Physiol Genomics 17: 292–299, 2004.
First published March 23, 2004; 10.1152/physiolgenomics.00227.2003.
Expression of an angiotensin-(1–7)-producing fusion protein produces
cardioprotective effects in rats
Robson A. S. Santos,1* Anderson J. Ferreira,1,2* Ana Paula Nadu,1 Aline N. G. Braga,1
Alvair Pinto de Almeida,1 Maria José Campagnole-Santos,1 Ovidiu Baltatu,2 Radu Iliescu,2
Timothy L. Reudelhuber,3 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; 2Max-Delbrück-Center for Molecular Medicine, Berlin-Buch 13125, Germany; and
3
Laboratory of Molecular Biochemistry of Hypertension, Clinical Research Institute of Montreal, Quebec H2W 1R7, Canada
Submitted 29 December 2003; accepted in final form 17 March 2004
Santos, Robson A. S., Anderson J. Ferreira, Ana Paula Nadu,
Aline N. G. Braga, Alvair Pinto de Almeida, Maria José Cam-
pagnole-Santos, Ovidiu Baltatu, Radu Iliescu, Timothy L. Reudel-
huber, and Michael Bader. Expression of an angiotensin-(1–7)-
producing fusion protein produces cardioprotective effects in rats.
Physiol Genomics 17: 292–299, 2004. First published March 23,
2004; 10.1152/physiolgenomics.00227.2003.—Angiotensin-(1–7)
[ANG-(1–7)] is a recently described heptapeptide product of the
renin-angiotensin system. Because biosynthesis of ANG-(1–7) in-
creases 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 cardioprotec-
tive 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 testic-
ular-specific expression of a cytomegalovirus promoter-driven trans-
gene, resulting in a doubling of circulating ANG-(1–7) compared with
nontransgenic control rats. Radiotelemetry hemodynamic measure-
ments 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 hypertro-
phy 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
THE HEPTAPEPTIDE angiotensin-(1–7) [ANG-(1–7)] is now con-
sidered 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), prolylendopep-
* R. A. S. Santos and A. J. Ferreira contributed equally to this work.
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 Biofı́sica, Av. Antônio Carlos, 6627-ICB-UFMG,
31 270-901, Belo Horizonte, MG, Brazil (E-mail: santos@icb.ufmg.br).
292 1094-8341/04 $5.00 Copyright © 2004 the American Physiological Society
Downloaded from www.physiology.org/journal/physiolgenomics (177.182.050.062) on January 31, 2020.
tidase, 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 nonpharmaco-
logical measures for treating hypertension and other cardiovas-
cular diseases produce increases in plasma ANG-(1–7) con-
centration (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
Generation of the TGR(A1–7)3292 transgenic rats. The transgene
construct contained the human prorenin signal peptide and the immu-
noglobulin 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⌬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 lib-
erated by XmnI and NruI from vector sequences and used for pro-
nuclear 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.
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.
 ANGIOTENSIN-(1–7)-PRODUCING FUSION PROTEIN
Fig. 1. Schematic representation of structural components of the engineered
fusion protein. ANG-(1–7) is released by the action of furin during secretion.
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 per-
formed following the TRIzol reagent method (Invitrogen Life Tech-
nologies) according to the manufacturer’s protocol. RNA samples (2
␮g) 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⬘-CATCAC-
CCATCGAGAGAACC-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 ␮g 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 ␤-actin gene. RNA samples were hybrid-
ized with ⬃40,000 cpm of the radiolabeled transgene antisense probe
and 25,000 cpm of the ␤-actin probe. The hybridized fragments, once
protected from RNase A ⫹ T1 digestion, were separated by electro-
phoresis 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 pro-
tein). Each assay was performed three times using organs from
different animals. The agarose beads were spun out in a microcentri-
fuge, 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
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293
␮mol/l p-hydroxymercuribenzoate, 131.50 ␮mol/l of 1,10-phenan-
throline, 0.90 ␮mol/l phenylmethylsulfonyl fluoride (PMSF), 1.75
␮mol/l pepstatin A, 0.032% EDTA, and 0.0043% protease-free bo-
vine serum albumin (BSA) and evaporated (n ⫽ 4–5 different ani-
mals). 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-phenanthro-
line, 1 mmol/l PMSF, 1 mmol/l pepstatin A, and 7.5% EDTA (50
␮l/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% heptaflu-
orobutyric 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 accord-
ing to Botelho et al. (5). Angiotensin-(1–7) levels were measured by
radioimmunoassay (RIA), as previously described (5). Protein con-
centration 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 accompany-
ing software (Dataquest ART, Gold 2.0) to store and analyze the data.
Before the experiments were started, the rats were housed in individ-
ual 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, normal-
ized 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 ␮m) were cut starting from the base area of the left
ventricle at intervals of 40 ␮m and dyed according to Rosenfeld (27).
Tissue sections (3–4 for each animal) were examined with a light
microscope (Axioplan 2, Zeiss) at 100⫻ magnification, photographed
(AxioCam digital camera, Zeiss), and analyzed with a Zeiss KS 400
3.0 software. Only digitized images of cardiomyocytes cut longitudi-
nally 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 nu-
cleus. 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
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294 ANGIOTENSIN-(1–7)-PRODUCING FUSION PROTEIN

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.

KCl, 1.2 KH2PO4, 1.2 MgSO4 䡠 7H2O, 2.5 CaCl2 䡠 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,

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.

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 ANGIOTENSIN-(1–7)-PRODUCING FUSION PROTEIN 295
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.

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 molec-
ular 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 indepen-
dent experiments.

ventricular tachycardia, and/or ventricular fibrillation after reperfu-

sion. 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 car-
diac 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

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.
Total RNA of testis, adrenal gland, heart, kidney, brain,
liver, and lung were used for RPA (Fig. 3). Transgene expres-
sion was detectable only in testis of the transgenic rats accord-
ing to profile observed in RT-PCR.
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
Fig. 5. HPLC followed by ANG-(1–7) radioimmunoassay (RIA) in C18
of ⬃32,000 Da. Goat anti-mouse IgG2b antibody presented a extracted samples of testis (A), venous plasma (B), and arterial plasma (C).
cross immunoreactivity to rat IgG in transgenic and normal Data are means ⫾ SE of 4 different animals. *Significant at P ⬍ 0.05
animals with a molecular mass of ⬃150,000 Da. compared with SD animals (Student’s t-test).

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296 ANGIOTENSIN-(1–7)-PRODUCING FUSION PROTEIN

Fig. 6. HPLC followed by ANG-(1–7) RIA in C18 extracted

samples of atrium, left ventricle, lung, adrenal gland, and kid-
ney. Data are means ⫾ SE of 4–5 different animals. P ⬍ 0.05
compared with SD animals (Student’s t-test).

17.6 ⫾ 0.9 pg/ml plasma, P ⬍ 0.05, Fig. 5C). In keeping with fluctuations of blood pressure and HR were also not changed in
the transgene expression data ANG-(1–7) levels in atrium, left transgenic rats (Table 1).
ventricle, lung, adrenal gland, and kidney were not signifi- Further physiological characterization of the transgenic an-
cantly changed in transgenic rats compared with normal ani- imals was performed using isoproterenol-induced hypertrophy
mals (Fig. 6). and isolated perfused hearts. Left ventricle/body weight ratio
Radiotelemetry hemodynamic measurements showed that of isoproterenol-treated SD rats was significantly increased
transgenic rats presented a significant increase in daily and compared with vehicle-treated rats (3.0 ⫾ 0.1 vs. 2.5 ⫾ 0.04
nocturnal HR (average for the 1-wk interval: 330 ⫾ 1.4 vs. mg/g, P ⬍ 0.001). In TG rats isoproterenol treatment also
309 ⫾ 1.9 beats/min, P ⬍ 0.001; and 389 ⫾ 0.7 vs. 363 ⫾ 1.8 induced left ventricular hypertrophy (2.7 ⫾ 0.06 vs. 2.4 ⫾ 0.03
beats/min, P ⬍ 0.001, respectively). In addition, transgenic rats mg/g, P ⬍ 0.01); however, the left ventricular hypertrophy
presented a significant increase in daily and nocturnal dP/dt induced by isoproterenol treatment in TG was significantly
(1.9 ⫾ 0.02 vs. 1.7 ⫾ 0.02 mmHg/ms, P ⬍ 0.001; and 2.0 ⫾ lower compared with normal rats (2.7 ⫾ 0.06 vs. 3.0 ⫾ 0.1
0.01 vs. 1.9 ⫾ 0.01 mmHg/ms, P ⬍ 0.001, respectively) (Table mg/g, P ⬍ 0.05, Fig. 7A). Cardiomyocyte cross-sectional area
1). These changes were not accompanied by significant was significantly increased in isoproterenol-treated rats com-
changes in systolic or diastolic blood pressure. Circadian pared with vehicle-treated rats in both groups (17.46 ⫾ 0.14 vs.
13.46 ⫾ 0.09 ␮m in vehicle-treated SD rat, P ⬍ 0.001; and
14.13 ⫾ 0.10 vs. 12.41 ⫾ 0.10 ␮m in vehicle-treated TG rat,
Table 1. Cardiovascular parameters of Sprague-Dawley and P ⬍ 0.01). However, the cardiomyocyte cross-sectional area
TGR(A1–7)3292 rats obtained by radiotelemetry was significantly lower in isoproterenol-treated TG rats com-
SD TG
pared with isoproterenol-treated SD rats (14.13 ⫾ 0.10 vs.
17.46 ⫾ 0.14 ␮m, P ⬍ 0.001, Fig. 7B).
Day Night Day Night Isolated hearts from transgenic rats perfused according to the
HR, beats/min 309⫾1.9 363⫾1.8* 330⫾1.4† 389⫾0.7*† Langendorff technique showed reduced duration of reperfusion
DAP, mmHg 88⫾0.1 93⫾0.2* 88⫾0.3 93⫾0.2* arrhythmias compared with hearts from age-matched SD rats
SAP, mmHg 125⫾0.4 130⫾0.4* 124⫾0.4 130⫾0.3* (ASI ⫽ 2.5 ⫾ 0.3 vs. 4.4 ⫾ 0.9 arbitrary units in SD rats, P ⬍
dP/dt, mmHg/ms 1.7⫾0.02 1.9⫾0.01* 1.9⫾0.02† 2.0⫾0.01*†
0.05, Fig. 8A). In addition, during reperfusion period there was
Data are means ⫾ SE of the heart rate (HR), diastolic arterial pressure a decrease in perfusion pressure and an increase in diastolic
(DAP), systolic arterial pressure (SAP), and dP/dt values sampled every 5 min tension. However, the postischemic function in hearts from TG
for 10 s/24 h for 1 wk. *Significantly different from respective daily period.
†Significantly different from SD rats to the same period. Data were analyzed
rats was preserved compared with hearts from SD rats. The
by two-way ANOVA followed by the Bonferroni test. P values of 0.05 or less perfusion pressure returned to basal levels at 5 min after
were considered significant (n ⫽ 6). TG, TGR(A1–7)3292 rats. reperfusion in TG rats (86.1 ⫾ 5.1% of the basal period) and
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 ANGIOTENSIN-(1–7)-PRODUCING FUSION PROTEIN 297
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 accor-
dance 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 trans-
gene integration site. HPLC-RIA analysis of testis homoge-
nates 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 differ-
ences 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 arte-
riovenous 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 in-

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.

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.
DISCUSSION

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
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 Bon-
ferroni test (n ⫽ 8, †P ⬍ 0.05 and *P ⬍ 0.001 vs. basal period).

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298 ANGIOTENSIN-(1–7)-PRODUCING FUSION PROTEIN
creased 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 ab-
sence 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 pro-
vided by Bevilaqua et al. (4). In this work ANG-(1–7) changed
acetylcholine release at the neuromuscular junction, evidenc-
ing 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 postis-
chemic 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 measure-
ments.
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 bio-
chemical 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 cardio-
protective effect of blockade (AT1 antagonists) or reduction
(ACE inhibitors) of ANG II in the heart.
We have observed that the isoproterenol-induced heart hy-
pertrophy 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 estab-
lished. Changes in testosterone levels as consequence of the
increase in ANG-(1–7) in testis are apparently not involved,
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because no significant changes in its plasma levels were ob-
served 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
This work was supported in part by Conselho Nacional de Desenvolvimento
Cientı́fico 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 Pes-
soal de Nı́vel Superior (CAPES).
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