypertension. Unfortunately, there was no consensus among researchers on this question over the past decades (24–27). Therefore, it becomes interesting to clarify whether the increased cardiac ACE1 activity is a cause or a consequence in the process of cardiac defects. In the present study, we generated a transgenic rat model with selective over-expression of human ACE1 in cardiac tissues and assessed the functions of the increased cardiac ACE1 under physiological and pathological conditions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of transgenic rats with selective cardiac expression of human ACE1
A 2.1 kb rat myosin light chain-2 (rMLC-2) promoter was used to direct the cardiac specific expression of hACE1 cDNA (Fig. 1A). Of the 12 pups developed from microinjected fertilized eggs, which were identified by Southern blot hybridization, four (1163, 1167, 1172 and 1173) were found to be transgenic (Fig. 1B). Among these, 1172 and 1173 successfully transmitted the transgene to their progeny in a Mendelian fashion, thus establishing two transgenic rat (Sprague–Dawley, SD) lines, referred to as TGR(rMLC2–hACE1)L1172 and -1173.



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  		Figure 1. Generation of transgenic rats. (A) Transgenic construct, rMLC-2: rat myosin light chain-2 promoter; hACE1 cDNA: human ACE1 cDNA and pl A: human growth factor poly(A) signal. (B) Southern blot analysis. EcoRI-digested plasmid pBSK(rMLC–hACE1) was used as a positive control. SD, 1163, 1167, 1172 and 1173 were SD control rat and transgenic founders. A 4.0 kb band represented the positive hybridization signal for the transgene. (C) RNase protection assay. HindIII-pRHA was used for generate probes, which produced two bands in RNase protection assay (150 nt for hACE1 and 200 nt for rACE1). Sk, skeletal; R, right and L, left; total RNA from lung and others were 5 and 50 µg, respectively. (D) ACE1 activity measurement. The net increase in ACE1 activity in transgenic rats was presented as ‘fold increase’ (ACE1F), which was calculated by a formula: ACE1F=(ACE1TG–ACE1SD)/ACE1SD, where ACE1TG and ACE1SD are the ACE1 activities from transgenic and from SD controls, respectively. The gray and black filled bars represent TGR(rMLC–2hACE1)L1172 and -L1173, respectively. Ao, Br, Ki, Li, Lu, SM, RV, LV and Se represent, respectively, aorta, brain, kidney, liver, lung, skeletal muscle, right ventricle, left ventricle and serum. (E) In situ hybridization. The purple staining was the positive signal for the expression of the human ACE1 transgene. The heart sections were from SD control (SD) and transgenic rat L1173 (TGR). R.A., right atrium, R.V., right ventricle and L.V., left ventricle.

 
To determine the tissue distribution of hACE1 expression, we performed RNase protection assay and demonstrated that the hACE1 transgene in TGR(rMLC2–hACE1)L1173 was highly expressed in the cardiac ventricles (Fig. 1C). A weak expression of transgene was also detected in kidney and skeletal muscle, but not in any other tissues tested (Fig. 1C). TGR(rMLC2–hACE1)L1172 shared the same expression patterns with TGR(rMLC2–hACE1)L1173 but had a lower expression level (data not shown). The measurement of ACE1 activity proved that in the left ventricle the total ACE1 activity was found to be increased by 13- and 50-fold in TGR(rMLC2–hACE1)L1172 and -1173, respectively (Fig. 1D). A negligible elevation of ACE1 was also detected in skeletal muscle and kidney. All other tissues tested did not show any increases in ACE1 activities. Notably, ACE1 activity in serum was not increased (Fig. 1D). In the transgenic heart, we demonstrated by in situ hybridization that the hACE1 transgene was localized exclusively in ventricles, not in atria (Fig. 1E). Thus, a transgenic rat model with selective cardiac ventricular expression of hACE1 was generated. TGR(rMLC2–hACE1)L1173 presented a higher expression level of hACE1, therefore, was characterized extensively in this study.

Phenotying of TGR(rMLC–2hACE1) under physiological conditions
We first determined whether the increased ACE1 in cardiac tissues could cause any increase in ANG II levels in both tissue and plasma. It was found that the ANG II levels in the plasma and ventricles of TGR(rMLC2–hACE1)L1173 remained unchanged compared with control (4.1±0.7 versus 3.9±0.5 pg/ml, P>0.05 in plasma; and 182±10 versus 185±9 pg/g, P>0.05 in ventricles). In order to study whether hACE1 transgene was functional, we tested the effects of ANG I on coronary artery flow (CAF) in the isolated perfused hearts from TGR(rMLC2–hACE1)L1173. Baseline CAF was similar in both the transgenic and control groups, and a single dose of ANG II perfusion elicited a similar decrease in CAF in both groups (data not shown). When ANG I was perfused, however, it caused a significant fall in CAF in the transgenic hearts compared with the control group with increase in dosages (10–10, 10–9, 3x10–9, 10–8, 3x10–8 M, Fig. 2A). This effect was reversible upon a co-infusion with an ACE1 inhibitor, quinaprilat (10–5 M, Fig. 2B).



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  		Figure 2. Characterization of transgenic rats. (A, B) ANG I perfusion in the isolated hearts. The CAF calibrated against baseline (%) was plotted upon the doses of ANG I in absence of quinaprilat (A) and in the presence of quiniaprilat (B). (C) Blood pressure measurement. SBP, systolic blood pressure; SD, He and Ho represent SD control, heterozygous and homozygous transgenic rats, respectively (L1173). (D) Determination of Lv/B; unfilled, gray and black filled bars were SD, TGR(rMLC–2hACE1)L1172 and -L1173, respectively. (E) RNase protection assay. SD and L1173 were, respectively, non-transgenic control and transgenic rats from TGR(rMLC–2hACE1)L1173. ANF and Col3 were determined in comparison with internal control GAPDH. An amount of 50 µg of total RNA was used in each lane. (F) Histological characterization: I and II; H&E staining; III and IV; Elastica van Gieson staining.

 
In both genders, no differences in systolic blood pressure (SBP) were observed between transgenic (heterozygous and homozygous) and littermate controls, as demonstrated by tail-cuff blood pressure measurement (Fig. 2C). Under standard living conditions, the left ventricle to body weight ratio (Lv/B) remained unchanged in both transgenic lines (L1172 and L1173) when compared with their littermate controls (Fig. 2D). The size of the cardiac myocytes was similar to that of the control rat (data not shown). The expressions of atrial natriuretic factor (ANF) and collagen III (Col3), markers for cardiac hypertrophy, were not altered by over-expression of hACE1, as suggested by the RNase protection assay (Fig. 2E). Neither morphological changes nor collagen deposition was observed between transgenic and control groups (Fig. 2F).

Effects of pressure overload on cardiac phenotype of TGR(rMLC–2hACE1)
After 10 weeks of suprarenal aortic banding, SBP was elevated significantly in comparison with sham operated groups (Fig. 3A). Notably, the elevation was not statistically different between the two banded groups [transgenic (n=8): 153±7; control (n=11): 158±8 mmHg, P>0.05, Fig. 3A]. In addition, we also did not detect any differences in plasma ACE1 activity between banded transgenic rats and their controls (42.3±0.11 versus 41.1±0.09 mU/ml, P>0.05). However, the increase in Lv/B was significantly higher in the banded TGR(rMLC2–hACE1)L1173 (n=8) when compared with the banded SD rats (n=11) (2.73±0.08 versus 2.4±0.1 mg/g, P<0.05) while the ratio in both banded groups were greater than their sham control rats (Fig. 3B). Severe cardiac hypertrophy in the banded transgenic rat as compared to the banded controls was proved by the further increase in the expression levels of ANF and Col3 after being calibrated with the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). As shown in Figure 3C and D, the expression of ANF and Col3 in sham groups was 0.45±0.09 and 1.8±0.25 in TGR(rMLC2–hACE1)L1173 (n=8) versus 0.62±0.1 and 1.96±0.3 in SD (n=11), P>0.05 for both; whereas in banded groups, the expression of ANF and Col3 was 2.3±0.3, and 6.8±1.3 in TGR(rMLC2–hACE1)L1173 (n=8) versus 1.7±0.2 and 3.1±0.7 in SD (n=11), P<0.01 for both, respectively. Using RNase protection assay, we also demonstrated that the gene expression of ANG II receptor type 1a (AT1a) was down-regulated dramatically in all banded groups (Fig. 3E), while the expression of bradykinin receptor type 2 (B2) was not different among the groups (Fig. 3F).



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  		Figure 3. Effects of pressure overload on transgenic heart (L1173). SD-sh and TGR-sh: sham operated SD and transgenic rats; SD-ab and TGR-ab: aortic banded SD and transgenic group. (A) SBP measurement after 10 weeks of aortic banding. (B) Determination of Lv/B. (C) RNase protection assay. ANF, Atrial natriuretic factor; Col3, collagen III; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. (D) The plot of (C). % GAPDH stands for the ratio of ANF or Col3 to GAPDH. (E, F) RNase protection assay. AT1a, angiotensin II receptor type 1a; B2, bradykinin receptor type 2. An amount of 50 µg total RNA was used in each lane.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
It has been documented that cardiac ACE1 is up-regulated in cardiac disorders such as myocardial hypertrophy. Although numerous studies have suggested that the cardiac effect of ACE1 inhibitors are, to a great extent, due to local inhibition of this enzyme and thereby to the local inhibition of both ANG II formation (28,29) and bradykinin degradation (30), there is no evidence that the increased ACE1 could directly cause cardiac disorders. Genetically, deletion mutation (D allele) in intron 16 of the human ACE1 results in an increased ACE1 activity in both plasma and cardiac tissues (22,26). This would provide a unique opportunity to address whether an increase in ACE1 could directly cause cardiac defects if an association could be established between the D allele and cardiac disorders as well as hypertension. However, there has been no consensus on this association over the past decades (24,26,31–34). One possible explanation for such debate was that the effect of increased cardiac ACE1 could be interfered or masked in humans by the differences in genders, genetic background, life style, medical intervention or even professions. To address this question, we developed two transgenic rat lines, referred to as TGR(rMLC2–hACE1)L1172 and -1173, with selective cardiac ventricular expression of human ACE1. We demonstrated that both transgenic lines at ages from 2 to 10 months show no fibrosis, necrosis, or elevation of ANF and Col3, which are associated typically with increased ANG II loading and are considered as molecular markers for cardiac hypertrophy (15,35), in cardiac tissues under standard living conditions although they presented a dramatic increase in ACE1 activity in their ventricles. This indicates that the increased ACE1 in cardiac tissues does not directly trigger the process of cardiac abnormalities. It is possible that cardiac ACE1, under normal circumstances, is not a rate-limiting factor in the generation of ANG II. This is supported by the absence of increased ANG II in the transgenic heart. However, these findings do not refute that inhibition of ACE1 decreases cardiac ANG II levels (36). To gain further insight into the role of the increased cardiac ACE1 under pathological conditions, suprarenal aortic banding was performed. Interestingly, a significant increase in cardiac hypertrophy was found in TGR(rMLC2–hACE1)L1173 under suprarenal aortic banding although both transgenic and non-transgenic groups showed similar SBP, indicating that increased ACE1 activity is functionally relevant in this experimental model of cardiac hypertrophy and dysfunction.

ACE1 converts ANG I into ANG II and metabolizes bradykinin into an inactive form. The beneficial effects of ACE1 inhibition in the treatment of cardiac hypertrophy are likely due to the inhibition of these two processes (37). To explore whether the ACE1 augments cardiac hypertrophy through ANG II or bradykinin pathway, we determined the expressions of receptor AT1a and B2. We found that the AT1a expression was decreased significantly in the cardiac tissues after 10 weeks of aortic banding. It has been reported that the cardiac expression of AT1a was down-regulated in the transgenic rat model TGR(mRen-2) that expresses mouse renin (Ren-2) gene and develops fulminant hypertension and cardiac hypertrophy (38) and in a cardiac hypertrophied model caused by aortic banding (39). The decrease in the AT1a expression likely results from a negative feedback owing to the local increased ANG II level (40,41). Previously, it has been documented that suprarenal aortic banding causes an elevation of renin (42–44). Thus, the rich angiotensinogen in plasma would make it possible for the increased renin to produce more ANG I; subsequently, ANG II may be over-produced by the increased ACE1 (45). It seems that ACE1 could eventually become a limiting factor under the circumstance that ANG I is over-produced in vivo, and participate in the pathological process. It was also noticed that the slightly increased ACE1 activities in kidney and skeletal muscle was also observed. Theoretically, the increased ACE1 in these tissues may elevate circulatory ANG II, which further contributes to the enhancement of the cardiac hypertrophy. However, such contribution is unlikely to be significant since the plasma ANG II is produced mainly by lung ACE1, and in the transgenic rats, the increase in ACE1 activities in both kidney and skeletal muscle is far less than that in ventricles. Additionally, we did not observe any differences in plasma ACE1 activities between banded transgenic and control groups, suggesting that plasma ACE1 may not be involved in the augmentation of the cardiac hypertrophy. The expression of B2 was not altered by either increased ACE1 or pressure overload. Our observation is supported by a recent report that B2 is only decreased at the early stage of banding, then is back to a normal level (46), suggesting that the role of bradykinin might be less important compared with ANG II in the maintenance of cardiac hypertrophy.

The striking finding from this study is that a severe increase in cardiac ACE1 activity does not induce cardiac abnormalities by itself, but such an increase can be deleterious to the heart under certain types of loading. This hypothesis is supported by reports from other groups which showed that the effect of blood pressure on the extent of cardiac hypertrophy in hypertension was enhanced in individuals with D/D allele (47–49). Furthermore, it has been observed that individuals homozygous for the D allele did not have a higher baseline in cardiac weight, but their hypertrophic response to strenuous exercise was augmented significantly (50,51). Our study possibly clarified a long-standing suspicion that an increase in cardiac ACE1 could be instrumental in the development of left ventricular hypertrophy. We show that ventricular hypertrophy is augmented after a particular form of left ventricular loading. Therefore, the increase in cardiac ACE1 activity has a functional relevance and should be considered as an additional risk factor in the presence of cardiac hypertrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of transgenic rats
A full length human somatic ACE1 cDNA (4.02 kb) (52) was inserted into pBSKMLC-II (53), forming a plasmid pBSK(rMLC–hACE1), in which the human ACE1 was under the control of a 2.1-kb rMLC-2 promoter and followed by a human growth factor poly(A) signal (Fig. 1A). A 6.2 kb fragment (XbaI–KpnI) was used for pronucleus microinjection (54,55). The fertilized eggs were collected from SD rats (Zentralinstitut für Versuchstierkunde, Hanover, Germany). The transgenic positive rats were identified by Southern blot analysis using the tail DNA digested with EcoRI and a 32P-dCTP labeled ACE1 cDNA as probe. The heterozygous transgenic rats were bred with non-transgenic rats to obtain the littermate control.

RNase protection assay
RNase protection assay was performed as described previously (1). The probes were cloned into pCR2.1 (Invitrogen) or pBSK(+) (Stratagene) by RT–PCR and sequenced to prove no mutations. The plasmid pCRhACE562, pCRrACE400, pBSKrCol3 and pCRrGA200 contained human ACE1 (562 bp: 5'-GGCTGAGGCCAGCAAGTTT-3'/5'-GGATGGTGTCTCGTACATA-3'), rat ACE1 (400 bp: 5'-AGTAAGCAGTGGCCAGAAGCC-3'/5'-TCAGGAGTGTATGAGCTCCA-3'), Col3 (492 bp: 5'-TAGGTCCTGCAGGTAACAGT-3'/5'-GACCCGTATCTCCTCTGTCA-3') and GAPDH cDNA (200 bp: 5'-GAAACCCATCACCATCTT-3'/5'-GGTT CACACCCATCACAA-3') fragments, respectively. The pHRA carried a fused human and rat ACE1 cDNA fragment, which was produced by replacing a 200 bp of HincII–NotI fragment from pCRrACE400 with a 150 bp of BbsI–NotI fragment from pCRhACE562. pG4ZrANF537 was a subclone from pGrANF that had the insert of rat ANF cDNA (generous gift from Dr Kansuka Otta) by a self-ligation after removing the XhoI–SalI fragment. pCR-rAT1a and pCR-rB2 contained rat ANG II AT1a and B2 cDNA fragments, respectively (generous gifts from Dr Ovidu Baltatu and Dr Joao Bosco Pesquero).

Total RNA was isolated from the tissues with Trizol reagent (Invitrogen). The linearized plasmids were used to generate radioactive cRNA probes by in vitro transcription (RNA transcription kit, Stratagene) with incorporation of [{alpha}-32P-UTP] (800 Ci/mmol, Amersham): (1) HindIII-pRHA produced a fused probe containing a 150 nt fragment of hACE1 and a 200 nt fragment of rACE1 cDNA with T7; (2) BglII-pG4ZrANF537 produced a 156 nt rat ANF probe with T7; (3) RsaI-pBSKrCol3 produced a 189 nt rat Col 3 probe with T3; (4) Sty1-pCRrGa200 produced a 100 nt rat GAPDH probe with T7. To obtain the probes with suitable radioactive specific activities, a hot to cold UTP ratio of 1 : 10 in the GAPDH labeling system and 1 : 1 in others was used. Prior to hybridization, 5–50 µg of total RNA was treated with DNAse 10 µg/U at 37°C for 30 min, followed by heating at 85°C for 10 min. Hybridization was carried out at 50°C overnight and the RNase digestion was performed according to manufacturer's instructions (Roche Diagnostics). The denatured polyacrylamide gel (5% 8 M urea) was employed to fragment protected probes. After the gel was dried, it was exposed to an image plate. The radioactive signal was scanned and quantified by Bio-Imaging Analyzer Bas 2000 (Fuji).

Measurement of hACE1 activity
The tissues were homogenized at 4°C in a 20-fold excess (v/v) of 20 mM sodium phosphate, pH 7.5, 0.25 M sucrose and 5 mM MgCl2. The crude homogenate was then centrifuged at 600g for 10 min (4°C), the subsequent supernatant was further centrifuged at 105 000g for 1 h (4°C) to yield the membrane pellet. The pellet was suspended in phosphate buffered saline containing 8 mM 3-[(cholamidopropyl-9-dimethylammonio]-1-propnesulphonic acid, sonicated (2x30 s, 4°C) and kept at 4°C for 1 h, finally centrifuged at 900g for 10 min, the supernatant containing soluble protein was subjected to ACE1 enzyme assay (56). One unit was defined as 1 µmol hippuric acid per minute. The net increase in ACE1 activity in transgenic rats was presented as ‘fold increase’ (ACE1F), which was calculated with a formula: ACE1F= (ACE1TG–ACE1SD)/ACE1SD, where, ACE1TG and ACE1SD are the ACE1 activities from transgenic and from SD control, respectively.

Tissue in situ hybridization and histological observation
In situ hybridization was performed as reported previously, with slight modification (57). In brief, hACE1 cRNA probe was made from BglII-linearized pCRhACE562 with incorporation of digoxygenin-11-UTP according to kit description (Roche). The DNA template was removed afterwards by addition of 2 U DNase I (RNase free, Roche) for 20 min at 37°C in the presence of 1 U/µl RNasin. The hearts were snap frozen in isopentane (–35°C) and sectioned at a 10 µm thickness in a cryostat (Leica Frigocat, Wetzlar, Germany). Following in situ hybridization, the slides were co-stained with 1% neutral red and covered with glycerine–gelatin (Merck, FRG).

For histological observation, the hearts were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned at 10 µm, and stained with hematoxylin and eosin (H&E) and Elastica van Gieson to observe the general morphology and total collagen deposition.

ANG II measurement
The left ventricles from male transgenic (n=4) and non-transgenic control rats (n=4) were collected, weighed and snap frozen in liquid nitrogen. Then, the frozen tissues were minced and homogenized in an iced solution of 0.1 M HCl/80% ethanol (1 : 10, w/v) with a Polytron (Kinematica, Littau, Switzerland) followed by sonification (2x10 s) with the Sonifier (Bandelin Sonoplus, Berlin, Germany). The homogenates were centrifuged at 20 000g for 20 min at 4°C. The supernatant was mixed with same volume of 0.1 M Tris–acetate buffer and centrifuged at 20 000g for 20 min at 4°C. The resultant supernatant was further diluted 1 : 1 with Tris–acetate buffer and adjusted to a pH of 7.4. The final solution was concentrated by phenylsilica cartridges (Bondelut, Analytichem). Absorbed angiotensins were eluted with methanol (3x0.5 ml). The eluted angiotensins were concentrated using Speedvac (Savant Instruments) and reconstituted with 500 µl of 0.1 M Tris–acetate buffer containing 0.1% bovine serum albumin and 0.1% Triton X-100, and then incubated with 50 µl tracer (125I-ANG II, 4000 cpm) plus 50 µl ANG II antiserum (a kind gift from P. Admiraal, Rotterdam) for 20 h at 4°C. The antibody-antigen complex was separated from free ANG II by addition of dextran-coated charcoal and subsequent centrifugation. Free radioactive ANG II was counted in a gamma counter (1470 wizard, Wallac ADL). A set of tubes with different amounts of ANG II were used as standard control and subjected to the same procedures above.

Intracoronary perfusion of ANG I in isolated heart
The heart was removed under Ketamin anesthesia and arrested in ice-cold 0.9% NaCl, after which the aorta was immediately perfused retrogradely as described by Langendorff. CAF was measured by an electromagnetic flow probe (TSE, Bad Homburg, Germany). Baseline CAF was measured after a 15 min equilibration period. Then, increasing doses of ANG I (10–10, 10–9, 3x10–9, 10–8, 3x10–8 M) were infused cumulatively into the perfusion buffer just before it reached the heart. After restabilizing, the protocol was repeated in the presence of the ACE1 inhibitor quinaprilat (10–5 M). Finally, a single dose of ANG II was given (3x10–9 M).

Measurement of blood pressure
The rats were anesthetized with ether, placed in a holder which lets the tail protrude. Vasodilation was achieved by local warming of the tail with an infrared bulb. Both cuff and transducer were put around the tail and the cuff was inflated until the pulse disappeared. When the cuff was deflated, the point of the pulse reappearance indicated the value of SBP. The measurement was repeated at least three times. The data reported were the mean of measurements.

Suprarenal aortic banding
The surgery was performed as described previously (58). In brief, male rats (12 in each group) were anesthetized with 4% chloralhydrate i.p. Then, the abdominal aorta was exposed through a midline abdominal incision, and a ligature was tied around a 0.7 mm needle, placed alongside the aorta. The ligature was placed just cranial of the left renal artery. In each rat, the left kidney turns fully pale after placing the ligature, and slowly regains color after removal of the needle. Thereafter, the rats were allowed to recover, and sacrificed for characterization after 10 weeks of banding. The blood pressure was measured through the carotid artery by a 2F Millar microtip catheter (TSE biosystems) before the rats were sacrificed.

Statistical analysis
Data were analyzed for statistical significances by STATISTICA software (StatSoft). The differences in Lv/B, blood pressure and cardiac functional parameters between transgenic rat and SD were evaluated by t-test for independent samples. The combined effects of both transgene and aortic banding were determined by factorial ANOVA followed by post hoc comparisons (Newman–Keuls test). P<0.05 was considered as significant difference. All data are shown as mean±SEM.


   ACKNOWLEDGEMENTS
 
The authors thank Dr F. Alhenc-Gelas (Paris) for providing the full-length hACE1 cDNA and Mrs Heidemaria Kistel for technical support. This study was supported by the following grants: the BIOMED-II program ‘Transgeneur’ from the European Community, a grant from the German Federal Ministry for Education, Technology and Research, the Clinical Pharmacology Network Berlin-Brandenburg, the National Cardiovascular Genome Research Network (NGFN) funded by the German Ministry for Science, Technology and Education (BMBF) to M.P. and an InterCardiology Institute Netherlands fellowship to Y.M.P.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Charite Medical School, Campus Benjamin Franklin, Institute for Clinical Pharmacology, Garystrasse 5, 14167 Berlin, Germany. Tel: +49 30450570251; Fax: +49 30450570952; Email: tianx{at}ccf.org


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Tian, X.L. and Paul, M. (2003) Species-specific splicing and expression of angiotensin converting enzyme. Biochem. Pharmacol., 66, 1037–1044.[CrossRef][Web of Science][Medline]

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