Expression of an angiotensin-(1–7)-producing fusion protein produces cardioprotective effects in rats

Robson A. S. Santos1,*, Anderson J. Ferreira1,2,*, Ana Paula Nadu1, Aline N. G. Braga1, Alvair Pinto de Almeida1, Maria José Campagnole-Santos1, Ovidiu Baltatu2, Radu Iliescu2, Timothy L. Reudelhuber3 and Michael Bader2

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 
Angiotensin-(1–7) [ANG-(1–7)] is a recently described heptapeptide product of the renin-angiotensin system. Because biosynthesis of ANG-(1–7) increases in animals treated with cardioprotective drugs and inactivation of the gene for angiotensin converting enzyme 2 [an enzyme involved in the biosynthesis of ANG-(1–7)] leads to the development of cardiac dysfunction, it has been suggested that ANG-(1–7) has cardioprotective properties. To directly test this possibility, we have generated transgenic rats that chronically overproduce ANG-(1–7) by using a novel fusion protein methodology. TGR(A1–7)3292 rats show testicular-specific expression of a cytomegalovirus promoter-driven transgene, resulting in a doubling of circulating ANG-(1–7) compared with nontransgenic control rats. Radiotelemetry hemodynamic measurements showed that transgenic rats presented a small but significant increase in daily and nocturnal heart rate and a slight but significant increase in daily and nocturnal cardiac contractility estimated by dP/d t measurements. Strikingly, TGR(A1–7)3292 rats were significantly more resistant than control animals to induction of cardiac hypertrophy by isoproterenol. In addition, transgenic rats showed a reduced duration of reperfusion arrhythmias and an improved postischemic function in isolated Langendorff heart preparations. These results support a cardioprotective role for circulating ANG-(1–7) and provide a novel tool for evaluating the functional role of ANG-(1–7).

engineered protein; renin-angiotensin system; heart hypertrophy


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 
THE HEPTAPEPTIDE angiotensin-(1–7) [ANG-(1–7)] is now considered one of the biologically active end products of the renin-angiotensin system (RAS) (9, 15, 16, 30). Contrasting with other members of the angiotensin peptide family (ANG III and ANG IV), several biological effects of ANG-(1–7) are opposite those elicited by ANG II (for review, see Refs. 16 and 30). ANG-(1–7) produces vasodilatation (30), antiproliferative effects in vascular smooth muscle cells (19), and increases baroreflex sensitivity (8, 24).

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-(19) 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.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 
Generation of the TGR(A1–7)3292 transgenic rats.
The transgene construct contained the human prorenin signal peptide and the immunoglobulin fragment from mouse IgG2b (22) linked to a portion of the human prorenin prosegment. The BglII site after the prorenin segment was used to insert a furin cleavage site and the coding sequence for ANG-(1–7) followed by a stop codon by the use of a double-stranded oligonucleotide. Furthermore, an intron and polyadenylation cassette of SV40 virus from p{Delta}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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Fig. 1. Schematic representation of structural components of the engineered fusion protein. ANG-(1–7) is released by the action of furin during secretion.

 
Animals.
One-month-old (isolated heart preparation) and 3-mo-old male Sprague-Dawley (SD) and TGR(A1–7)3292 (TG) rats were used. All rats were obtained from the transgenic animal facilities at Laboratório de Hipertensão, Universidade Federal de Minas Gerais. All experimental protocols were performed in accordance with the guidelines for the human use of laboratory animals of our institute and approved by local authorities.

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 
To generate an animal model with increased ANG-(1–7) production, transgenic rats were established with the DNA-construct that codes for a protein liberating ANG-(1–7), shown in Fig. 1, during its constitutive secretion from cells.

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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Fig. 2. Representative ethidium bromide-stained agarose gels of PCR reactions. RNA was isolated from ventricle, kidney, testis, liver, atrium, brain, adrenal gland, lung, and aorta excised from either Sprague-Dawley (SD) or TGR(A1–7)3292 (TG) rats. Top: PCR reactions using RNA to exclude genomic DNA contamination in the samples. Bottom: transgene expression assessed by PCR after reverse transcription of RNA. Genomic DNA from transgenic animals was used as a positive control, and PCR reactions without either RNA (top) or cDNA (bottom) were used as a negative control. The 450-bp fragment corresponds to the transgenic mRNA. Each test was performed three times using organs from different animals.

 
Total RNA of testis, adrenal gland, heart, kidney, brain, liver, and lung were used for RPA (Fig. 3). Transgene expression was detectable only in testis of the transgenic rats according to profile observed in RT-PCR.



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Fig. 3. RNase protection assay of adrenal gland, testis, heart, kidney, brain, liver, and lung using a specific probe (~500 bases) for the transgene RNA (protected fragment of ~450 bases). The presence of transgene mRNA was demonstrated only in testis. Each organ was tested in three different transgenic animals.

 
Further confirmation of the transgene expression in testis was performed using Western Blot analysis (Fig. 4). The engineered protein migrates with an apparent molecular mass of ~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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Fig. 4. Western blot was used for detection of the transgenic proteins. Testes were homogenized in 10 mmol/l phosphate buffer pH 7.4. Proteins were immunoprecipitated with protein A-agarose and analyzed by SDS-PAGE gel for electrophoresis. The engineered protein migrates with an apparent molecular mass of ~32,000 Da. Goat anti-mouse IgG2b antibody presented a cross immunoreactivity to rat IgG with a molecular mass of ~150,000 Da in transgenic and normal animals. Results are representative for three independent experiments.

 
Radioimmunoassay measurements revealed that transgene expression leads to significantly increased levels of ANG-(1–7) in testis of transgenic rats compared with control animals resulting in 4.5-fold higher ANG-(1–7) concentration in TG rats (1.3 ± 0.4 vs. 0.3 ± 0.07 pg/mg protein in age-matched SD, P < 0.05, Fig. 5A). A significant increase in venous ANG-(1–7) plasma concentration was also observed (31.5 ± 2.7 vs. 14.1 ± 5.9 pg/ml plasma, P < 0.05, Fig. 5B), indicating that in this model the testes are functioning as ANG-(1–7) infusion pumps. In addition, there was a significant increase in arterial ANG-(1–7) plasma concentration (43.6 ± 13.1 vs. 17.6 ± 0.9 pg/ml plasma, P < 0.05, Fig. 5C). In keeping with the transgene expression data ANG-(1–7) levels in atrium, left ventricle, lung, adrenal gland, and kidney were not significantly changed in transgenic rats compared with normal animals (Fig. 6).



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Fig. 5. HPLC followed by ANG-(1–7) radioimmunoassay (RIA) in C18 extracted samples of testis (A), venous plasma (B), and arterial plasma (C). Data are means ± SE of 4 different animals. *Significant at P < 0.05 compared with SD animals (Student’s t-test).

 


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Fig. 6. HPLC followed by ANG-(1–7) RIA in C18 extracted samples of atrium, left ventricle, lung, adrenal gland, and kidney. Data are means ± SE of 4–5 different animals. P < 0.05 compared with SD animals (Student’s t-test).

 
Radiotelemetry hemodynamic measurements showed that transgenic rats presented a significant increase in daily and nocturnal HR (average for the 1-wk interval: 330 ± 1.4 vs. 309 ± 1.9 beats/min, P < 0.001; and 389 ± 0.7 vs. 363 ± 1.8 beats/min, P < 0.001, respectively). In addition, transgenic rats presented a significant increase in daily and nocturnal dP/dt (1.9 ± 0.02 vs. 1.7 ± 0.02 mmHg/ms, P < 0.001; and 2.0 ± 0.01 vs. 1.9 ± 0.01 mmHg/ms, P < 0.001, respectively) (Table 1). These changes were not accompanied by significant changes in systolic or diastolic blood pressure. Circadian fluctuations of blood pressure and HR were also not changed in transgenic rats (Table 1).


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Table 1. Cardiovascular parameters of Sprague-Dawley and TGR(A1–7)3292 rats obtained by radiotelemetry

 
Further physiological characterization of the transgenic animals was performed using isoproterenol-induced hypertrophy and isolated perfused hearts. Left ventricle/body weight ratio of isoproterenol-treated SD rats was significantly increased compared with vehicle-treated rats (3.0 ± 0.1 vs. 2.5 ± 0.04 mg/g, P < 0.001). In TG rats isoproterenol treatment also induced left ventricular hypertrophy (2.7 ± 0.06 vs. 2.4 ± 0.03 mg/g, P < 0.01); however, the left ventricular hypertrophy induced by isoproterenol treatment in TG was significantly lower compared with normal rats (2.7 ± 0.06 vs. 3.0 ± 0.1 mg/g, P < 0.05, Fig. 7A). Cardiomyocyte cross-sectional area was significantly increased in isoproterenol-treated rats compared with vehicle-treated rats in both groups (17.46 ± 0.14 vs. 13.46 ± 0.09 µm in vehicle-treated SD rat, P < 0.001; and 14.13 ± 0.10 vs. 12.41 ± 0.10 µm in vehicle-treated TG rat, P < 0.01). However, the cardiomyocyte cross-sectional area was significantly lower in isoproterenol-treated TG rats compared with isoproterenol-treated SD rats (14.13 ± 0.10 vs. 17.46 ± 0.14 µm, P < 0.001, Fig. 7B).



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Fig. 7. Averaged ventricular mass index (A) and morphometry (B) in control and isoproterenol-treated rats. 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 at 0.1 ml/100 g ip, for 7 days). Values are means ± SE of 6–8 different animals for averaged ventricular mass index and 4–6 different animals for morphometry. *P < 0.05, **P < 0.01, and ***P < 0.001 determined by two-way ANOVA followed by the Bonferroni test.

 
Isolated hearts from transgenic rats perfused according to the Langendorff technique showed reduced duration of reperfusion arrhythmias compared with hearts from age-matched SD rats (ASI = 2.5 ± 0.3 vs. 4.4 ± 0.9 arbitrary units in SD rats, P < 0.05, Fig. 8A). In addition, during reperfusion period there was a decrease in perfusion pressure and an increase in diastolic tension. However, the postischemic function in hearts from TG rats was preserved compared with hearts from SD rats. The perfusion pressure returned to basal levels at 5 min after reperfusion in TG rats (86.1 ± 5.1% of the basal period) and at 20 min after reperfusion in SD rats (91.1 ± 5.4% of the basal period, Fig. 8B). Moreover, diastolic tension did not change after reperfusion in TG rats, but in SD rats diastolic tension increased with 1 and 5 min of reperfusion, returning to basal levels only at 10 min (Fig. 8C). Basal intrinsic HR was increased in isolated hearts from transgenic rats (250.0 ± 0.5 vs. 214.1 ± 2.2 beats/min in SD rats, n = 8, two-way ANOVA followed by the Bonferroni test). Furthermore, upon coronary occlusion there was a marked decrease in HR in isolated hearts from SD rats (43%, at 5 min after coronary occlusion), whereas a significantly smaller decrease occurred in isolated hearts from TG rats (11%, at 5 min after coronary occlusion). HR returned to basal levels within 20 and 30 min in TG and SD rats, respectively. No significant differences were observed for maximum and minimum dT/dt.



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Fig. 8. Averaged arrhythmia severity index (ASI) (A), percentage of the basal perfusion pressure (B), and percentage of the basal diastolic tension (C) upon reperfusion after 30 min of global ischemia in isolated perfused rat hearts. ASI was analyzed by Student’s t-test (n = 8, *P < 0.05). Perfusion pressure and diastolic tension were analyzed by two-way ANOVA followed by the Bonferroni test (n = 8, {dagger}P < 0.05 and *P < 0.001 vs. basal period).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 
Using the strategy initially described by Methot et al. (22), we have produced a transgenic rat expressing an ANG-(1–7)-producing fusion protein. The expression of the transgene in the TGR(A1–7)3292 line is apparently restricted to the testis, since no evidence for transgene mRNA was found in several other tissues examined (kidney, adrenal gland, lung, atria, ventricle, liver, brain, and aorta). The expression of transgenes under the control of the CMV promoter/enhancer seems to be very variable. On the one hand, expression in 24 of 28 tissues examined has been reported (31), but also the restriction of transgene mRNA exclusively to testis was observed in accordance to this study (14). This variability may be dependent on the methylation status of the transgene (14), which is probably influenced by the region in the genome adjacent to the transgene integration site. HPLC-RIA analysis of testis homogenates revealed a 4.5-fold increase in ANG-(1–7) concentration. The ~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.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 
This work was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico-Programa de Grupos de Excelência (CNPq-PRONEX). A. J. Ferreira is a recipient of a PhD degree fellowship from CNPq and a "Sandwich-fellowship" from Coodenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

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. Back


    References
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 

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