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1 Laboratory of Hypertension, Department of Physiology and Biophysics, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, MG, 31270-901 Brazil
2 Max-Delbrück-Center for Molecular Medicine, Berlin-Buch 13125, Germany
3 Laboratory of Molecular Biochemistry of Hypertension, Clinical Research Institute of Montreal, Quebec H2W 1R7, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSReferences |
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engineered protein; renin-angiotensin system; heart hypertrophy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSReferences |
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ANG-(1–7) can be formed from ANG II through hydrolysis by angiotensin converting enzyme 2 (ACE2), prolylendopeptidase, or prolylcarboxypeptidase (9, 16, 30, 12, 34) or directly from ANG I through hydrolysis by prolylendopeptidase and endopeptidase 24.11 (9, 15, 29, 35, 37). An indirect pathway involving hydrolysis of ANG I to ANG-(1–9) by ACE2 with subsequent conversion to ANG-(1–7) by ACE has also been suggested (13).
There are few studies examining the effects of chronic increases in plasma ANG-(1–7) concentration (2, 7, 32). In these studies ANG-(1–7) was administrated using osmotic mini-pumps for periods no longer than 15 days (2, 7, 36). Thus information on the effects of chronic increases in ANG-(1–7) is still missing. Data in this regard are particularly important considering that several pharmacological and nonpharmacological measures for treating hypertension and other cardiovascular diseases produce increases in plasma ANG-(1–7) concentration (10).
It has been recently described that peptides can be directly released within specific tissues from an engineered fusion protein by proteolytic action of the furin enzyme (22, 23). This technique provided a possibility to increase the release of peptides to defined tissues or a particular cell line (23). With this strategy, transgenic mice have been produced expressing an ANG II-producing fusion protein exclusively in cardiac myocytes or astrocytes (20, 33).
In this study we tested the possibility of application of the biological pumps concept for production of transgenic rats expressing an ANG-(1–7)-producing protein.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSReferences |
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Lux (25) was inserted 3' of the stop codon (see Fig. 1). Finally, the construct was cloned into the pcDNA3.1 vector (Invitrogen, Karlsruhe, Germany) to set it under the control of the cytomegalovirus (CMV) promoter/enhancer. The transgene was liberated by XmnI and NruI from vector sequences and used for pronuclear microinjection into fertilized rat zygotes as described (26). The offspring were analyzed for the integration of the transgene into the genome by a PCR specific for the SV40 DNA fragment in the construct.
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RNA isolation and RT-PCR.
The organs (heart, lung, liver, brain, testis, kidney, aorta, and adrenal gland) were isolated from SD and TG rats and frozen immediately. Each test was performed three times using organs from different animals. Total RNA isolation was performed following the TRIzol reagent method (Invitrogen Life Technologies) according to the manufacturer’s protocol. RNA samples (2 µg) were treated with DNase to eliminate genomic DNA present in the samples. Transgene expression was assessed by PCR after reverse transcription of RNA (RT-PCR). Total RNA treated with DNase was reverse transcribed using random primers. A 450-bp fragment of the single-stranded cDNA was amplified by PCR using 56°C as annealing temperature and 30 cycles and the forward primer IG5 (5'-CATCACCCATCGAGAGAACC-3') located in IgG fragment and the reverse primer hRENEX (5'-GGACCAAGCCTGGCCATGTCC-3') located in the human prorenin fragment of the transgene.
RNase protection assay.
Transgene expression was analyzed by RNase protection assay (RPA) using commercially available Ambion RPA II kits (AMS Biotechnology, Witney, UK), according to the protocol of the manufacturer. Forty micrograms total RNA of testis, adrenal gland, heart, kidney, brain, liver, and lung and 50 µg of yeast as a control were used for RPA. Each organ was tested in three different transgenic animals. cDNA sequences generated by PCR were subcloned in a T-vector (Promega, Mannheim, Germany). A T7-polymerase reaction transcribed a 
500-base radioactive probe complementary to 450 nucleotides of the transgene mRNA and a 200-base radioactive probe complementary to 150 nucleotides of the mRNA encoding the rat ß-actin gene. RNA samples were hybridized with 40,000 cpm of the radiolabeled transgene antisense probe and 25,000 cpm of the ß-actin probe. The hybridized fragments, once protected from RNase A + T1 digestion, were separated by electrophoresis on a denaturing gel (5% polyacrylamide, 8 M urea) and analyzed using a Fujix BAS 2000 phosphoimager system (Raytest, Straubenhardt, Germany).
Western blot analysis.
Testes were homogenized in 10 mmol/l phosphate buffer pH 7.4 (5 ml to ± 1 g of the tissue), and proteins were immunoprecipitated with protein A-agarose (protein A-agarose binds tightly to the immunoglobulin domain of the engineered protein). Each assay was performed three times using organs from different animals. The agarose beads were spun out in a microcentrifuge, rinsed, boiled in sample buffer, and loaded onto a 12.5% SDS-PAGE gel for electrophoresis. After electrophoresis, proteins were transferred to a nylon membrane, blocked with 5% nonfat milk solution for 1 h, and incubated for 2 h with an anti-mouse IgG2b goat antibody at 1:1,000 dilution (Sigma-Aldrich). Membranes were washed three times (20 min each wash) and stained using an anti-goat IgG conjugated with peroxidase at 1:2,000 dilution (Sigma-Aldrich) for 1 h. Finally, membranes were washed three times with 10 mmol/l phosphate-buffered saline, pH 7.4, 0.05% Tween 20 (20 min each wash) and exposed to Hyperfilm ECL film (Amersham International). The engineered protein migrates with an apparent molecular mass of 32,000 Da.
Angiotensin-(1–7) measurement.
The organs were homogenized with 0.045 N HCl in ethanol (10 ml/g of tissue) containing 0.90 µmol/l p-hydroxymercuribenzoate, 131.50 µmol/l of 1,10-phenanthroline, 0.90 µmol/l phenylmethylsulfonyl fluoride (PMSF), 1.75 µmol/l pepstatin A, 0.032% EDTA, and 0.0043% protease-free bovine serum albumin (BSA) and evaporated (n = 4–5 different animals). After evaporation, the samples were dissolved in 0.003% trifluoracetic acid (TFA). Blood samples were collected from the carotid artery or jugular vein through a cannula. Immediately after collection the blood was transferred to polypropylene tubes containing 1 mmol/l p-hydroxymercuribenzoate, 30 mmol/l of 1,10-phenanthroline, 1 mmol/l PMSF, 1 mmol/l pepstatin A, and 7.5% EDTA (50 µl/ml of blood). After centrifugation, plasma samples were frozen in dry ice and stored at –80°C. Peptides were extracted onto a BondElut phenylsilane cartridge (Varian). The columns were preactivated by sequential washes with 10 ml of 99.9% acetonitrile/0.1% heptafluorobutyric acid (HFBA) and 10 ml of 0.1% HFBA. Sequential washes with 10 ml of 99.9% acetonitrile/0.1% HFBA, 10 ml of 0.1% HFBA, 3 ml of 0.1% HFBA containing 0.1% BSA, 10 ml of 10% acetonitrile/0.1% HFBA, and 3 ml of 0.1% HFBA were used to activate the columns. After sample application, the columns were washed with 20 ml of 0.1% HFBA and 3 ml of 20% acetonitrile/0.1% HFBA. The adsorbed peptides were eluted with 3 ml of 99.9% acetonitrile/0.1% HFBA into polypropylene tubes rinsed with 0.1% fat-free BSA. After evaporation, the samples were analyzed using a HPLC system according to Botelho et al. (5). Angiotensin-(1–7) levels were measured by radioimmunoassay (RIA), as previously described (5). Protein concentration in the crude homogenates was determined by the Bradford method (6).
Radiotelemetry monitoring of blood pressure and heart rate.
A telemetry system was used for measuring systolic pressure, diastolic pressure, dP/d t, and heart rate (HR). This monitoring system consists of radio frequency transducers model TA11PA-C40, receivers, a matrix, and an IBM-compatible personal computer with accompanying software (Dataquest ART, Gold 2.0) to store and analyze the data. Before the experiments were started, the rats were housed in individual cages until the telemetry tracings indicated reestablishment of regular 24-h oscillations of blood pressure (BP) and HR. Thereafter, data were sampled (200 Hz) every 5 min for 10 s/24 h for 1 wk (n = 6 different animals).
Heart hypertrophy.
Heart hypertrophy was induced in male SD and TG rats by daily injection of isoproterenol (2 mg/kg ip, for 7 days). Control groups received daily injections of vehicle (0.9% NaCl, 0.1 ml/100 g ip, for 7 days). At the end of the 7-day period, the rats were killed by decapitation and the hearts were immediately removed. The atria and right ventricle were dissected free from the left ventricle and discarded. Wet weights of the left ventricles were recorded, normalized for body weight, and expressed as ventricular mass index (mg/g) (n = 6 to 8 different animals). In addition, left ventricles were left in 4% formalin in 0.1 M phosphate buffer ph 7.4 for 24 h at room temperature. The tissues were dehydrated by sequential washes with 70% ethanol, 80% ethanol, 90% ethanol, and 100% ethanol and embedded in glycidyl methacrylate (JB-4, Polysciences). Transversal sections (2 µm) were cut starting from the base area of the left ventricle at intervals of 40 µm and dyed according to Rosenfeld (27). Tissue sections (3–4 for each animal) were examined with a light microscope (Axioplan 2, Zeiss) at 100x magnification, photographed (AxioCam digital camera, Zeiss), and analyzed with a Zeiss KS 400 3.0 software. Only digitized images of cardiomyocytes cut longitudinally with nuclei and cellular limits visible were used for analysis (an average of 30 cardiomyocytes for each slice). The diameter of each myocyte was measured across the region corresponding to the nucleus. We analyzed 50–100 cardiomyocytes for each animal (n = 4–6 different animals).
Isolated rat heart technique.
Male SD and TG rats were decapitated 10–15 min after intraperitoneal injection of 400 IU heparin (n = 8 different animals). The thorax was opened, and the heart was carefully dissected and perfused through a 1.0 ± 0.3 cm aortic stump with Krebs-Ringer solution (KRS) containing (in mmol/l) 118.4 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4·7H2O, 2.5 CaCl2·2H2O, 11.7 glucose, and 26.5 NaHCO3. The perfusion flow was maintained constant (9.0 ml/min) at 37 ± 1°C and constant oxygenation (5% CO2 and 95% O2). A force transducer was attached through a heart clip to the apex of the ventricles to record the contractile force (tension, g) on a computer, through a data-acquisition system (Biopac System, Santa Barbara, CA). A diastolic tension of 0.5–1.0 g was applied to the hearts. Electrical activity was recorded by using the data-acquisition system with the aid of two cotton wicks placed directly on the surface of the right atrium and left ventricle (bipolar lead). The HR was calculated from the electrocardiograph records. Coronary perfusion pressure was measured by means of a pressure transducer connected to the aortic cannula and coupled to the recording system. After an equilibration period of 30 min, the hearts were subjected to 30 min of global ischemia, followed by 30 min of reperfusion with KRS. Cardiac arrhythmias were defined as the presence of extra systoles, ventricular tachycardia, and/or ventricular fibrillation after reperfusion. To obtain a quantitative measurement, the arrhythmias were graded arbitrarily according to their duration considering duration of 30 min as irreversible arrhythmia. Therefore, the occurrence of cardiac arrhythmias for up to 3 min was assigned the factor 2; 3 to 6 min was assigned the factor 4; 6 to 10 min was assigned the factor 6; 10 to 15 min was assigned the factor 8; 15 to 20 min was assigned the factor 10; 20 to 25 min was assigned the factor 11; and 25 to 30 min was assigned the factor 12. A value of 0–12 was thus obtained in each experiment and was denoted as "arrhythmia severity index" (ASI) (3, 17).
Statistical analysis.
Data are reported as means ± SE. Statistical analysis of the peptide levels and ASI was performed by Student’s t-test. Data obtained in the isoproterenol-induced hypertrophy and cardiac function in isolated hearts were analyzed by two-way ANOVA followed by the Bonferroni test. For telemetry statistical analysis, 72 values for every 12 h (1 value at each 10 min) for each rat were computed and a mean value (day and night) was calculated. These individual data were averaged (n = 6 for each group) and analyzed by two-way ANOVA followed by the Bonferroni test. P values of 0.05 or less were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSReferences |
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First, we investigated the transgene gene expression in various organs of the transgenic animals using PCR after reverse transcription of RNA (RT-PCR). Figure 2 shows that the transgene mRNA is exclusively expressed in testis of the transgenic animals.
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32,000 Da. Goat anti-mouse IgG2b antibody presented a cross immunoreactivity to rat IgG in transgenic and normal animals with a molecular mass of 150,000 Da.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSReferences |
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2.5-fold increase in venous and arterial blood ANG-(1–7) concentration indicates that in these animals the testes are acting as a biological ANG-(1–7) infusion pump. The HPLC data also show that ANG-(1–7) concentration did not change in several tissues a finding that is in keeping with the expression data. Interestingly, there were no obvious arteriovenous differences in ANG-(1–7) concentration in both SD and TG rats. Previous studies showed that ACE is a major ANG-(1–7) metabolizing enzyme (1, 11). Therefore, the absence of arteriovenous differences for ANG-(1–7) concentration suggests that the pulmonary vascular bed is a source of circulating ANG-(1–7). This possibility warrants further investigation.
There were no differences of arterial pressure between SD and TG rats. However, HR and dP/dt were significantly increased in TG rats. The absence of major changes in AP was expected (28). Chronic or acute infusion of ANG-(1–7) in rats produces only slight changes in mean arterial pressure (MAP). This could be due to an increase in cardiac output as recently described in anesthetized rats by Sampaio and coworkers (28). The increased cardiac output could mask a decrease in total peripheral resistance produced by ANG-(1–7) leading to absence of MAP changes. Whether this is also in true for the TG rats remains to be elucidated.
The increased HR in TG rats is in contrast to a recent study in which we observed that infusion of ANG-(1–7) for 7 days produced a slight but significant bradycardia in Wistar rats (7). Besides strain differences, the short-term administration of the peptide compared with the chronic increase in ANG-(1–7) in the TG rats may account for these divergent observations. The increased HR in TG rats could be part of a compensatory mechanism or to a direct cardiac or central effect of the peptide at areas deficient in blood-brain barrier. The fact that HR of isolated hearts taken from TG rats was higher than those from SD rats suggests that chronic increases of ANG-(1–7) could produce changes in pacemaker ionic currents. An evidence for an effect of ANG-(1–7) on ionic currents was recently provided by Bevilaqua et al. (4). In this work ANG-(1–7) changed acetylcholine release at the neuromuscular junction, evidencing a neuromodulatory action for this peptide.
We have previously described that at low concentration (220 fmol/l) ANG-(1–7) decreased the incidence and duration of cardiac reperfusion arrhythmias (17). An improvement of postischemic systolic function was also found (18). Accordingly, a severe systolic cardiac dysfunction was observed in mice with deletion of ACE2 gene expression (12). In keeping with these recent findings TG rats presented a decreased duration of reperfusion arrhythmias and an improved myocardial function after reperfusion, mainly by showing an attenuated increase in diastolic tension. In addition, an increase in daily and nocturnal dP/dt was observed in radiotelemetry hemodynamic measurements.
It should be mentioned that the cardioprotective effects observed in isolated hearts of transgenic rats indicate that the increase in plasma ANG-(1–7) levels induced sustained biochemical and functional alterations leading to an improved cardiac postischemic function. We have observed that Wistar rats infused for 7 days with ANG-(1–7) and the TG rats presented a marked reduction in the ANG II levels in the left ventricle (A. C. Mendes et al., unpublished observation). This change may be one of the major factors for the cardioprotection observed in the TG rats, considering the well-known cardioprotective effect of blockade (AT1 antagonists) or reduction (ACE inhibitors) of ANG II in the heart.
We have observed that the isoproterenol-induced heart hypertrophy was attenuated in TG rats. In line with these findings, chronic administration (8 wk) of ANG-(1–7) using osmotic minipumps has been recently reported to preserve cardiac function, coronary perfusion, and aortic endothelial function in a rat model for heart failure produced by ligation of the left coronary artery (21). Whether these cardioprotective effects of ANG-(1–7) are due to a direct tissue action through a paracrine or autocrine mechanism or to an indirect mechanism, such as reduction of ANG II as discussed above, remains to be established. Changes in testosterone levels as consequence of the increase in ANG-(1–7) in testis are apparently not involved, because no significant changes in its plasma levels were observed in the transgenic rats (data not shown).
In summary, using the engineered fusion protein strategy, we have produced a new transgenic model expressing an ANG-(1–7)-producing fusion protein. Using this model, we have provided further evidence for an important role of ANG-(1–7) in cardiac function. The TGR(A1–7)3292 rats would be a useful and important model to clarify several aspects of the biological role of this heptapeptide.
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GRANTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSReferences |
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. A. S. Santos, Departamento de Fisiologia e Biofísica, Av. Antônio Carlos, 6627-ICB-UFMG, 31 270-901, Belo Horizonte, MG, Brazil (E-mail: santos{at}icb.ufmg.br).
10.1152/physiolgenomics.00227.2003.
* R. A. S. Santos and A. J. Ferreira contributed equally to this work. 
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSReferences |
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P < 0.05 and *P < 0.001 vs. basal period).