Download Cardiac Hypertrophy Is Associated With Decreased eNOS

Cardiac Hypertrophy Is Associated With Decreased eNOS
Expression in Angiotensin AT2 Receptor–Deficient Mice
Marc Brede, Wilhelm Roell, Oliver Ritter, Frank Wiesmann, Roland Jahns, Axel Haase,
Bernd K. Fleischmann, Lutz Hein
Abstract—Angiotensin II receptors play an essential role in cardiovascular physiology and disease. The significance of
angiotensin type II (AT2) receptors in cardiac disease still remains elusive. Thus, we tested in gene-targeted mice
whether AT2 receptors modulate cardiac function and remodeling after experimental myocardial injury. To generate
myocardial infarcts of reproducible size, a cryolesion was generated at the free wall of the left ventricle of wild-type
mice (Agtr2⫹/Y) and mice carrying a deletion of the AT2 receptor gene (Agtr2-/Y). Postinjury remodeling was followed
up for 4 weeks after cryoinjury. The cryoprocedure led to an increased heart weight/body weight ratio and heart
weight/tibia length ratio in AT2-deficient mice compared with control mice. Morphometric analysis revealed a
significant increase in myocyte cross-sectional area after cardiac injury (infarct vs sham Agtr2⫹/Y, ⫹53%; vs Agtr2-/Y,
⫹95%). Expression of endothelial nitric oxide synthase (eNOS) was significantly lower in hearts from Agtr2-/Y than
from Agtr2⫹/Y mice. eNOS downregulation was accompanied by a decrease in cardiac cGMP levels in Agtr2-/Y mice.
In isolated murine cardiomyocytes, angiotensin II induced eNOS expression through AT2 receptors, and inhibition of
NO production by NG-nitro-L-arginine methyl ester abolished the antihypertrophic effect of AT2 on cardiac myocytes.
Our results demonstrate in a genetic mouse model that angiotensin II AT2 receptors exert an antihypertrophic effect in
cardiac remodeling after myocardial cryoinjury and link the expression of cardiac eNOS to AT2 receptor activation.
(Hypertension. 2003;42:1177-1182.)
Key Words: myocardial infarction 䡲 cryoinjury 䡲 animals, transgenic 䡲 mice 䡲 nitric oxide synthase
A
antihypertrophic effects7,8 or mediates part of the protective
effects of AT1 receptor antagonists.9 In contrast, transgenic
overexpression of the AT2 receptor in cardiac myocytes in
vivo resulted in enhanced hypertrophy 10 or dilated
cardiomyopathy.11
Several signaling pathways have been proposed to mediate
the intracellular actions of AT2 receptor activation in the
heart.12–14 We have recently identified that AT2 receptors in
rat cardiac myocytes induce expression of the endothelial
nitric oxide (eNOS) isoform through a calcineurin-dependent
pathway.15 Thus, the aim of this study was to determine
whether AT2 receptor activation and eNOS expression are
also linked in the murine heart in vivo and whether AT2dependent eNOS regulation affects cardiac myocyte hypertrophy. Because pressure-overload and coronary artery ligation models have yielded divergent results, part of which
might be due to variability of the initial insults, we chose a
cryoinfarction model with precisely defined cardiac injury to
assess the effects of AT2 gene deletion on postinjury cardiac
remodeling in vivo. We also investigated expression of NOS
isoforms in this model in vivo and in vitro. Our data show that
growing body of experimental evidence suggests that
the angiotensin II receptor subtypes, AT1 and AT2, exert
opposing effects in the cardiovascular system. Activation of
AT2 receptors might cause antigrowth effects on cells of the
cardiovascular system and thus, counteract the growthpromoting action of AT1 receptors.1–3 However, the pathophysiologic relevance of AT2 receptors for cardiac remodeling has not yet been firmly established. Recently, transgenic
and gene-targeted mouse models with overexpression or
deficiency in angiotensin II receptor subtypes have significantly advanced our understanding of the renin-angiotensin
system. Unfortunately, data obtained from AT2 receptor–
deficient mice have been contradictory with respect to the
specific function of this receptor subtype for cardiac hypertrophy in vivo. In mouse models of left ventricular pressure
overload, either AT2 receptors were required for hypertrophic
remodeling4 or they did not affect cardiac hypertrophy.5 After
experimental coronary ligation in mice, one report concluded
that AT2 receptor activation enhanced cardiac hypertrophy.6
However, the majority of the myocardial infarction studies
performed with AT2-deficient mice document that AT2 has
Received August 20, 2003; first decision September 9, 2003; revision accepted September 30, 2003.
From the Institut für Pharmakologie und Toxikologie (M.B., R.J., L.H.), Medizinische Klinik (O.R., F.W.), Medizinische Poliklinik (R.J.), and
Physikalisches Institut (F.W., A.H.), Universität Würzburg, Würzburg; the Klinik und Poliklinik für Herzchirurgie (W.R.), Universität Bonn, Bonn; and
Institut für Neurophysiologie (B.K.F.), Universität Köln, Köln, Germany.
Correspondence to Lutz Hein, MD, Institut für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Strasse 9, 97078 Würzburg,
Germany. E-mail [email protected]
© 2003 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000100445.80029.8E
1177
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AT2-deficient mice display an enhanced hypertrophic response to myocardial cryoinjury, which was associated with
lower cardiac eNOS and cGMP levels. In isolated myocytes,
AT2 activation was sufficient to induce eNOS expression, and
inhibition of NO production abolished the antihypertrophic
effects of AT2.
Methods
AT2 Receptor–Deficient Mice and
Myocardial Cryoinjury
Since the initial generation of AT2-deficient mice,16 the Agtr2-/Y
deletion was crossed back onto an FVB/N background for 10
generations. For the present study, male littermates with intact
(Agtr2⫹/Y) or deleted (Agtr2-/Y) AT2 genes were used, which were
derived from crosses of heterozygous female (Agtr2⫹/-) and wildtype male FVB/N mice.17 All animal procedures were approved by
the responsible authorities of the University of Würzburg and the
Government of Unterfranken (protocol No. 621-2531.01-28/01).
Left ventricular infarcts were induced at the free wall of the left
ventricle after thoracotomy under isoflurane anesthesia by a copper
rod (4-mm diameter), which was precooled in liquid N2.18 Altogether, 23 Agtr2⫹/Y and 20 Agtr2-/Y mice were subjected to the
cryoinjury. Ninety-three percent of the animals survived the operation and the first 48 hours of the postoperative period. Sixteen mice
(8 Agtr2⫹/Y, 8 Agtr2-/Y) were sham-operated. Mice that died during
hemodynamic analysis (2 Agtr2⫹/Y and 1 Agtr2-/Y) were not
included for further analysis.
␮mol/L N␻-nitro-L-arginine methyl ester (L-NAME), and cells were
stimulated with for 4 hours with 100 nmol/L angiotensin II. Proteins
were precipitated in 10% trichloroacetic acid and resuspended in 1%
sodium dodecyl sulfate. Radioactivity was measured in a ␤-counter
and normalized to DNA content of the samples.
Statistical Analysis
For all experiments, 1- or 2-way ANOVA followed by appropriate
post hoc tests or t tests was used to determine statistical significance
(P⬍0.05) with the use of Prism 3.0cx software (GraphPad).
Results
Cardiac Remodeling After Cryoinjury
To obtain left ventricular infarcts of reproducible size, we
chose a cryoinjury model that has been used previously for
cellular cardiomyoplasty.18,23 In brief, the hearts of anesthetized mice were exposed through a small thoracotomy, and a
copper rod (4-mm diameter) that had been precooled in LN2
was placed on the free wall of the left ventricle to injure a
defined area of myocardium. The overall mortality of mice
during this procedure was 7%: 2 of 23 wild-type mice
(Agtr2⫹/Y) and 1 of 20 AT2-deficient mice (Agtr2-/Y) died
within 48 hours after the operation. During the following
Hemodynamic Measurements
For magnetic resonance imaging (MRI) of the heart, mice were
anesthetized with 2% isoflurane in 1 L/min oxygen flow through a
nose cone. MRI was performed 4 weeks after myocardial cryoinjury
in 6 Agtr2⫹/Y and 6 Agtr2-/Y mice, and 5 sham-operated mice per
genotype were used for comparison. Cardiac images were taken with
a 7.05-T BIOSPEC 70/20 scanner.19 Left ventricular dimensions,
ejection fraction, and cardiac output were derived from 3D reconstructions of sequential short-axis slices as described.19 For direct
cardiac catheterization with a 1.4F high-fidelity pressure-volume
catheter (Millar Instruments), mice were anesthetized with tribromoethanol (13 ␮L of 2.5% solution per g body weight).17 For evaluation
of cardiac contractility, hemodynamic parameters were determined
10 to 12 minutes after induction of anesthesia in all mice.20
Histology
Hearts were fixed in 4% paraformaldehyd in phosphate-buffered
saline, embedded in paraffin, stained with Sirius red, and analyzed
by computer-assisted morphometry.21 Midequatorial sections from a
total of 51 hearts (sham, 8 Agtr2⫹/Y and 8 Agtr2-/Y; infarct, 19
Agtr2⫹/Y and 16 Agtr2-/Y) were used for determination of interstitial
fibrosis and myocyte cross-sectional area (16 to 24 nucleated
myocytes per section).
NOS Expression
To analyze protein expression by Western blotting, cardiac tissue
was prepared and analyzed as described.17 Monoclonal antibodies
against eNOS, neuronal NOS, inducible NOS (all from Transduction
Laboratories), ␤-actin, or histone H1 (Santa Cruz Biotechnology)
were used. Left ventricular cGMP levels were determined with a
cGMP immunoassay (R&D Systems).
Isolated Cardiac Myocytes
Neonatal cardiac myocytes were isolated from Agtr2⫹/Y and
Agtr2-/Y mice as described previously.22 To assess eNOS expression
in cardiac myocytes, cells were stimulated for 48 hour with 100
nmol/L angiotensin II in minimal essential medium-1. For determination of protein synthesis, [3H]leucine (2.5 ␮Ci/mL) was added to
isolated myocytes, and cells were incubated for 4 hours at 37°C. For
some experiments, NO production was inhibited by addition of 200
Figure 1. Cardiac hypertrophy after myocardial infarction. a and b,
Sirius red staining of midventricular tissue sections from shamoperated (a) or cryolesioned (b) Agtr2-/Y mice 4 weeks after operation. Area of the cryolesion is visible as a fibrotic scar (b, arrows),
which covers 40% of the left ventricular internal circumference. LV
indicates left ventricle; RV, right ventricle. Bars⫽1 mm. Inserts
show myocyte cross sections with hypertrophy in operated
Agtr2-/Y mice (bars⫽20 ␮m). c, Heart weight– body weight ratios
were significantly increased after cryoinjury. In AT2-deficient mice,
the magnitude of hypertrophy after cryoinjury was greater than in
Agtr2⫹/Y mice. d, Morphometric analysis of left ventricular tissue
sections revealed enhanced cardiac myocyte hypertrophy, ie,
cross sectional areas, in Agtr2-/Y mice 4 weeks after the cryolesion compared with wild-type mice. Data are mean⫾SEM, n⫽8 to
19 mice per group. *P⬍0.05, Agtr2-/Y vs Agtr2⫹/Y. †P⬍0.05,
infarct vs sham.
Brede et al
Angiotensin AT2 Receptors and Cardiac Hypertrophy
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Cardiovascular Parameters of Wild-Type and Agtr2-/Y Mice After Cryoinjury
Agtr2⫹/Y Sham
(n⫽8)
Parameter
Body weight, g
Heart weight, mg
Heart weight/tibia length, mg/mm
LV collagen volume, %
Infarct area, mm2
Infarct scar thickness, ␮m
LV catheterization
⫺1
Agtr2-/Y Sham
(n⫽8)
Agtr2⫹/Y Infarct
(n⫽19)
Agtr2-/Y Infarct
(n⫽16)
31.8⫾1.9
30.5⫾1.1
32.4⫾0.7
32.6⫾0.5
116.1⫾5.4
115.5⫾3.6
132.0⫾4.2†
145.7⫾4.4*†
6.25⫾0.21
6.34⫾0.19
1.5⫾0.3
2.1⫾0.3
7.14⫾0.23†
8.07⫾0.25*†
2.8⫾0.4†
4.5⫾0.8†
䡠䡠䡠
䡠䡠䡠
8.0⫾0.3
8.3⫾0.4
䡠䡠䡠
n⫽8
䡠䡠䡠
n⫽8
238⫾18
254⫾7
n⫽16
n⫽15
Heart rate, min
441⫾19
462⫾18
517⫾16†
529⫾15†
Systolic pressure, mm Hg
96.5⫾2.3
99.7⫾3.9
96.0⫾3.6
101.5⫾4.6
dP/dtmax, mm Hg/s
8468⫾605
dP/dtmin, mm Hg/s
⫺6923⫾1020
LV MRI
Stroke volume, ␮L
8296⫾501
⫺6617⫾837
6373⫾377†
⫺6080⫾350
7874⫾555*
⫺6172⫾961
n⫽5
n⫽5
n⫽6
n⫽6
56.4⫾4.4
59.5⫾6.0
36.2⫾3.6†
37.4⫾1.3†
Cardiac output, mL/min
28.4⫾3.1
27.7⫾2.6
19.1⫾2.1†
19.9⫾1.6†
End-diastolic volume, ␮L
82.9⫾5.3
87.5⫾4.1
101.2⫾9.1†
97.4⫾5.9†
LV indicates left ventricle.
*P⬍0.05, Agtr2-/Y vs Agtr2⫹/Y; †P⬍0.05, infarct vs sham-operated.
4-week observation period, none of the infarcted or shamoperated Agtr2⫹/Y and Agtr2-/Y mice died. Thus, there was
no selection bias between genotypes caused by differences in
survival during or after cryoinjury.
Cryoinjury of the left ventricle led to a transmural necrosis,
which was replaced by a solid fibrotic scar (Figure 1a and
1b). The infarct covered ⬇40% of the internal circumference
of the left ventricle. Total infarct area and scar thickness were
identical between wild-type and AT2-deficient mice (Table).
Cryoinjury caused cardiac remodeling, which was apparent as
left ventricular hypertrophy and mild interstitial fibrosis
(Figure 1 and the Table). Several parameters indicated that
postinjury remodeling was enhanced in Agtr2-/Y mice compared with that in wild-type control animals. Heart weight
normalized to body weight or tibia length was significantly
greater in AT2-deficient mice than in wild-type mice after
injury (Figure 1c and the Table). At the microscopic level,
cardiac myocyte cross-sectional area was increased by 53%
in Agtr2⫹/Y mice and by 95% in Agtr2-/Y mice after
cryoinjury (Figure 1d). Whereas myocytes were completely
replaced by fibrous tissue in the area of cryoinjury, interstitial
collagen content had increased only mildly in the noninfarcted area of the septum (Table). The degree of fibrosis did
not differ significantly between AT2-deficient and wild-type
mice (Table). In sham-operated mice, no differences in
cardiac weight and myocyte structure were detected between
wild-type and AT2-deficient mice. Thus, genetic deletion of
the AT2 receptor gene was associated with increased cardiac
hypertrophy after myocardial injury.
Cardiac Function After Cryoinjury
Four weeks after infarction, left ventricular function was
assessed by rapid MRI and by direct cardiac catheterization
of anesthetized mice. MRI revealed significant dilation of
Agtr2⫹/Y and Agtr2-/Y left ventricles after infarction (Figure
2a). End-systolic and end-diastolic volumes were elevated
after cryoinjury, resulting in a decrease of left ventricular
ejection fraction from nearly 70% in sham-operated mice to
35% to 38% in infarcted mice (Figures 2b through 2d). Left
ventricular volumes, ejection fractions, and cardiac output as
determined by MRI did not differ between genotypes after
cryoinjury. In contrast, the maximal rate of left ventricular
contraction (dP/dtmax) was significantly lower in wild-type
mice than in AT2-deficient animals after cryolesioning (Figure 2e). Left ventricular systolic and end-diastolic pressures
and heart rate were similar in Agtr2⫹/Y and Agtr2-/Y mice
after the infarction (Table), indicating that hemodynamic load
did not differ between genotypes.
Cardiac eNOS Expression
Because activation of NO signaling has been suggested to
mediate part of the biologic actions of AT2 receptors,24 we
next determined the levels of expression of NOS isoforms in
hearts of wild-type and AT2 receptor– deficient mice. In
sham-operated animals, left ventricular eNOS expression was
reduced by 35% in Agtr2-/Y mice compared with Agtr2⫹/Y
mice (Figure 3a). After cryoinjury, hearts from Agtr2⫹/Y
mice showed an increase in cardiac eNOS expression
(⫹24%), which was not observed in Agtr2-/Y mice (Figure
3a). Similar results were obtained when eNOS expression
levels were normalized to histone H1 (sham Agtr2⫹/Y,
100⫾5%; sham Agtr2-/Y, 77⫾8%; infarcted Agtr2⫹/Y,
124⫾8%; and infarcted Agtr2-/Y, 83⫾4%). No significant
differences were detectable in cardiac expression levels of
neuronal NOS or inducible NOS isoforms (Figures 3b and
3c). Altered eNOS expression in hearts from AT2-deficient
mice was also correlated with decreased cardiac cGMP levels
(Figure 3d). After cryoinjury, cGMP concentration in the
noninfarcted remote myocardium was decreased by 80% in
Agtr2-/Y hearts compared with Agtr2⫹/Y ventricles.
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Figure 2. Hemodynamic function of mice after cardiac cryoinjury. a, High-resolution end-systolic MRIs (fast low-angle shot)
of sham-operated (upper) and cryoinjured (lower) hearts 4
weeks after operation. Bar-1.5 mm. Arrows mark infarct borders. After cryoinjury, left ventricular end-systolic (b) and end-diastolic (c) volumes were increased and ejection fractions (d)
were severely decreased in Agtr2⫹/Y and Agtr2-/Y mice. Left
ventricular maximal rate of contraction (dP/dtmax) was lower in
Agtr2⫹/Y mice than in Agtr2-/Y mice after cryoinjury. *P⬍0.05,
Agtr2-/Y vs Agtr2⫹/Y. †P⬍0.05, infarct vs sham.
We have previously shown that AT2 receptor activation in
isolated rat cardiac myocytes regulates eNOS expression
through a calcineurin-dependent pathway.15 To test whether
AT2 receptor activation in mice is also directly linked to
eNOS expression, cardiac myocytes were isolated from
newborn Agtr2⫹/Y and Agtr2-/Y mice. Stimulation of these
cardiac myocytes with angiotensin II for 48 hours increased
eNOS protein levels in Agtr2⫹/Y but not in Agtr2-/Y myocytes (Figure 4), indicating that AT2 receptors participate in
the control of cardiac myocyte eNOS levels. To assess
whether decreased eNOS expression is causally linked to
cardiac myocyte hypertrophy after Agtr2 gene deletion, we
determined protein synthesis in cardiac myocytes in vitro. In
Figure 3. Decreased eNOS expression in Agtr2-/Y mice after
cryoinfarction. a, Left ventricular levels of eNOS as detected by
Western blotting were lower in hearts from Agtr2-/Y than from
Agtr2⫹/Y mice. After cryoinjury, this difference in eNOS expression between genotypes was even more pronounced. b and c,
Cardiac protein levels of neuronal (n) or inducible (i) NOS did
not differ between genotypes. Upper panels show representative
Western blots for cardiac eNOS, nNOS, iNOS, and ␤-actin of
Agtr2⫹/Y and Agtr2-/Y mice 4 weeks after cryoinjury. d, In
hearts from AT2-deficient mice, cardiac cGMP levels were significantly decreased compared with those in Agtr2⫹/Y hearts.
Data are mean⫾SEM, n⫽5 to 13 per group. *P⬍0.05, Agtr2-/Y
vs Agtr2⫹/Y. †P⬍0.05, infarct vs sham.
unstimulated myocytes, incorporation of [3H]leucine did not
differ between myocytes isolated from AT2-deficient or
wild-type hearts (Figure 5). After stimulation with angiotensin II, protein synthesis increased by 56% in wild-type
myocytes and by 113% in AT2-deficient cells. On addition of
angiotensin II and the NO synthesis inhibitor L-NAME,
protein synthesis was further increased, but the difference
between genotypes was abolished (Figure 5), indicating that
altered NO signaling after disruption of the Agtr2 gene and
myocyte hypertrophy are directly linked.
Discussion
In the present study, we provide experimental evidence that
angiotensin II AT2 receptors exert an antihypertrophic effect
during cardiac remodeling after myocardial infarction and
that AT2 receptor activation is directly linked to cardiac
eNOS expression.
Previous experiments in mouse models of pressure overload–induced hypertrophy or angiotensin II–induced hypertension have yielded controversial results with respect to the
Brede et al
Angiotensin AT2 Receptors and Cardiac Hypertrophy
Figure 4. Activation of AT2 receptors increases eNOS expression in isolated mouse cardiac myocytes. Stimulation of isolated
cardiac myocytes with angiotensin II (100 nmol/L for 48 hours)
caused a significant increase in eNOS expression in cells from
Agtr2⫹/Y hearts (a) but not in cells obtained from Agtr2-/Y
hearts (b). c, Summary of results obtained from 6 independent
determinations. *P⬍0.05, angiotensin II vs control.
function of cardiac AT2 receptors.4,5,25 In a most recent report,
coronary artery ligation caused lethal cardiac rupture in 64%
of the Agtr2-/Y mice compared with 24% of the wild-type
mice within the first week after myocardial infarction.6 To
avoid an initial selection bias because of different mortality
rates in Agtr2⫹/Y and Agtr2-/Y mice in the early postopera-
Figure 5. Protein synthesis in cardiac myocytes isolated from
wild-type and AT2-receptor– deficient mice. Incorporation of
[3H]leucine was determined in cardiac myocytes for 4 hours in
the absence or presence of angiotensin II (100 nmol/L) or the
NO synthesis inhibitor L-NAME (200 ␮mol/L). Angiotensin II
stimulated protein synthesis more effectively in AT2-deficient
myocytes than in wild-type cells, but this difference was abolished in the presence of L-NAME *P⬍0.05, Agtr2-/Y vs
Agtr2⫹/Y. †P⬍0.05, vs untreated control (mean⫾SEM, n⫽6).
1181
tive period, we chose a cryomodel of cardiac injury.18 After
cryoinjury, overall mortality of the animals was 7% and
infarct sizes were absolutely identical between genotypes.
Cardiac cryoinjury was associated with a 50% decline in left
ventricular ejection fraction and structural remodeling of the
left ventricle. However, in contrast to coronary artery ligation6,9 or aortic constriction models,4,5 cardiac hypertrophy
was still modest 4 weeks after cryoinjury (15% increase in
heart weight and 50% increase in left ventricular cardiac
myocyte cross section; see the Table).
Previous studies applying cardiac injury models to study
the function of AT2 receptors in gene-targeted mice might
also have yielded conflicting data, because mice used for
these studies were maintained on different genetic backgrounds. When crossed onto a C57BL/6J background, AT2deficient mice did not show cardiac hypertrophy after cardiac
pressure overload4 or angiotensin II–induced hypertension.25
In contrast, our FVB/N mice did not display differences in
cardiac hypertrophy after pressure overload between wildtype and AT2-deficient mice.5 After coronary artery ligation,
hypertrophic remodeling of Agtr2-/Y mice was decreased in 1
report6 or indistinguishable from the phenotype observed in
wild-type mice in the second study.9 However, in the presence of an AT1 receptor antagonist, postinfarction hypertrophy was significantly greater in AT2-deficient mice than in
wild-type mice,9 which corresponds well with our current
results after cryoinjury. These data suggest that, at least in
mice, AT2 receptors play an important role in the therapeutic
effect of AT1 receptor antagonists.9
NO is an important regulator of cardiac function,26 and many
studies have suggested multiple interactions between the reninangiotensin system and NO signaling. Here we report that
AT2-deficient mice show decreased cardiac eNOS expression
resulting in diminished left ventricular cGMP levels. Most
important, we provide a biochemical link between cardiac AT2
receptors and expression of eNOS (NOS3). Recently, we have
shown that myocardial AT2 receptors induced eNOS expression
by way of calcineurin.15 Thus, AT2 receptors might stimulate
NO production through 2 different pathways: (1) AT2 upregulates eNOS expression by calcineurin–nuclear factor-AT signaling15 or (2) AT2 might increase NO production by a bradykinin
B2 receptor– dependent mechanism.24
Our findings suggest that reduced eNOS-mediated NO production might, at least in part, be responsible for the exacerbated
hypertrophic response of AT2-deficient mice after cryoinjury. In
isolated cardiomyocytes, inhibition of NO production abolished
the difference in protein synthesis between wild-type and AT2deficient cells. Owing to vascular hypertrophy in AT2-deficient
mice,17 we could not perform similar experiments in vivo
without significantly altering the hemodynamic load of the heart.
eNOS-knockout mice have systemic27,28 and pulmonary29 hypertension, but at baseline cardiac contractility was reported to
be normal in eNOS-deficient mice.30 However, with increasing
age, both eNOS- and neuronal NOS– deficient mice develop
cardiac hypertrophy.31,32 In an ischemia/reperfusion model,
eNOS-deficient mice had increased infarct sizes.33 eNOS deficiency was associated with exacerbated left ventricular remodeling and dysfunction and increased mortality after myocardial
infarction by ligation of the left anterior descending coronary
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artery.34 Importantly, this phenotype was not affected by pharmacologic normalization of blood pressure. Thus, eNOS-derived
NO limits deleterious effects of ischemia by an afterloadindependent mechanism, possibly by increasing capillary density and/or by decreasing myocyte hypertrophy in the remote
myocardium.
Perspectives
Cardiac angiotensin II AT2 receptors exert an antihypertrophic effect after myocardial injury and are linked to the
regulation of cardiac eNOS expression. Thus, selective activation of AT2 receptors might provide further therapeutic
benefit in the treatment of postmyocardial infarction remodeling. Future experiments are required to test whether activation of AT2 receptors in other tissues mediates biologic
effects through upregulation of eNOS-NO signaling.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft
(SFB355), the EURCAR program of the European Union, and the
BONFOR program (in support of W.R.).
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