(1-7) Receptor Mas Knockout Mice

Impairment of In Vitro and In Vivo Heart Function in
Angiotensin-(1-7) Receptor Mas Knockout Mice
Robson A.S. Santos, Carlos H. Castro, Elisandra Gava, Sérgio V.B. Pinheiro, Alvair P. Almeida,
Renata Dutra de Paula, Jader S. Cruz, Anderson S. Ramos, Kaleizu T. Rosa, Maria C. Irigoyen,
Michael Bader, Natalia Alenina, Gregory T. Kitten, Anderson J. Ferreira
Abstract—In this study we investigated the effects of the genetic deletion of the angiotensin (Ang)-(1-7) receptor Mas on
heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(1-7).
Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results
obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed
in Mas⫺/⫺ mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and
fibronectin were analyzed. Ang-(1-7) binding showed that Mas is localized in cardiomyocytes of the mouse heart.
Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: 1.4⫾0.09 versus
2.1⫾0.03 g in Mas⫹/⫹ mice), ⫾dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed
in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased
fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left
ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial
performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes
in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in
Mas-deficient mice. These results indicate that Ang-(1-7)-Mas axis plays a key role in the maintenance of the structure
and function of the heart. (Hypertension. 2006;47:996-1002.)
Key Words: receptors, angiotensin 䡲 extracellular matrix 䡲 echocardiography 䡲 heart failure 䡲 cardiac function
T
producing fusion protein, an improvement in cardiac function
was observed.16 Ang-(1-7) also reduces the overall growth of
cardiomyocytes.16 –18 Concerning the effects in the coronary
vasculature, it has been shown that Ang-(1-7) elicits vasorelaxation in isolated canine8 and porcine19 coronary artery rings. In
isolated perfused mouse hearts, Ang-(1-7) produced complex
vascular effects involving interaction of its receptor with angiotension II type 1 (AT1) and AT2 receptor–related mechanisms,
leading to vasodilation.20
We have reported recently that Ang-(1-7) is an endogenous
ligand for the G protein-coupled receptor Mas.21 The genetic
deletion of Mas abolished the binding of Ang-(1-7) to mouse
kidneys and abrogated the antidiuretic effect of Ang-(1-7) in
mice after acute water load. In addition, aortic rings from
Mas-deficient mice lack the endothelium-dependent
Ang-(1-7)-induced relaxation. Ang-(1-7) also induced an
increase of 3H-arachidonic acid release and NO release from
Mas-transfected cells that were blocked by the specific
Ang-(1-7) receptor Mas antagonist, A-779, but not by AT1 or
AT2 antagonists.21,22
he renin–angiotensin system (RAS), a potent regulator of
blood pressure (BP), plays a major role in the pathogenesis of cardiovascular diseases.1–3 Although angiotensin
(Ang) II is the major effector of this system, several other
Angs are now recognized as being biologically active. The
heptapeptide Ang-(1-7) is particularly interesting, because it
appears to counterbalance most of the Ang II effects. Thus,
Ang-(1-7) may have a pivotal role in the cardiovascular system.4 –7 Accordingly, Ang-(1-7) has been reported to present
important effects in coronary vessels and heart function.8 –14 This
peptide produced a significant increase in cardiac output and
stroke volume in anesthetized Wistar rats,15 decreased the
incidence and duration of ischemia-reperfusion arrhythmias,11
apparently by activating the sodium pump,14 and improved the
postischemic contractile function12 in isolated perfused rat
hearts. In keeping with these data, chronic infusion of Ang-(1-7)
improved endothelial aortic function and coronary perfusion and
preserved cardiac function in a rat model of heart failure.10 More
recently, using a transgenic rat that expresses an Ang-(1-7)-
Received November 30, 2005; first decision December 24, 2005; revision accepted February 14, 2006.
From the Departments of Physiology and Biophysics (R.A.S.S., C.H.C., A.P.A., R.D.d.P.), Morphology (E.G., G.T.K., A.J.F.), and Biochemistry and
Immunology (J.S.C., A.S.R.), Biological Sciences Institute, Belo Horizonte, MG, Brazil; Department of Pediatrics (S.V.B.P.), Federal University of Minas
Gerais, Belo Horizonte, MG, Brazil; Hypertension Unit (K.T.R., M.C.I.), Heart Institute, University of Sao Paulo, Sao Paulo, Brazil; and
Max-Delbrück-Center for Molecular Medicine (M.B., N.A.), Berlin-Buch, Germany.
Correspondence to Robson A.S. Santos, Departments of Physiology and Biophysics, Av. Antônio Carlos, 6627 Biological Sciences Institute, UFMG,
31 270-901 Belo Horizonte, MG, Brazil. E-mail [email protected]
© 2006 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000215289.51180.5c
996
Santos et al
In rodents, Mas mRNA has been detected in the heart in
addition to other tissues, such as testis, kidney, and brain.23
Recent in vitro studies have suggested that Ang-(1-7) induces
antifibrotic and antitrophic effects in cardiac cells when it
binds to cardiac fibroblasts17 and reduces the growth of
cardiomyocytes through activation of the Mas receptor.18
However, there are little data available concerning the potential role of Mas in the heart. Thus, the aim of this study was
to investigate the effects of genetic deletion of Mas on cardiac
function and coronary vascular resistance. In addition, the
effect of genetic deletion of Mas on the expression of
extracellular matrix protein in the heart was also investigated.
Methods
An expanded Methods section detailing the techniques and procedures mentioned here is available in an online supplement at
http://www.hypertensionaha.org.
Mas-Knockout Mice
Wild-type (Mas⫹/⫹) and Mas knockout (Mas⫺/⫺)24 C57BL/6 mice
were obtained from the transgenic animal facilities of the Laboratory
of Hypertension, Federal University of Minas Gerais. All of the
experimental protocols were performed in accordance with the
guidelines for the humane use of laboratory animals established at
our institution.
In Vitro Fluorescent–Labeled Ang-(1-7) Binding
Hearts from 3-month– old Mas⫹/⫹ (n⫽3) and Mas⫺/⫺ (n⫽2) mice
were snap-frozen in cooled isopentane. Cryostat sections (6 ␮m)
were serially cut, mounted on gelatin-coated slides, and dried at 4°C,
before the assay. The slices were preincubated in the assay buffer
(10⫺2 M Na-phosphate buffer, pH 7.4; 1.2⫻10⫺1 M NaCl; 5 mmol/L
MgCl2; 0.2% BSA; and 0.005% bacitracin) for 30 minutes. The
experiments were performed using assay buffer containing 2.5⫻10⫺5
M phenylmethylsulfonyl fluoride; 6⫻10⫺4 ␮M 1,10-phenanthroline;
and 2⫻10⫺9 M rhodamine-labeled Ang-(1-7) in the presence (nonspecific binding) or absence (specific binding) of Ang-(1-7) (10⫺6
mol/L) for 1 hour at 22°C to 24°C. Relative fluorescence measurements were performed on a Zeiss 510 Meta confocal microscope
using a ⫻63 oil-immersion objective lens.
Ang-(1-7) Receptor Mas in the Heart
997
BP Measurement
A polyethylene catheter was inserted into right carotid artery of
Mas⫹/⫹ and Mas⫺/⫺ mice (6 months old, n⫽3) for BP measurements.
All of the surgical procedures, basal mean arterial pressure, and heart
rate (HR) measurements were performed under halothane anesthesia
(0.75 to 1.25%, 1 L/min). An additional measurement was made in
conscious mice after recovery of anesthesia. BP and HR (derived
from the BP measurement) were recorded on a data acquisition
system (MP 100, Biopac Systems).
Statistical Analysis
Data are reported as mean⫾SEM. Statistical analyses for the binding
assay, echocardiography, electrophysiological studies, and BP measurements were performed using unpaired Student t test. Immunohistochemistry statistical analysis was performed using unpaired
Student t test followed by the Mann Whitney test. Statistical
significance for isolated perfused heart experiments was estimated
using 2-way ANOVA followed by Bonferroni test. Pⱕ0.05 was
considered significant.
Results
Fluorescent-labeled Ang-(1-7) binding was found in the
cardiomyocytes of Mas⫹/⫹ mice as displayed by confocal
analysis (Figure 1). Figure 1A shows total binding in Mas⫹/⫹
cardiomyocytes, which presented a punctuated staining pattern as would be expected for a G protein– coupled receptor.
Conversely, specific binding of rhodamine-labeled Ang-(1-7)
to heart slices from Mas⫺/⫺ evaluated by quantitative confocal
microscopy was essentially abolished (Figure 1E).
Isolated Mouse Heart Technique
Seven male Mas⫹/⫹ and 4 Mas⫺/⫺ mice (3 months old) were used for
isolated hearts experiments as described previously.11
Echocardiography Studies
Echocardiographic features were obtained using the recommendations of American Society of Echocardiography. All of the transthoracic echocardiograms were performed in male Mas⫹/⫹ and Mas⫺/⫺
mice (6 months old; n⫽10) by a single, blinded observer with the use
of a Sequoia 512 (ACUSON Corp), which offers a 10- to 14-MHz
multifrequencial linear transducer.
Electrophysiological Analysis
Individual mouse ventricular myocytes (n⫽8 cardiomyocytes from 3
different animals) were isolated from Mas⫹/⫹ and Mas⫺/⫺ animals
from the same littermates at 3 months of age. All of the experiments
were performed using conventional whole-cell patch-clamp techniques at room temperature.25,26
Immunohistochemical Analysis
Immunofluorescence labeling and quantitative confocal microscopy
were used to investigate the distribution and quantity of collagen
types I, III, and VI and fibronectin present in Mas⫹/⫹ and Mas⫺/⫺
mice hearts.
Figure 1. In vitro localization of rhodamine–Ang-(1-7) binding in
mice left ventricle slices. Figure shows total binding in Mas⫹/⫹
cardiomyocytes (A), nonspecific binding in Mas⫹/⫹ cardiomyocytes (B), total binding in Mas⫺/⫺ cardiomyocytes (C), and nonspecific binding in Mas⫺/⫺ cardiomyocytes (D). Concentration of
rhodamine-Ang-(1-7) was 2⫻10⫺9 mol/L in the presence (nonspecific binding) or absence (specific binding) of Ang-(1-7) (10⫺6
mol/L). Quantification of relative fluorescence in Mas⫹/⫹ and
Mas⫺/⫺ cardiomyocytes (E). Data are shown as the SD. *P⬍0.01
(unpaired Student t test.).
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Figure 2. Time course of systolic tension
(A), diastolic tension (B), ⫹dT/dt (C),
⫺dT/dt (D), HR (E), and coronary perfusion pressure (F) in isolated perfused
mouse heart. The hearts were perfused
according to Langendorff technique.
**P⬍0.001, ***P⬍0.0001 (2-way ANOVA
followed by Bonferroni test).
Mas⫺/⫺ mice presented an impairment of in vitro heart
function. As shown in Figure 2A, systolic tension was
significantly lower in hearts from Mas⫺/⫺ mice (average:
1.4⫾0.09 versus 2.1⫾0.03 g in age-matched Mas⫹/⫹ mice;
P⬍0.0001). Diastolic tension was similar in both groups
(Figure 2B). Mas-deficient mice presented a lower ⫹dT/dt
and ⫺dT/dt (average: 49.5⫾1.3 versus 65.2⫾0.3 and
49.5⫾1.5 versus 64.2⫾1.0 g/s in age-matched Mas⫹/⫹ mice,
P⬍0.0001, respectively; Figure 2C and 2D) and a lower HR
(average: 250.8⫾9.5 versus 333.3⫾2.6 bpm in age-matched
Mas⫹/⫹ mice; P⬍0.0001; Figure 2E). In addition, isolated
hearts from Mas⫺/⫺ mice presented a significantly higher
coronary perfusion pressure (average of the 30-minute recording: 172.7⫾3.0 versus 126.9⫾2.8 mm Hg in agematched Mas⫹/⫹ mice; P⬍0.001; Figure 2F).
The Table summarizes the echocardiographic parameters
obtained in Mas⫹/⫹ and Mas⫺/⫺ mice. In agreement with the
results obtained with isolated hearts, intact hearts in Masdeficient mice showed a significantly lower fractional shortening
(19.01⫾0.71 versus 22.90⫾0.25% in Mas⫹/⫹ mice; P⬍0.001).
Morphologically, Mas⫺/⫺ mice displayed a significantly lower
posterior wall thickness in systole (0.085⫾0.001 versus
0.090⫾0.001 mm in Mas⫹/⫹ mice; P⬍0.05) and left ventricle
end-diastolic dimension (0.387⫾0.001 versus 0.395⫾0.003 cm
in Mas⫹/⫹ mice; P⬍0.05) and a higher left ventricle end-systolic
dimension (0.313⫾0.002 versus 0.305⫾0.002 cm in Mas⫹/⫹
mice; P⬍0.01). A substantially lower global ventricular function, expressed by a higher myocardial performance index,
was also observed (0.73⫾0.05 versus 0.54⫾0.03 in Mas⫹/⫹
mice; P⬍0.01; Figure 3).
To elucidate the possible mechanisms underlying the
cardiac function impairment, we analyzed the whole-cell
L-type Ca2⫹ current in individual cardiomyocytes and cardiac
expression of collagen types I, III, and VI and fibronectin.
Although it is now generally accepted that L-type Ca2⫹
current is the major trigger for sarcoplasmic reticulum Ca2⫹
release, no significant differences were observed between
L-type Ca2⫹ current density from age-matched Mas⫹/⫹ and
Mas⫺/⫺ mice (Figure 4A through 4D). Interestingly, the time
to the peak of Ca2⫹ current was statistically different when
Santos et al
Echocardiographic Parameters Obtained in Masⴙ/ⴙ and
Masⴚ/ⴚ Mice
Echocardiographic Parameters
Mas⫹/⫹
Mas⫺/⫺
Fractional shortening, %
22.90⫾0.25
19.01⫾0.71*
Posterior wall thickness in
diastole, mm
0.068⫾0.001
0.066⫾0.001
Posterior wall thickness in
systole, mm
0.090⫾0.001
0.085⫾0.001†
Interventricular septal
thickness in diastole, mm
0.067⫾0.001
0.066⫾0.001
Interventricular septal
thickness in systole, mm
0.089⫾0.001
0.084⫾0.001
Left ventricle end-diastolic
dimension, cm
0.395⫾0.003
0.387⫾0.001†
Left ventricle end-systolic
dimension, cm
0.305⫾0.002
0.313⫾0.002‡
Myocardial performance index
0.54⫾0.03
0.73⫾0.05‡
Isovolumic relaxation time, ms
19.50⫾1.18
Isovolumic relaxation time/HR
1.80⫾0.10
Ang-(1-7) Receptor Mas in the Heart
999
No major differences were detected in the atria (data not
shown, E. Gava and G.T. Kitten, unpublished data, 2005). A
similar change was observed in the left ventricle of neonate
mice (Figures I and II, available online).
To further discard the possibility that the changes in collagen
expression in cardiac function in vivo were secondary to
increased BP, an additional group of animals at the same age of
those used for echocardiograph measurements (6 months) were
instrumented for acute BP recordings. As expected,24,27 no
differences in BP between Mas⫺/⫺ and Mas⫹/⫹ mice were
observed (BP: 79.3⫾4.9 versus 90.3⫾6.4 mm Hg, P⫽0.2438;
HR: 467.0⫾56.1 versus 511.3⫾24.2 bpm, P⫽0.5086 in agematched Mas⫹/⫹ mice under halothane anesthesia and BP:
100.0⫾3.0 versus 104.3⫾2.9 mm Hg, P⫽0.3622; HR:
559.7⫾21.2 versus 613.3⫾27.2 bpm, P⫽0.1957 in Mas⫹/⫹ mice
after recovery of anesthesia). Actually, halothane-anesthetized
Mas⫺/⫺ animals presented slight lower BP in comparison to
Mas⫹/⫹, probably resulting from the lower cardiac output.
20.60⫾0.88
1.88⫾0.07
Discussion
Heart rate, bpm
512.9⫾9.4
501.8⫾10.5
Left ventricular mass, g
0.085⫾0.002
0.082⫾0.003
In this study, we have unmasked a key functional role of the
Ang-(1-7) receptor Mas in the heart. We observed that
systolic tension, ⫹dT/dt, and ⫺dT/dt were significantly
lower in isolated hearts of Mas-deficient mice. Accordingly,
an impaired heart function was observed in vivo by means of
echocardiography. Echocardiographic measurements revealed a decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and
a higher left ventricle end-systolic dimension. A markedly
lower global ventricular function, as defined by a higher
myocardial performance index, was observed. In addition,
Mas⫺/⫺ mice presented a higher coronary perfusion pressure
compared with Mas⫹/⫹ mice. These findings are in keeping
with several studies suggesting that Ang-(1-7) has beneficial
effects in myocardial function.8 –13,16,28 We have shown that
Ang-(1-7), at subnanomolar concentration, has an antiarrhythmogenic effect on reperfusion11 and that it preserves the
cardiac function in the postischemic period12 in isolated
perfused rat hearts. These effects were completely blocked by
the selective Ang-(1-7) receptor Mas antagonist A-779,
suggesting that these Ang-(1-7) actions in the heart are
mediated by its specific receptor. One possible mechanism
that may contribute to the beneficial effects of Ang-(1-7) in
the heart is the modulation of cardiac Ang II levels, as
described recently by Mendes et al.29 The present results
indicate that the Ang-(1-7) receptor Mas mediates the effects
of Ang-(1-7) on the cardiac function.
The higher perfusion pressure observed in the isolated
heart preparation is in agreement with the previous reports of
a vasorelaxant activity of Ang-(1-7) in the coronary bed.8,19
Brosnihan et al9 found that this heptapeptide elicits a vasodilator response in canine coronary arteries. This effect was
completely blocked by the nonselective Ang antagonist,
[Sar1Thr8]-Ang II, but not by either an AT1 receptor antagonist or an AT2 receptor antagonist, suggesting that the
Ang-(1-7)-induced vasodilation in coronary arteries is mediated by a non-AT1/AT2 receptor. Our data indicate that the
Ang-(1-7) receptor Mas may account for Ang-(1-7)-induced
vasodilation in the coronary bed. Accordingly, the Mas
Values are mean⫾SEM. Transthoracic echocardiogram was performed in
male Mas⫹/⫹ and Mas⫺/⫺ mice (n⫽10 for each group). Data were analyzed by
unpaired Student t test.
*P⬍0.0001; †P⬍0.05; ‡P⬍0.01.
comparing the 2 groups (8.5⫾0.4 ms for the Mas⫹/⫹ versus
10.8⫾0.6 ms for the Mas⫺/⫺ at ⫺10 mV; P⬍0.01; and
7.3⫾0.6 ms for the Mas⫹/⫹ versus 9.4⫾0.4 ms for the Mas⫺/⫺
at 0 mV; P⬍0.01). This change could contribute to the
cardiac dysfunction described.
Strikingly, we observed that the expression of several
matrix proteins were significantly and markedly higher in the
ventricles of adult Mas⫺/⫺ mice hearts compared with Mas⫹/⫹
control mice (Figure 5A and 5B): that is, type I collagen
94.22⫾4.90 versus 59.00⫾2.35 in Mas⫹/⫹ mice (P⬍0.0001);
type collagen III 111.20⫾8.09 versus 45.67⫾2.21 in Mas⫹/⫹
mice (P⬍0.001); and fibronectin 78.89⫾2.90 versus
45.67⫾2.21 in Mas⫹/⫹ mice (P⬍0.0001). Conversely, the
expression of type VI collagen in Mas⫺/⫺ mice was decreased
20.78⫾2.29 versus 43.22⫾3.29 in Mas⫹/⫹ mice (P⬍0.0001).
Figure 3. Representative echocardiographic measurements of
contracting hearts from Mas⫹/⫹ and Mas⫺/⫺. LVESD indicates
left ventricle end-systolic dimension; LVEDD, left ventricle enddiastolic dimension.
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May 2006
Figure 4. Electrophysiological analysis.
Representative L-type Ca2⫹ currents in
ventricular cardiomyocytes from Mas⫹/⫹
(A) and Mas⫺/⫺ (B). (C) voltage dependence of Ica,L, plotted as current density
(A/F) obtained for test potentials from
⫺50 to ⫹60 mV. Values represent mean
⫾SEM. (D) membrane capacitance (pF)
measured in Mas⫹/⫹ (䡺) and Mas⫺/⫺ (f)
ventricular myocytes. Bar graph values
are 157.5⫾12.9 (Mas⫹/⫹, n⫽8) and
145.0⫾13.0 (Mas⫺/⫺, n⫽8).
receptor antagonist A-779 abolished the potentiating effect of
Ang-(1-7) on bradykinin-induced vasodilation in an isolated
perfused rat heart preparation30 and the vasodilation induced
by Ang-(1-7) in losartan-treated isolated mouse heart.20 This
later effect was also abolished in isolated hearts of Masdeficient mice.20
Recent reports have suggested that Ang-(1-7) could be a
modulator of ion channel functioning similar to Ang II.31–33
Both peptides augment the release of [3H] norepinephrine
elicited by nerve stimulation in isolated rat atria.31 At the
neuromuscular junction, Ang-(1-7) increased acetylcholine
release in a dose-dependent manner.33 Thus, the lower contractility observed in Mas⫺/⫺ mice provides additional evidence that interactions between Ang-(1-7) and Mas receptor
play an important modulatory role in calcium homeostasis in
the heart tissue. Our data also indicate that the time to the
peak of Ca2⫹ current measured at ⫺10 and 0 mV for the
L-type Ca2⫹ current was slower for the Mas⫺/⫺ cells, which
could impair the sarcoplasmic reticulum refilling process and,
thus, lead to less Ca2⫹ being released during excitation.
We have suggested that Ang-(1-7) could produce changes
in pacemaker ionic currents. Isolated hearts from transgenic
rats [TGR(A1-7)3292] that chronically overproduce Ang(1-7) resulting in 2.5-fold increase in circulating Ang-(1-7)
concentration showed a significantly higher basal intrinsic
HR observed in vivo and in ex vivo conditions.16 On the other
hand, in Mas⫺/⫺ mice, the basal intrinsic HR was significantly
lower compared with Mas⫹/⫹ mice. In line with these data, De
Mello14 reported that Ang-(1-7) activates the sodium pump,
hyperpolarizes the heart cell, and re-establishes the impulse
conduction during ischemia/reperfusion, indicating that the
antiarrhythmogenic effect of Ang-(1-7) could be because of
changes in ionic currents. These observations suggest that
Ang-(1-7) could actually modulate some component of the
pacemaker ionic currents through Ang-(1-7) Mas receptor
stimulation. However, the exact mechanism involved in the
change in HR in Mas-deficient mice remains to be
established.
The changes in time to the peak of Ca2⫹ current could be
related to the actual amount of Ca2⫹ that is mobilized during
an action potential, but because we did not measure the global
Ca2⫹ transients elicited by electrical stimulation, we cannot
address this point. However, a major mechanism for the
impairment in heart function in Mas⫺/⫺ appears to be the
strikingly changes in extracellular matrix proteins. Our results
provide strong evidence for a previously unexpected key role
for the Ang-(1-7)-Mas axis in the control of expression of
extracellular matrix proteins. The fact that collagen VI
undergoes an opposite change as that observed for collagen I,
III, and fibronectin suggests that this influence is selective.
These observations are in accordance with a recent report by
Iwata et al,17 showing that Ang-(1-7) produces antifibrotic
effects. As expected,24,27 a contribution of BP to the changes
in collagen expression or in cardiac function could be
excluded by the direct BP measurements in halothane-anesthetized and in nonanesthetized mice. Actually, under halothane anesthesia, the mean arterial pressure of Mas⫺/⫺ mice
was slightly lower than that of Mas⫹/⫹ mice, probably because
of the lower cardiac output. Because the expression of
extracellular matrix proteins presented a similar pattern in
neonate mice, we can further exclude a contribution of BP or
systemic vascular resistance in Mas⫺/⫺ as a primary cause for
the cardiac phenotype observed. Furthermore, Walther et al24
showed that Mas⫺/⫺ are healthy, grow normally, and show no
obvious developmental abnormalities. They also reported no
difference in drinking behavior. It is well known that fibrosis
leads to alterations in diastolic function. Accordingly, associated with the important deficit of systolic function, we have
also found a significantly lower dimension of the left ventricle at the end of diastole and a decreased ⫺dT/dt in isolated
Santos et al
Ang-(1-7) Receptor Mas in the Heart
1001
Another limitation was the need for anesthetic to perform
echocardiography. The anesthetic of choice to make echocardiograms in mice is always a complex issue, because most of
them influence HR, which alters cardiac function.34 Because
the mice HR is normally very high, choosing an anesthetic
regimen that keeps it near to physiological levels should be an
important concern. In this study, halothane has been used to
perform echocardiogram, because it is more convenient and
reliable with respect to rate of induction, reversibility, and
control of anesthetic depth.34 In addition, HR is less affected
with inhaled anesthetic than with intraperitoneal ones. Although further studies with other anesthetics, such as isoflurane, would be interesting to confirm our findings with
halothane, the similarity between our in vivo and in vitro data
provides a very strong evidence for a key role of the
Ang-(1-7)-Mas axis in heart function.
Perspectives
Figure 5. Immunofluorescent localization of matrix proteins in
the left ventricles of adult Mas⫹/⫹ and Mas⫺/⫺ mice hearts.
Expression of type I collagen, type III collagen, and fibronectin
were increased in the left ventricles of Mas⫺/⫺ compared with
Mas⫹/⫹ mice, whereas the expression of type VI collagen was
decreased. The images in the control column show the level of
immunostaining obtained when the primary antibody is omitted
from the incubation procedure (A). Quantification of extracellular
matrix proteins in the left ventricles of Mas⫹/⫹ and Mas⫺/⫺ mice
(B). Data are shown as the SEM. *P⬍0.001; **P⬍0.0001. A.U.
indicates arbitrary unit.
hearts of Mas⫺/⫺. Walther et al27 have reported an increased
BP variability and indirect evidence for an increased sympathetic tonus in male Mas⫺/⫺ mice as compared with control
mice (presumably C57BL/6 mice) using spectral analysis of
BP data sampled at a relatively low frequency (250 Hz).
Whether these changes contribute or are consequence or
adaptative responses to the lower cardiac function remains to
be established. Taken all together, these data indicate a
BP-independent fibrosis as the main cause of heart dysfunction in Mas-deficient mice.
One of the limitations of our study is related to the
echocardiogram measurements. Reporting of E and A peaks
and E/A relation, as well as E deceleration time would add an
important information. However, because the halothane anesthetic regimen keeps HR near physiological levels and the
HR of mice is very high, peak E fuses with peak A. Besides,
values of fused peak E and A did not differ between the
groups, in accordance with isovolumic relaxation time.
The changes in extracellular matrix proteins to a profibrotic
profile in Mas⫺/⫺ mice provide additional evidence that the
Ang-(1-7)-Mas axis plays a key role in the beneficial effects
of angiotensin-converting enzyme (ACE) inhibitors, AT1
receptor blockers (ARBs), and aldosterone antagonists in the
heart. It has been shown that chronic treatment with ACE
inhibitors or ARBs increases the activity of Ang-(1-7)–
forming enzymes,35–37 in particular, heart ACE2.36 More
recently, it has been demonstrated in humans that treatment
with aldosterone antagonists substantially increased ACE2
activity.38 The prominent decrease in cardiac function in
Mas⫺/⫺ mice, which can be correlated with the marked
changes in extracellular matrix proteins and the antihypertrophic effects of Ang-(1-7),16,17 apparently mediated by its
interaction with Mas,18 suggest that activation of the Ang-(17)–forming pathways by RAS blockade produces beneficial
cardiac effects through activation of Mas. One could argue
that ACE2 knockout mice39 showed more pronounced cardiac
function impairment than Mas knockout mice. However, the
ACE2 knockout mice presented a higher Ang II level, which
may contribute to the cardiac phenotype. Of note is the fact
that, in ACE2 knockout mice, the cardiac dysfunctions
appeared only after 6 months of age, whereas the cardiac
fibrotic phenotype in Mas⫺/⫺ mice was already observed in
the neonatal period.
In conclusion, our findings suggest that Ang-(1-7)-Mas
axis plays a key role in the heart. Furthermore, our data also
suggest that this RAS axis may be a potential target for the
development of new cardiovascular drugs.7
Acknowledgments
This work was supported in part by Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico–Programa de Grupos de Excelência and Fundação de Amparo à Pesquisa de Minas Gerais. We are
thankful to José Roberto da Silva. The Zeiss confocal microscope is
located in the Centro de Microscopia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais.
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