© 1995 American Heart Association, Inc.
Articles |
Angiotensin II–Induced Growth Responses in Isolated Adult Rat Hearts
Evidence for Load-Independent Induction of Cardiac Protein Synthesis by Angiotensin II
From the Charles A. Dana Research Institute and Harvard-Thorndike Laboratory of Beth Israel Hospital, Department of Internal Medicine, Cardiovascular Division, Beth Israel Hospital, Harvard Medical School (J.S., Y.K., E.O.W., S.I., B.H.L.), Boston, Mass, and Medizinische Klinik II, University of Regensburg (Germany) (H.S., T.C., G.R.).
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Abstract |
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Abstract Cardiac myocyte hypertrophy often occurs in response to both hemodynamic and neurohumoral factors. To study whether activation of the renin-angiotensin system by itself may induce a cardiac growth response, the acute effects of angiotensin II on cardiac protein synthesis were studied in isolated rat hearts. New protein synthesis in isolated buffer-perfused adult rat hearts was measured by incorporation of [3H]phenylalanine into cardiac proteins during a 3-hour perfusion protocol. Angiotensin II (1x10-8 mol/L), administered alone or in combination with the
1-blocker prazosin (1x10-7 mol/L),
stimulated protein synthesis in both ventricles. The rate of
[3H]phenylalanine incorporation into cardiac proteins was
3.9-fold (P<.005) and 2.6-fold (P<.01) higher
in angiotensin II–perfused (n=6) than in vehicle-perfused (n=6) left
and right ventricles, respectively. The induction of new protein
synthesis by angiotensin II was blocked by the angiotensin II type 1
(AT1) receptor antagonist losartan (1x10-7
mol/L, n=5). To study the pathways of angiotensin signal transduction,
protein kinase C (PKC)-
as well as cardiac c-fos and
c-jun mRNA levels were analyzed. Angiotensin II
(1x10-8 mol/L, n=20) resulted in a transient
translocation of PKC- from the cytosol to the cellular membrane.
However, compared with phorbol ester stimulation (phorbol 12-myristate
13-acetate [PMA], 1x10-7 mol/L; n=20), angiotensin II
effects on PKC translocation were significantly less pronounced and
required a more prolonged stimulation. There was no effect of
angiotensin II in concentrations from 10-9 to
10-6 mol/L (n=22) on c-fos and c-jun
mRNA levels in intact adult rat hearts studied over a time course from
15 to 120 minutes of perfusion. In contrast, norepinephrine
(10-6 mol/L, n=6), phorbol ester (PMA, 10-7
mol/L; n=5), and calcium ionophore (A23187, 2.5x10-6
mol/L; n=5) infusion as well as elevated left ventricular systolic wall
stress (n=6) were all followed by a threefold to fourfold induction of
cardiac c-fos and c-jun mRNA levels
(P<.005) compared with respective angiotensin II–infused
or vehicle-infused rat hearts (n=12). In contrast, administration of
angiotensin II in concentrations >10-9 mol/L caused a
significant induction of c-fos in adult and neonatal cardiac
myocytes. In conclusion, angiotensin II acutely stimulates protein
synthesis in cultured adult isolated perfused rat hearts. Angiotensin
II–activated signal transduction appears to involve AT1
receptors and activation of PKC. However, further downstream signaling
mechanisms remain elusive, since angiotensin II may stimulate protein
synthesis in the adult intact heart without preceding c-fos
and c-jun proto-oncogene induction.
Key Words: angiotensin II • protein synthesis • c-fos • c-jun • proto-oncogenes
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Introduction |
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Cardiac load and, in particular, systolic wall stress have been identified as important mechanisms regulating the growth of the heart in vivo.1 2 3 However, the cardiac growth in response to increased loading is not uniform. For example, patients with moderate arterial hypertension present with a wide variation of left ventricular muscle mass, ranging from normal heart weight to severe hypertrophy.4 5 Therefore, other factors, such as neuroendocrine activation, have been implicated to modulate the cardiac growth response. In particular, the renin-angiotensin system may contribute to cardiac hypertrophy.3 6 Angiotensin II mediates both arterial and venous vasoconstriction, resulting in elevation of preload and afterload of the heart. Direct cardiac effects of angiotensin II include positive inotropy7 and, in rats with left ventricular hypertrophy, negative lusitropy.8 9 10 In addition, chronic angiotensin II infusion experiments in rats,11 12 13 rabbits,14 or pigs15 have revealed that the vasoactive peptide can also increase the heart weight in these species. More evidence that the renin-angiotensin system may be involved in the regulation of cardiac growth comes from pharmacological studies using inhibitors of the system. Our recent study16 and work by others17 18 have shown that chronic angiotensin-converting enzyme (ACE) inhibition in experimental animals with cardiac pressure overload or in hypertensive patients may result in regression or prevention of cardiac hypertrophy. Unfortunately, in these studies differentiation of possible direct effects of angiotensin II on protein synthesis and cardiac growth from indirect effects mediated by a rise in blood pressure and cardiac load is difficult. These studies also do not clarify whether the effects of ACE inhibition are related to blunted activation of angiotensin II receptors or to effects of ACE inhibition on bradykinin metabolism.19
Evidence that angiotensin II may exert load-independent effects on cardiac myocyte growth comes from recent studies on isolated embryonic chick20 and rat21 22 myocytes. These studies demonstrated that incubation of cardiac myocytes with angiotensin II in vitro resulted in a significant stimulation of amino acid incorporation, suggesting a direct angiotensin II effect on the cardiac muscle growth response. Intracellular cations,23 24 25 26 inositol phosphates,25 26 and protein kinase C (PKC)27 have been implicated as potential second messengers of angiotensin II stimulation in cultured cardiac myocytes. Following the intracellular signaling pathway, possible targets of these second messengers are the rapidly activated transcription factors c-fos and c-jun.28 29 Evidence suggests that these proto-oncogenes can be activated by angiotensin II in neonatal cultured cardiac myocytes,22 29 but their role in mediating an increase in protein synthesis in the adult heart has not been determined.
The goal of the present study was to examine load-independent effects of angiotensin II on amino acid incorporation and its signaling pathway in ex vivo adult rat hearts. We present data that demonstrate a load-independent angiotensin II type 1 (AT1) receptor–mediated effect on cardiac protein synthesis that does not require induction of proto-oncogenes such as c-fos and c-jun. Furthermore, the present study provides support that the signaling pathway of angiotensin II–mediated protein synthesis may differ in the intact beating heart compared with isolated dissociated cultured myocytes.
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Materials and Methods |
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Perfusion of Isolated Hearts
Male Wistar rats (450 to 500 g) were obtained from the Charles River Breeding Laboratories, Wilmington, Del. Perfusion techniques using a modified Langendorff apparatus in the isolated rat heart have been described before.8 9 The perfusate consisted of modified Krebs-Henseleit buffer (mmol/L): NaCl 118, KCl 4.7, CaCl2 2.0, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 5.5, and lactate 1.0. For measurements of protein synthesis, the buffer contained 0.5% albumin and a mixture of amino acids in the following concentrations (µmol/L): aspartic acid 38, asparagine 64, glutamic acid 207, glutamine 656, glycine 328, alanine 559, valine 226, leucine 184, isoleucine 99, serine 285, threonine 371, methionine 57, proline 246, phenylalanine 400, tyrosine 119, tryptophan 84, histidine 77, lysine 532, and arginine 157.30 31 The perfusate was equilibrated with a 5% CO2/95% O2 gas mixture. After coronary perfusion was initiated, the left ventricles were decompressed by apical puncture and the insertion of a short apical drain to vent left ventricular thebesian flow. Temperature of the hearts was kept constant at 35°C and was monitored with a thermistor probe. Before hearts were subjected to the experimental protocol, the cardiac performance was allowed to stabilize, and the perfusion pressure was adjusted to 80 mm Hg.
Experimental Protocols in Isolated Hearts
Effects of Angiotensin II, Norepinephrine, and Vehicle on Amino
Acid Incorporation in Isolated Perfused Rat Hearts
Groups of six hearts were perfused with (1) a mixture of
angiotensin II (1x10-8 mol/L) and prazosin
(1x10-7 mol/L) (Sigma Chemical Co), (2) norepinephrine
(1x10-6 mol/L) (Sigma), or (3) vehicle. Angiotensin II
was combined with the 1-blocker prazosin to prevent any
indirect stimulation of protein synthesis or proto-oncogenes via
activation of the postsynaptic sympathetic nervous
system.32 After 60 minutes, administration of the agonists
was stopped, and hearts were perfused for another 120 minutes with the
modified Krebs-Henseleit buffer to which 0.5 mCi/L radiolabeled
phenylalanine was added. Thus, the hearts were allowed to incorporate
tritiated phenylalanine into newly synthesized proteins for 2 hours.
Unlabeled phenylalanine, present in a defined concentration in the
buffer, was used for calculation of the incorporation of phenylalanine
into cardiac proteins on a molar basis.23 30 31
Phenylalanine incorporation was assumed to be linear over the 2-hour
period of incorporation, and data are expressed as nanomoles of
phenylalanine per gram protein per hour.
Effects of Angiotensin II on PKC Translocation
Groups of four hearts were perfused with either (1) angiotensin
II (1x10-8 mol/L) or (2) phorbol ester (phorbol
12-myristate 13-acetate [PMA], 1x10-7 mol/L) for 0, 3,
7, 15, and 30 minutes. Immediately after perfusion, hearts were
freeze-clamped and stored at -70°C for PKC determination.
Effects of Angiotensin II Receptor Blockade on Amino Acid
Incorporation in Isolated Perfused Rat Hearts
Groups of five hearts were perfused with (1) a mixture of
angiotensin II (1x10-8 mol/L) and vehicle, (2) a mixture
of angiotensin II (1x10-8 mol/L) and the AT1
receptor antagonist losartan (Dup 753, 1x10-7 mol/L)
(Dupont), or (3) vehicle alone. After 60 minutes, administration of
agonist and receptor antagonist was stopped, and hearts were perfused
for another 120 minutes with the modified Krebs-Henseleit buffer plus
0.5 mCi/L radiolabeled phenylalanine as described above.
Effects of Angiotensin II on c-fos and
c-jun Proto-oncogene Expression in Isolated Perfused Rat
Hearts
Groups of hearts were perfused with (1) angiotensin II
(1x10-9 to 1x10-6 mol/L, n=22), (2)
norepinephrine (1x10-6 mol/L, n=6), (3) elevated systolic
wall stress of 550x103 dyne/cm2 (by insertion
of a fluid-filled latex balloon in the cavity of the left ventricle to
achieve a left ventricular systolic pressure of 120 mm Hg as
previously described33 ) (n=6), or (4) vehicle
(n=12) for a total of 60 minutes. Angiotensin II (10-8
mol/L) was infused for 15, 30, 60, 90, or 120 minutes to characterize a
possible time course of gene expression. A dose-finding experiment was
carried out by using angiotensin II at 10-9,
10-8, 10-7, 10-6,
and 3x10-5 mol/L concentrations for 60 minutes.
Additional hearts were perfused for 60 minutes with the phorbol ester
(PMA, 10-7 mol/L) in the absence (n=5) or presence (n=5)
of angiotensin II (10-8 mol/L). Similarly, hearts were
perfused for 60 minutes with the calcium ionophore (A23187,
2.5x10-6 mol/L) in the absence (n=5) or presence (n=5) of
angiotensin II (10-8 mol/L).
Preparation and Culture of Left Ventricular Myocytes
Twelve-week-old male Wistar rats were anesthetized, and the
hearts were perfused with a calcium-free Krebs-Henseleit buffer with
electrolyte concentrations other than calcium, as described above.
After 3 minutes of perfusion, the perfusate was changed to a
recirculating calcium-free Krebs-Henseleit buffer supplemented with 0.6
mg/mL collagenase (class II, Worthington Biochemical Corp) and 0.04
mg/mL protease (type XIV, Sigma) for 20 minutes. The left ventricle was
then dissected from the other chambers, cut into small pieces, and
dispersed into single cells by gentle agitation through a serological
pipette in Krebs-Henseleit buffer containing 100 mmol/L
CaCl2 and 0.1% bovine serum albumin. The resulting
suspension was then gently forced through a 450-µm nylon screen
filtration cloth into a 50-mL plastic tube, rinsed twice, and
transferred to a culture dish. Cardiac myocytes were cultured in
serum-free Dulbecco's modified Eagle's medium/F-12 medium
supplemented with 3 mmol/L pyruvic acid, 100 µmol/L ascorbic acid,
and 100 µg/mL ampicillin. The culture medium was once changed to the
same serum-free medium 12 hours after seeding. To selectively remove
nonmyocytes, dissociated cells were preplated for 1 hour. Nonadherent
cells (enriched in myocytes) were collected and replated. The
percentage of myocytes was estimated to be 95%, as judged by the
characteristic rod-shaped morphology of adult myocytes. Stimulation
with angiotensin II was performed 12 hours thereafter. Preparation of
neonatal myocyte cultures and estimation of the myocyte population by
immunochemistry were performed as previously
described.22
Effects of Angiotensin II on c-fos and
c-jun Proto-oncogene Expression in Cultured Ventricular Rat
Myocytes
Ventricular myocytes of 12-week-old male Wistar rats were
stimulated with (1) angiotensin II (1x10-9 to
1x10-6 mol/L, n=9) or (2) vehicle (n=3) for a total of 30
minutes. In separate experiments, the dose-response effect of
angiotensin II on c-fos expression over a concentration
ranging from 10-11 to 10-6 mol/L was measured
in adult and neonatal myocytes. Cells were harvested with 4 mol/L
guanidine thiocyanate solution for RNA extraction and Northern
blotting.
Biochemical Analyses
Protein Synthesis
After the perfusion protocols, the atria and great
vessels were quickly removed. Left and right ventricles were blotted
dry, weighed, and snap-frozen in liquid nitrogen. For measurement of
protein synthesis, the methods of Morgan et al31 with
modifications by Kent et al2 were used. An aliquot (
100
mg) was minced and homogenized in 1 mL ice-cold 5% perchloric acid to
denature proteins and to remove unincorporated
[3H]phenylalanine. After centrifugation, the pellet was
washed with 5% perchlorate, resuspended, and heated to 80°C to
remove RNA-bound [3H]phenylalanine. After
centrifugation, the pellet was washed with 5% perchlorate and then
resuspended in 0.2N NaOH. A small aliquot (50 µL) of this solution
was used for protein assay, and a second aliquot (500 µL) was used
for liquid scintillation counting. Data were corrected for quenching by
extrapolation. The net protein synthesis by left or right ventricles
during the 120 minutes of perfusion with
[3H]phenylalanine was calculated as follows:
phenylalanine incorporation (in moles per gram protein per hour)=
phenylalanine (in disintegrations per minute [dpm] per gram protein
per hour) perfusate phenylalanine specific activity (in dpm per mole).
Since all hearts were collected after 120 minutes of
[3H]phenylalanine perfusion, the dpm per gram per hour
values were divided by 2, and data were expressed as moles per gram
protein per hour.
RNA Measurements
RNA extraction using a cesium chloride gradient, standard
Northern blot analysis using the formaldehyde-agarose method, and
hybridization conditions have been described previously in great
detail.8 33 34
Western Blot for PKC-
Cytosolic and membrane fractions were prepared according to the
protocol by Yuan et al.35 Samples containing 50 µg
protein were separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis36 by use of a 10% (wt/vol) acrylamide
separating gel and a 6% (wt/vol) acrylamide stacking gel. Gels were
electroblotted to a polyvinylidine difluoride membrane (Millipore,
Inc). Detection of PKC- was carried out by using an anti–PKC-
antibody (Life Technologies) that corresponds to the C-terminus of
PKC- and an ECL–Western blotting analysis system (Amersham
International) according to the manufacturer's instructions. PKC-
was quantified by laser densitometric analysis.
Statistical Analysis
All data are presented as mean±SEM. Phenylalanine
incorporation or proto-oncogene–to–glyceraldehyde-3-phosphate
dehydrogenase mRNA ratios were directly compared by Student's unpaired
t tests. Two-way ANOVA and Fisher's exact test for post hoc
analyses were used for multiple comparisons in case of three or more
comparisons between groups. Significance was accepted at
P<.05.
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Hemodynamics
To address confounding effects of hemodynamics on the induction of protein synthesis37 and proto-oncogenes,38 we carefully monitored coronary perfusion pressure and coronary flow in the isolated perfused hearts. Coronary perfusion pressure was adjusted to 80 mm Hg in all hearts at the end of the stabilization period. The infusion of angiotensin II was accompanied by an increase of coronary perfusion pressure to 110±5 mm Hg at 60 minutes and 115±11 mm Hg at 180 minutes. A similar rise was seen with norepinephrine (100±8 mm Hg at 60 minutes and 138±5 mm Hg at 180 minutes). In the vehicle-perfused control hearts, these pressures were matched by slightly increasing coronary flow to achieve similar levels of coronary perfusion pressure (110±5 mm Hg at 60 minutes and 111±12 mm Hg at 180 minutes). At 60 and 180 minutes, coronary flow was 21±1 and 21±1 mL/min in the angiotensin group, 21±1 and 21±1 mL/min in the norepinephrine group, and 24±2 and 26±1 mL/min in the vehicle control group. With the exception of a higher flow rate in vehicle-perfused hearts, there was no statistical difference in hemodynamic parameters between the groups. To avoid the generation of left ventricular systolic wall stress,33 thebesian flow was vented, and the left ventricles were flaccid, such that no pressure (<10 mm Hg) was generated by the perfused hearts.
Effects of Angiotensin II, Norepinephrine, and Vehicle on Amino
Acid Incorporation in Isolated Perfused Rat Hearts
During vehicle perfusion, phenylalanine was incorporated into
newly synthesized proteins of isolated adult rat hearts at a rate of
138±33 nmol phenylalanine per gram protein per hour in the left
ventricles and 145±43 nmol phenylalanine per gram protein per hour in
the right ventricles. Sixty minutes of angiotensin II/prazosin infusion
followed by 120 minutes of vehicle perfusion resulted in a 3.9-fold
increase of phenylalanine incorporation in the left ventricles compared
with vehicle control hearts (P<.005) (Fig 1
). In the right ventricles, angiotensin II resulted in
a 2.6-fold increase of phenylalanine incorporation (P<.01)
(Fig 1). Norepinephrine, infused for 60 minutes and followed by 120
minutes of vehicle perfusion, also resulted in an increase of
phenylalanine incorporation, albeit smaller than that seen with
angiotensin II (Fig 1). Compared with vehicle-perfused hearts, the
induction of protein synthesis was 2.1-fold in left ventricles
(P<.05) and 1.6-fold in right ventricles (P=NS)
infused with norepinephrine (Fig 1).
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Effects of Angiotensin II Receptor Blockade on Amino Acid
Incorporation in Isolated Perfused Rat Hearts
Additional hearts were studied to examine the effect of
angiotensin II receptor blockage on novel protein synthesis in these
isolated perfused hearts. As in the first protocol, angiotensin II
infusion resulted in a significant induction of phenylalanine
incorporation compared with vehicle-perfused hearts (Fig 2). In contrast, when losartan, an AT1
receptor antagonist, was infused in parallel with angiotensin II, the
rate of phenylalanine incorporation remained at baseline levels and was
not different from that in vehicle-perfused hearts (Fig 2).
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Effects of Angiotensin II on PKC Translocation in Isolated Perfused
Rat Hearts
Western blot analysis of cardiac proteins extracted from
isolated perfused rat hearts after stimulation with angiotensin II
(1x10-8 mol/L) demonstrated a transient translocation of
PKC- from the cytosolic (Fig 3A and 3C) to the
membrane fraction (Fig 3B and 3D). Thus, the data suggest that
angiotensin II activates PKC in adult rat hearts. Interestingly,
compared with angiotensin II, the effects of phorbol ester (PMA,
1x10-7 mol/L) on PKC translocation were evident at an
earlier time point (3 versus 15 minutes), reached a higher maximum
(percent translocation), and lasted longer (>30 versus 15 minutes)
(Fig 3A through 3D), suggesting differences with regard to the kinetics
of PKC activation after stimulation with the two agonists.
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Effects of Angiotensin II on c-fos and
c-jun Proto-oncogene Expression in Isolated Perfused Rat
Hearts
To study whether the angiotensin II–mediated stimulation of the
proto-oncogenes c-fos and c-jun accompanies the
induction of protein synthesis in intact adult hearts, additional rat
hearts were perfused with angiotensin II at concentrations from
10-9 to 10-6 mol/L (Fig 4). In
the intact hearts, angiotensin II failed to stimulate c-fos
and c-jun mRNA expression (Figs 4 and 5) when
compared with respective vehicle-perfused control groups. The lack of
an angiotensin II–related induction of these proto-oncogenes was
evident over the entire time course studied (15, 30, 60, 90, and 120
minutes [Fig 6]). In contrast, hearts perfused with
norepinephrine (Fig 5) or exposed to a high left ventricular systolic
wall stress (Fig 6) expressed high levels of c-fos and
c-jun mRNA. In addition, angiotensin II did not interfere
with phorbol ester (10-7 mol/L)– and calcium ionophore
(2.5x10-6 mol/L)–related proto-oncogene induction (Fig 7).
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Effects of Angiotensin II on c-fos Proto-oncogene
Expression in Cultured Left Ventricular Adult Cardiac Myocytes
To compare the effects of angiotensin II on proto-oncogene
expression in neonatal and adult rat myocytes versus the intact heart,
we studied the dose-response effect of angiotensin II on the induction
of the proto-oncogene c-fos in primary cultured neonatal rat
ventricular myocytes and adult rat myocytes. Over the identical time
course, maximum angiotensin II–induced c-fos expression was
greater in primary cultured neonatal myocytes than in adult cardiac
myocytes (Fig 8A). The dose-response curve of relative
c-fos expression in adult and neonatal myocytes is also
shown (Fig 8B). Although the EC50 differed in neonatal and
adult cells, angiotensin II at concentrations >10-9 mol/L
caused a significant induction of c-fos mRNA levels in adult
isolated myocytes.
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Discussion |
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In cultured vascular and neonatal cardiac myocytes, angiotensin II has been shown to be a potent stimulus for c-fos, c-jun, and c-myc proto-oncogene induction.22 29 39 40 In light of the characterized molecular function of these transcription factors, namely, to form nuclear binding proteins that regulate expression of various genes,41 42 43 44 45 it is intriguing to speculate that angiotensin II may act as a growth factor via mandatory activation of these proto-oncogenes.28 Nevertheless, so far, there is no direct evidence that the effects of angiotensin II or any other growth factors in adult cardiac cells on the cardiac growth response are mediated by c-fos or c-jun.1 The present study was performed to examine the effects of angiotensin II on cardiac protein synthesis as well as c-fos and c-jun proto-oncogenes in adult rat hearts and in adult and neonatal cultured cardiac myocytes. The data indicate that perfusion of isolated hearts with angiotensin II is associated with a significant induction of protein synthesis as measured by the rate of [3H]phenylalanine incorporation into cardiac proteins. Since systemic hemodynamic effects of angiotensin II do not interfere in this isolated rat heart preparation, the data suggest that the angiotensin II–mediated induction of cardiac protein synthesis can occur independently from changes in cardiac loading conditions. The data are in agreement with reports by Khairallah and colleagues11 12 and Geenen et al,13 who studied the effects of angiotensin II on cardiac protein synthesis in isolated rat atria12 and in vivo in adult rats infused with angiotensin II.11 13 Similar effects of angiotensin II have been observed in studies carried out on cultured neonatal and adult cardiac myocytes, which clearly demonstrated growth-promoting effects of this octapeptide.20 22 23
The mechanisms of angiotensin II–induced protein synthesis can
be extended, given the present data, by analysis of the
parallel use of receptor agonists and antagonists. The stimulation of
protein synthesis by angiotensin II was not linked to the
1-receptor, since coadministration of prazosin had no
effect on [3H]phenylalanine incorporation into cardiac
proteins. Furthermore, the data of the present study and others
suggest that the effect of angiotensin II on new protein synthesis is
mainly mediated by the AT1 receptor
subtype.46 47 Blockade of this receptor by the
AT1 receptor antagonist losartan abolished the stimulatory
effects of angiotensin II on protein synthesis. Interestingly, a recent
study of our group demonstrated that in normal rat left ventricles the
majority of angiotensin II binding sites refer to the AT1
receptor, whereas rat hearts with established pressure-overload
hypertrophy express an increased proportion of AT2
receptors.48
To study whether angiotensin II–mediated induction of protein synthesis in the intact heart is associated with the activation of proto-oncogenes, we examined the effects of angiotensin II administration on c-fos and c-jun mRNA levels. The data obtained in isolated neonatal and adult cardiac myocytes suggest that angiotensin II is a potent stimulus for cardiac c-fos expression. On the other hand, angiotensin II failed to stimulate these proto-oncogenes in the intact ex vivo–perfused adult rat heart. The range of angiotensin II concentrations used should be considered to be sufficient. First, similar doses were used to demonstrate hemodynamic effects of angiotensin II, such as coronary vasoconstriction, positive inotropy, and diastolic dysfunction in normal and hypertrophied rat hearts under similar experimental conditions.8 9 49 Second, stimulation of protein synthesis was detected at a dose that was used in hearts that showed no induction of c-fos and c-jun proto-oncogenes. Furthermore, one may consider that the perfusion conditions may repress the inducibility of c-fos and c-jun. However, two positive control groups, eg, norepinephrine-stimulated and wall stress–stimulated rat hearts, expressed high levels of these proto-oncogenes when perfused ex vivo in an identical fashion, corroborating our previous observations of the effects of these stimuli on proto-oncogene induction in intact perfused hearts.33 To study whether angiotensin II might exert an inhibitory effect on proto-oncogene expression by induction of a repressing element,50 we studied the effects of coadministration of angiotensin II with stimuli known to induce c-fos expression by well-defined pathways.33 PKC stimulation and calcium ionophore administration both resulted in high levels of c-fos and c-jun irrespective of the presence or absence of angiotensin II. Thus, the data suggest that downstream intracellular signaling pathways resulting in c-fos and c-jun expression are intact in angiotensin II–perfused rat hearts.
The discrepancy of angiotensin II–mediated cardiac proto-oncogene induction in isolated myocytes versus the intact heart is consistent with prior studies. Prior studies of angiotensin II effects in cultured cardiac cells, mostly using isolated neonatal cardiocytes, demonstrated that incubation of rat or chick cardiac myocytes with angiotensin II at doses similar to that used in the present experiments resulted in induction of c-fos, c-jun, and c-myc mRNAs.21 22 29 However, differing observations have been made from in vivo studies of angiotensin II–mediated cardiac proto-oncogene induction. In particular, Moalic et al51 demonstrated that phenylephrine or vasopressin infusions in adult rats were accompanied by high levels of cardiac c-fos and c-myc expression, whereas angiotensin II infusion had no effect on induction of these cardiac proto-oncogenes.
Several explanations may account for the discrepant observations in studies on angiotensin II–mediated proto-oncogene induction in cultured myocytes compared with intact hearts. First, studies on cultured myocytes are often carried out on fetal or neonatal cells.21 22 23 29 52 Therefore, angiotensin II–mediated proto-oncogene induction may characterize the early stages of cardiac cell development. This is not surprising, since cardiac mRNA levels of c-fos and c-myc proto-oncogenes are fairly abundant during embryonic development and rapidly decrease in postnatal life.53 Furthermore, expression of cardiac plasma membrane and soluble angiotensin II receptors,54 55 56 57 as well as angiotensin II–induced intracellular signaling pathways, may be developmentally regulated.54 55 56 57 Thus, maturation of cardiac myocytes may affect angiotensin II–mediated stimulation of c-fos and c-jun proto-oncogenes. Another possible explanation for the different effects of angiotensin II on cardiac proto-oncogene expression in cultured cells compared with intact hearts may relate to the experimental conditions of cardiac myocytes under cell culture. For example, studies of Claycomb and Lanson58 demonstrated that c-fos and c-myc mRNAs are more abundantly expressed in cultured fetal or adult cardiac myocytes compared with fetal or adult cardiac tissue. Furthermore, Woodcock et al59 demonstrated substantial differences between intact rat hearts and isolated myocytes with regard to the phosphatidylinositol metabolism, a pathway that is an integral part of the signaling cascade that results in the angiotensin II–related proto-oncogene induction in isolated myocytes.29 Our present finding that angiotensin II–mediated PKC translocation in intact hearts was significantly less pronounced and markedly delayed compared with phorbol ester stimulation corroborates this notion, since the same comparison made in isolated myocytes resulted in similar kinetics of PKC activation after administration of the two agonists.29 Taken together, these data suggest that culturing conditions or loss of cell-to-cell contact may influence the inducibility of these proto-oncogenes in response to various stimuli such as angiotensin II.
In summary, these studies demonstrate the potential difference between using cultured cardiac cells and the intact heart to clarify fundamental mechanisms of cardiac growth regulation or gene expression in the intact heart.58 59 On one hand, studies on intact cardiac tissue are performed on a heterogeneous cell population consisting of myocytes as well as interstitial, vascular, and blood-derived cells. Thus, quantifications of extracted mRNAs or proteins may be affected to a various extent by nonmyocyte cells. This limitation of studies on intact cardiac tissue applies to the present experiments. However, given the present data, it seems reasonable to conclude that angiotensin II stimulation of protein synthesis in adult rat hearts is not associated with an overall stimulation of cardiac c-fos and c-jun mRNA levels. Our observations also indicate that isolated adult cardiac myocytes may reconstitute signaling the neonatal pathway, resulting in c-fos and c-jun proto-oncogene expression in response to angiotensin II in experimental conditions and possibly in vivo under pathological conditions.
Recent data suggest that stretching of cultured neonatal cardiac
myocytes causes a release of angiotensin II, which in turn mediates
early stretch-induced hypertrophy.34 Our observation
confirms the hypothesis that angiotensin II is a potent stimulus of
protein synthesis in adult rat hearts as well. However, in the
present study, no induction of c-fos and
c-jun proto-oncogenes was observed before angiotensin
II–mediated stimulation of protein synthesis, whereas elevated wall
stress resulted in proto-oncogene and protein synthesis
induction.33 60 Thus, in intact isolated perfused rat
hearts, induction of these proto-oncogenes does not seem to be an
obligatory phenomenon in the signaling cascade preceding the rapid
stimulation of cardiac protein synthesis after administration of
angiotensin II. Likewise, in the intact heart, it remains to be
demonstrated whether angiotensin II acts as a second messenger in the
induction of proto-oncogenes and protein synthesis after elevation of
wall stress.60 Thus, the intracellular signal transduction
resulting in angiotensin II–mediated induction of protein synthesis in
the intact heart is unclear and may differ from that observed in
isolated neonatal and adult myocytes. However, these findings do not
rule out the possibility that c-fos and c-jun
proto-oncogenes may be important modulators of gene expression in
adaptive hypertrophy, eg, after stimulation with -adrenergic
agonists or elevated cardiac load.33 60
In summary, the present study indicates that angiotensin II is a potent stimulus of protein synthesis induction in isolated rat hearts, which is predominantly mediated by activation of cardiac AT1 receptors. In contrast to neonatal and adult cardiac myocytes, activation of c-fos and c-jun proto-oncogenes does not precede angiotensin II–mediated protein synthesis induction in intact adult rat hearts.
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Acknowledgments |
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This study was supported by a Bayer Award for Cardiovascular Research and an Established Investigatorship of the American Heart Association (Dr Izumo), the Deutsche Forschungsgemeinschaft and an Astra Award for Cardiovascular Research (Dr Schunkert), a Fellowship of the American Heart Association, Massachusetts Affiliate, Inc (Dr Sadoshima), and Program Project grant HL-38189 of the National Heart, Lung, and Blood Institute and an Established Investigatorship and Grant-in-Aid of the American Heart Association (Dr Lorell). We thank Drs Inder M. Verma and Daniel Nathans for providing the cDNA probes and Dr Victor J. Dzau for critical discussion and support of this work. We also thank Drs Armin Kurtz, Reinhold Büttner, and Anthony Ware for support of these studies and Barbara Zillman for assistance in the preparation of this manuscript.
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Footnotes |
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Reprint requests to Beverly H. Lorell, MD, Cardiovascular Division, Beth Israel Hospital, Harvard Medical School, 330 Brookline Ave, Boston, MA 02215, or Heribert Schunkert, MD, Klinik und Poliklinik für Innere Medizin II, Universität Regensburg, Franz-Josef Strauss Allee, D-8400 Regensburg, FRG.
Received March 22, 1994; accepted November 28, 1994.
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Y. Takeishi, A. Bhagwat, N. A. Ball, D. L. Kirkpatrick, M. Periasamy, and R. A. Walsh Effect of angiotensin-converting enzyme inhibition on protein kinase C and SR proteins in heart failure Am J Physiol Heart Circ Physiol, January 1, 1999; 276(1): H53 - H62. [Abstract] [Full Text] [PDF]
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M. O. Gray, C. S. Long, J. E. Kalinyak, H.-T. Li, and J. S. Karliner Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-{beta}1 and endothelin-1 from fibroblasts Cardiovasc Res, November 1, 1998; 40(2): 352 - 363. [Abstract] [Full Text] [PDF]
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T. A. Fischer, K. Singh, D. S. O'Hara, D. M. Kaye, and R. A. Kelly Role of AT1 and AT2 receptors in regulation of MAPKs and MKP-1 by ANG II in adult cardiac myocytes Am J Physiol Heart Circ Physiol, September 1, 1998; 275(3): H906 - H916. [Abstract] [Full Text] [PDF]
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M. Pfeifer, G. Bruckschlegel, S. R Holmer, M. Paul, A.J.G. Riegger, and H. Schunkert Reciprocal regulation of pulmonary and cardiac angiotensin-converting enzyme in rats with severe left ventricular hypertrophy Cardiovasc Res, April 1, 1998; 38(1): 125 - 132. [Abstract] [Full Text] [PDF]
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M. C. C. de Hurtado, B. V. Alvarez, N. G. Perez, I. L. Ennis, and H. E. Cingolani Angiotensin II Activates Na+-Independent Cl--HCO3- Exchange in Ventricular Myocardium Circ. Res., March 9, 1998; 82(4): 473 - 481. [Abstract] [Full Text] [PDF]
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M. Hamawaki, T. M. Coffman, A. Lashus, M. Koide, M. R. Zile, M. I. Oliverio, G. Defreyte, G. Cooper IV, and B. A. Carabello Pressure-overload hypertrophy is unabated in mice devoid of AT1A receptors Am J Physiol Heart Circ Physiol, March 1, 1998; 274(3): H868 - H873. [Abstract] [Full Text] [PDF]
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H. Kodama, K. Fukuda, J. Pan, S. Makino, M. Sano, T. Takahashi, S. Hori, and S. Ogawa Biphasic Activation of the JAK/STAT Pathway by Angiotensin II in Rat Cardiomyocytes Circ. Res., February 9, 1998; 82(2): 244 - 250. [Abstract] [Full Text] [PDF]
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A. Malhotra, D. Reich, D. Reich, A. Nakouzi, V. Sanghi, D. L. Geenen, and P. M. Buttrick Experimental Diabetes Is Associated With Functional Activation of Protein Kinase C{epsilon} and Phosphorylation of Troponin I in the Heart, Which Are Prevented by Angiotensin II Receptor Blockade Circ. Res., December 19, 1997; 81(6): 1027 - 1033. [Abstract] [Full Text]
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T. Cornelius, S. R. Holmer, F. U. Muller, G. A. J. Riegger, and H. Schunkert Regulation of the Rat Atrial Natriuretic Peptide Gene After Acute Imposition of Left Ventricular Pressure Overload Hypertension, December 1, 1997; 30(6): 1348 - 1355. [Abstract] [Full Text]
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D. G. Peters, H. L. Mitchell, S. A. McCune, S. Park, J. H. Williams, and S. C. Kandarian Skeletal Muscle Sarcoplasmic Reticulum Ca2+-ATPase Gene Expression in Congestive Heart Failure Circ. Res., November 19, 1997; 81(5): 703 - 710. [Abstract] [Full Text]
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J. Magga, O. Vuolteenaho, M. Marttila, and H. Ruskoaho Endothelin-1 Is Involved in Stretch-Induced Early Activation of B-Type Natriuretic Peptide Gene Expression in Atrial but Not in Ventricular Myocytes : Acute Effects of Mixed ETA/ETB and AT1 Receptor Antagonists In Vivo and In Vitro Circulation, November 4, 1997; 96(9): 3053 - 3062. [Abstract] [Full Text]
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W. Linz, T. Jessen, R. H. A. Becker, B. A. Scholkens, and G. Wiemer Long-term ACE Inhibition Doubles Lifespan of Hypertensive Rats Circulation, November 4, 1997; 96(9): 3164 - 3172. [Abstract] [Full Text]
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J. Fareh, R. M. Touyz, E. L. Schiffrin, and G. Thibault Cardiac Type-1 Angiotensin II Receptor Status in Deoxycorticosterone Acetate–Salt Hypertension in Rats Hypertension, November 1, 1997; 30(5): 1253 - 1259. [Abstract] [Full Text]
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J. Pan, K. Fukuda, H. Kodama, S. Makino, T. Takahashi, M. Sano, S. Hori, and S. Ogawa Role of Angiotensin II in Activation of the JAK/STAT Pathway Induced by Acute Pressure Overload in the Rat Heart Circ. Res., October 19, 1997; 81(4): 611 - 617. [Abstract] [Full Text]
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J. L. Segar, T. D. Scholz, K. A. Bedell, O. M. Smith, D. J. Huss, and E. N. Guillery Angiotensin AT1 receptor blockade fails to attenuate pressure-overload cardiac hypertrophy in fetal sheep Am J Physiol Regulatory Integrative Comp Physiol, October 1, 1997; 273(4): R1501 - R1508. [Abstract] [Full Text] [PDF]
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N. Makino, M. Sugano, S. Otsuka, and T. Hata Molecular Mechanism of Angiotensin II Type I and Type II Receptors in Cardiac Hypertrophy of Spontaneously Hypertensive Rats Hypertension, October 1, 1997; 30(4): 796 - 802. [Abstract] [Full Text]
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M. O. Boluyt, J.-S. Zheng, A. Younes, X. Long, L. O'Neill, H. Silverman, E. G. Lakatta, and M. T. Crow Rapamycin Inhibits {alpha}1-Adrenergic Receptor–Stimulated Cardiac Myocyte Hypertrophy but Not Activation of Hypertrophy-Associated Genes : Evidence for Involvement of p70 S6 Kinase Circ. Res., August 19, 1997; 81(2): 176 - 186. [Abstract] [Full Text]
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D. D. D'Angelo, Y. Sakata, J. N. Lorenz, G. P. Boivin, R. A. Walsh, S. B. Liggett, and G. W. Dorn II Transgenic Galpha q overexpression induces cardiac contractile failure in mice PNAS, July 22, 1997; 94(15): 8121 - 8126. [Abstract] [Full Text] [PDF]
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V. Regitz-Zagrosek, J. Fielitz, R Dreysse, A. G Hildebrandt, and E. Fleck Angiotensin receptor type 1 mRNA in human right ventricular endomyocardial biopsies: downregulation in heart failure Cardiovasc Res, July 1, 1997; 35(1): 99 - 105. [Abstract] [Full Text] [PDF]
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C. D. Thienelt, E. O. Weinberg, J. Bartunek, and B. H. Lorell Load-Induced Growth Responses in Isolated Adult Rat Hearts : Role of the AT1 Receptor Circulation, June 17, 1997; 95(12): 2677 - 2683. [Abstract] [Full Text]
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A. Benetos, P. Lacolley, and M.E. Safar Prevention of Aortic Fibrosis by Spironolactone in Spontaneously Hypertensive Rats Arterioscler Thromb Vasc Biol, June 1, 1997; 17(6): 1152 - 1156. [Abstract] [Full Text]
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E. O. Weinberg, M. A. Lee, M. Weigner, K. Lindpaintner, S. P. Bishop, C. R. Benedict, K. K. L. Ho, P. S. Douglas, E. Chafizadeh, and B. H. Lorell Angiotensin AT1 Receptor Inhibition : Effects on Hypertrophic Remodeling and ACE Expression in Rats With Pressure-Overload Hypertrophy due to Ascending Aortic Stenosis Circulation, March 18, 1997; 95(6): 1592 - 1600. [Abstract] [Full Text]
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J. Osaki, T. Haneda, H. Sakai, and K. Kikuchi cAMP-mediated c-fos expression in pressure-overloaded acceleration of protein synthesis in adult rat heart Cardiovasc Res, March 1, 1997; 33(3): 631 - 640. [Abstract] [PDF]
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S. Hokimoto, H. Yasue, K. Fujimoto, H. Yamamoto, K. Nakao, K. Kaikita, R. Sakata, and E. Miyamoto Expression of Angiotensin-Converting Enzyme in Remaining Viable Myocytes of Human Ventricles After Myocardial Infarction Circulation, October 1, 1996; 94(7): 1513 - 1518. [Abstract] [Full Text]
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R. L. Kent and P. J. McDermott Passive Load and Angiotensin II Evoke Differential Responses of Gene Expression and Protein Synthesis in Cardiac Myocytes Circ. Res., May 1, 1996; 78(5): 829 - 838. [Abstract] [Full Text]
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J. Sadoshima and S. Izumo Rapamycin Selectively Inhibits Angiotensin II–Induced Increase in Protein Synthesis in Cardiac Myocytes In Vitro : Potential Role of 70-kD S6 Kinase in Angiotensin II– Induced Cardiac Hypertrophy Circ. Res., December 1, 1995; 77(6): 1040 - 1052. [Abstract] [Full Text]
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G. Wu, T. Toyokawa, H. Hahn, and G. W. Dorn II epsilon Protein Kinase C in Pathological Myocardial Hypertrophy. ANALYSIS BY COMBINED TRANSGENIC EXPRESSION OF TRANSLOCATION MODIFIERS AND Galpha q J. Biol. Chem., September 22, 2000; 275(39): 29927 - 29930. [Abstract] [Full Text] [PDF]
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A. I M Al-Shafei, R G Wise, G A Gresham, G Bronns, T A Carpenter, L D Hall, and C. L-H Huang Non-invasive magnetic resonance imaging assessment of myocardial changes and the effects of angiotensin-converting enzyme inhibition in diabetic rats J. Physiol., January 15, 2002; 538(2): 541 - 553. [Abstract] [Full Text] [PDF]
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B. V. Alvarez, J. Fujinaga, and J. R. Casey Molecular Basis for Angiotensin II-Induced Increase of Chloride/Bicarbonate Exchange in the Myocardium Circ. Res., December 7, 2001; 89(12): 1246 - 1253. [Abstract] [Full Text] [PDF]
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