© 2001 American Heart Association, Inc.
Cellular Biology |
Canine Ventricular Myocytes Possess a Renin-Angiotensin System That Is Upregulated With Heart Failure
From the Departments of Medicine (L.B., A.L., F.F., J.K., B.N.-G., P.A.) and Physiology (H.T., T.H.H.), New York Medical College, Valhalla, NY; Division of Molecular Cardiology (D.E.D.), The Texas A&M University, Temple, Tex; and Istituto Ricerche Farmacologiche Mario Negri (F.F.), Milano, Italy.
Correspondence to Piero Anversa, MD, Department of Medicine, Vosburgh Pavilion, Room 302, New York Medical College, Valhalla, NY 10595. E-mail piero_anversa{at}nymc.edu
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Abstract |
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Abstract—Ventricular pacing leads to a dilated myopathy in which cell death and myocyte hypertrophy predominate. Because angiotensin II (Ang II) stimulates myocyte growth and triggers apoptosis, we tested whether canine myocytes express the components of the renin-angiotensin system (RAS) and whether the local RAS is upregulated with heart failure. p53 modulates transcription of angiotensinogen (Aogen) and AT1 receptors in myocytes, raising the possibility that enhanced p53 function in the decompensated heart potentiates Ang II synthesis and Ang II–mediated responses. Therefore, the presence of mRNA transcripts for Aogen, renin, angiotensin-converting enzyme, chymase, and AT1 and AT2 receptors was evaluated by reverse transcriptase–polymerase chain reaction in myocytes. Changes in the protein expression of these genes were then determined by Western blot in myocytes from control dogs and dogs affected by congestive heart failure. p53 binding to the promoter of Aogen and AT1 receptor was also determined. Ang II in myocytes was measured by ELISA and by immunocytochemistry and confocal microscopy. Myocytes expressed mRNAs for all the constituents of RAS, and heart failure was characterized by increased p53 DNA binding to Aogen and AT1. Additionally, protein levels of Aogen, renin, cathepsin D, angiotensin-converting enzyme, and AT1 were markedly increased in paced myocytes. Conversely, chymase and AT2 proteins were not altered. Ang II quantity and labeling of myocytes increased significantly with cardiac decompensation. In conclusion, dog myocytes synthesize Ang II, and activation of p53 function with ventricular pacing upregulates the myocyte RAS and the generation and secretion of Ang II. Ang II may promote myocyte growth and death, contributing to the development of heart failure.
Key Words: pacing • myocyte renin-angiotensin system • p53 function • heart failure
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myocyte death and myocyte hypertrophy characterize dilated cardiomyopathy in humans.1 2 Cardiomegaly, ventricular dilation, myocyte apoptosis, and myocyte hypertrophy develop with rapid ventricular pacing,3 4 5 mimicking the human disease. Angiotensin (Ang) II is implicated in the activation of several cellular responses, including myocyte apoptosis and hypertrophy.6 7 8 Interference with the systemic and local renin-angiotensin system (RAS) limits myocyte growth and the expansion in cavitary volume, improving ventricular performance and the lifespan of patients with cardiac failure.9 10 11 Experimentally, similar interventions inhibit apoptotic cell death in the stressed myocardium preventing or attenuating decompensation.12 13 Rat ventricular myocytes possess the various components of RAS, and an upregulation of renin, Aogen, Ang I, angiotensin-converting enzyme (ACE), Ang II, and AT1 and AT2 receptors occurs in myocytes of the overloaded rat heart.14 15 16 17 Whether canine myocytes synthesize Ang II, influencing directly cellular adaptations via an autocrine mechanism, remains to be demonstrated. This is a relevant issue, because other pathways in the formation of Ang II have been identified in the dog myocardium.18 19
Chymostatin-sensitive angiotensin-producing enzyme, ie, heart chymase, has been implicated as the major source of Ang II accumulation in the canine,18 19 baboon,20 and human21 22 hearts. These observations tend to minimize the role of ACE in the conversion of Ang I to Ang II in large mammals and raise questions on the existence of a myocyte RAS because of the claimed absence of chymase in this cell population.18 19 20 21 22 However, opposite results have also been reported.23 One of the problems with these findings is that little attention has been given to myocytes even though the entire myocardium or the properties of the interstitial fluid have been analyzed.18 19 20 21 22 23 24 25 In view of these uncertainties, the expression of the multiple constituents of RAS was measured at the mRNA and protein levels in myocytes of normal and paced dog hearts. Additionally, p53 function was analyzed because the tumor suppressor transactivates Aogen and AT1 receptor.6 7 26 27 28 Aogen is the limiting factor in the synthesis of Ang II,28 and binding to AT1 conditions myocyte growth and death.6 7 Thus, we established whether canine myocytes have the capability of generating Ang II, whether Ang II formation is enhanced in the overloaded ventricle, and whether p53 transcriptional potential correlates with the response of stressed myocytes. Positive results would imply that Ang II plays a critical role in myocyte growth, myocyte death, and the terminal evolution of the failing heart.
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Materials and Methods |
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Ventricular Function
Mongrel dogs were instrumented with corkscrew electrode in the left ventricle (LV) attached to an external pacemaker.4 5 26 29 Hearts were paced at 210 bpm for 1 (n=6) and 3 (n=6) weeks and at 240 bpm for an additional week (n=7). Control dogs were instrumented but not paced (n=7). Protocols were approved by New York Medical College. Cardiac function was measured in conscious dogs with the pacemaker turned off.26 29
Myocyte Isolation
Myocytes were enzymatically dissociated
(Figure 1
) from 6 control hearts and 4, 4, and 6 hearts paced
for 1, 3, and 4 weeks,
respectively.4 5 26
Purity of the preparation and the distinction of myocytes from other
cells is described in the online data supplement available at
http://www.circresaha.org.
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Reverse Transcriptase–Polymerase Chain
Reaction
Total RNA was extracted with Trizol reagent (Gibco
Life Technologies). Total RNA 5 µg was reverse transcribed with 200 U
of Superscript II reverse transcriptase (RT) (Gibco Life Technologies)
according to the supplier protocol. The resulting cDNA was amplified
using 1.5 U Taq DNA polymerase
(Boehringer Mannheim) as described in the supplier protocol. Sense and
antisense primers and polymerase chain reaction (PCR) amplification
cycles used for Aogen, renin, chymase, ACE, AT1
and AT2 receptors, and ß-actin are described
in the online data supplement. PCR products were separated on a 2%
agarose gel.
Electrophoretic Mobility Shift Assay
Oligonucleotides were prepared as recently
described.6 7 26 27 28
Nuclear extracts, 40 µg, were incubated with 2 µL of
[32P]ATP–end-labeled probe and subjected
to electrophoresis. Control for specificity included the exposure of
nuclear extracts to anti-p53, 0.5 µg of PAb 240, or an irrelevant
antibody, 0.5 µg of mouse anti-c-Jun.
Western Blot
Equivalents of 50 to 75 µg of proteins were
separated by SDS-PAGE, transferred on nitrocellulose membranes, and
exposed to mouse anti-rat Aogen, mouse anti-rat renin, mouse monoclonal
anti-human cathepsin D, mouse monoclonal anti-rat
ACE,30 mouse monoclonal
anti-human chymase, rabbit polyclonal anti-human
AT1 receptor, and rabbit polyclonal anti-rat
AT2
receptor31 antibodies. Rat
kidney and purified human ACE were used as positive controls for ACE.
Rat adrenal gland was used as a positive control for
AT2.
Ang II Labeling
Myocardial sections were fixed in formaldehyde and
incubated with Ang II
antiserum.6 7
Specificity was determined by preabsorption of 10 µL of antibody with
0.05 mg of antigen. Nonimmune rabbit serum was used as an additional
control.6 7 Myocyte
cytoplasm was identified by 
-sarcomeric actin
antibody.6 7 Ang
II–positive myocytes and number of Ang II sites per myocyte profile
were measured by confocal microscopy.
Ang II Concentration
Ang II levels in myocytes were obtained by ELISA
using the methodology previously
described.9 27
Data Analysis
Results are mean±SD. Autoradiograms were analyzed by
an image analyzer. Each set of samples in each gel included all time
points after pacing and controls. Statistical significance was
determined by Student’s t test
or Bonferroni method.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Ventricular Hemodynamics
Alterations in cardiac function in this animal model have been described.4 5 26 27 With 4 weeks of pacing, heart rate increased 42% (P<0.001), from 86±12 to 122±15 bpm, and mean arterial pressure decreased 18% (P<0.01), from 106±9 to 87±9 mm Hg. Similarly, LV systolic pressure decreased 24% (P<0.001), from 132±8 to 100±11 mm Hg, and LV positive dP/dt decreased 48% (P<0.001), from 2970±457 to 1546±351 mm Hg/s. Conversely, LV end-diastolic pressure increased 333% (P<0.001), from 6±2 to 26±6 mm Hg. Tachypnea, ascites, pulmonary congestion, and pleural effusion were present in these animals. There were no significant hemodynamic changes at 1 week of pacing, whereas at 3 weeks, LV end-diastolic pressure was increased 133% (P<0.01), to 14±3 mm Hg.
RT-PCR of Aogen, Renin, ACE, Chymase, and
AT1 and AT2
Receptors
The expression of the components of the local RAS in
control and paced canine myocytes was detected by RT-PCR
(Figure 2). Four determinations were obtained for each gene.
The use of RT-PCR did not permit quantitative measurements of the
changes in mRNAs with the duration of pacing. These difficulties in the
interpretation of mRNA levels are inherent in this methodology.
However, the purpose of this analysis was to document that a myocyte
RAS was present at baseline and that ventricular dysfunction and
failure with prolonged pacing did not impair the ability of dog
myocytes to express these transcripts.
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p53 DNA Binding
p53 is implicated in the upregulation of the myocyte
RAS, leading to the synthesis and secretion of Ang
II.6 7 28 A
gel-retardation assay was performed using oligonucleotides, including
the consensus site for p53 binding to the Aogen and
AT1 promoter, respectively. On the basis of the
results described above, this assay was restricted to myocytes from
control and failing hearts at 4 weeks of pacing, to establish whether a
correlation existed between p53 function and RAS activation in the
terminal phases of the dilated myopathy.
Figure 3A illustrates that the Aogen oligonucleotide
resulted in the formation of a p53-shifted complex in nuclear extracts
from myocytes of sham-operated and failing dogs. However, the optical
density (OD) of the p53 band was increased in cells from paced hearts
(controls, OD=1±0.2, n=6; paced, OD=2.9±1.1, n=6;
P<0.005). The specificity of
the assay was confirmed by demonstrating that the addition of an excess
of unlabeled self-oligonucleotide or of a monoclonal p53 antibody to
nuclear extracts from paced myocytes opposed the appearance of a p53
band. Conversely, preincubation with an irrelevant antibody did not
affect p53 DNA binding. The effects of pacing on p53 binding to the
promoter of AT1 receptor are shown in
Figure 3B. Two specific bands were identified, but the OD of
the shifted complexes was significantly greater in myocytes from paced
hearts (OD=1.4±0.4, n=6; paced, OD=3.5±0.8, n=6;
P<0.001).
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Western Blot of Aogen, Renin, Cathepsin D, ACE,
Chymase, and AT1 and AT2
Receptors
The protein levels of the myocyte RAS were analyzed in
control and failing hearts at 4 weeks of pacing. Expression of Aogen,
renin, and cathepsin D in myocytes is illustrated in
Figure 4. Aogen was represented by 2 bands at 54 and 56 kDa,
but the higher molecular-weight protein prevailed
(Figure 4A). Cardiac failure resulted in a 2.5-fold
(P<0.001) increase in Aogen
expression in myocytes, from a baseline OD value of 3.3±0.7 (n=6) to a
value of 8.1±1.6 (n=6). Similarly, renin was detected as 2 distinct
bands at 36 and 37 kDa, respectively
(Figure 4B). With pacing, renin increased 3.6-fold (controls,
OD=1.2±0.4, n=6; paced, OD=4.3±1.1, n=6,
P<0.001), paralleling the
changes in Aogen. Myocyte cathepsin D appeared as 3 bands at 52, 43,
and 34 kDa
(Figure 4C). However, the mature form of cathepsin D
corresponds to 43 kDa and is the most visible on Western blot.
Quantitatively, only the 43-kDa band was measured, because the 52-kDa
protein is an inactive precursor, and the 34-kDa protein is an inactive
degradation product. Pacing was associated with a 3-fold
(P<0.001) increase of the
active 43-kDa enzyme (controls, OD=1.1±0.3, n=6; paced, OD=3.3±0.7,
n=6).
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Two forms of ACE were identified at 134 and 170 kDa
(Figure 5A). The higher molecular weight reflected the
glycosylated enzyme; the other form corresponded to nonglycosylated
ACE. Both proteins increased with heart failure. The 134-kDa band
increased 1.45-fold (P<0.005),
and the 170-kDa band increased 1.44-fold
(P<0.002). In contrast,
chymase, detected at 29 kDa, did not change
(P=0.28) in nonpaced and paced
animals
(Figure 5A). AT2 receptor increased
42%
(Figure 5B) with pacing, but this change was not significant
(P<0.06). Conversely,
AT1 receptor in myocytes increased
significantly, 1.7-fold
(P<0.001) at 4 weeks after
pacing
(Figure 6) (controls, OD=3.1±0.75, n=6; paced, OD=5.4±0.98,
n=6).
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Data by Western blot were not always consistent with the levels of expression obtained by RT-PCR. These differences reflected the nonquantitative aspects of RT-PCR and the fact that changes in mRNAs are not necessarily identical to those in the quantity of proteins. Conversely, immunoblotting has inherent limitations in the precise identification of the size of each protein. Different molecular weight markers were used to minimize this possibility.
Ang II Quantity in Myocytes
Ang II was measured in left ventricular myocytes by
ELISA. Pacing resulted in a 2.2-fold
(P<0.004) increase in the
quantity of Ang II in myocytes (controls, 20±11 pg/mg protein, n=5;
paced, 44±7 pg/mg protein, n=5). Two additional approaches were used:
quantitative evaluation of the percentage of myocytes labeled by Ang II
antibody and measurements of Ang II sites per unit area of myocyte
cytoplasm by confocal microscopy.
Figure 7A illustrates by green fluorescence the discrete
sites of Ang II labeling, and
Figure 7B depicts by red fluorescence myocytes stained by
-sarcomeric actin. These two images are shown together in
Figure 7C; the fluorescent dots correspond to the
localization of Ang II in the myocardium. Myocyte profiles, defined by
laminin staining, contained a minimum of 1 to a maximum of 14 stained
sites per cell. Preabsorption of the primary Ang II antibody with Ang
II resulted in the absence of immunostaining
(Figure 7D). Similarly, substitution of the Ang II antibody
with nonimmune rabbit serum was characterized by the lack of staining
in the myocardium (not shown). An average of 180 ventricular myocytes
were examined at random in each of 6 nonpaced and 6 paced dogs, for a
total of 1086 and 1056 cells in the 2 groups of animals. This analysis
demonstrated that 22±4% and 45±7% of myocytes were labeled in
nonpaced and paced hearts, respectively. Cardiac failure resulted in a
2-fold (P<0.001) increase in
the fraction of myocytes containing Ang II. Additionally, the number of
Ang II–positive sites per mm2 of myocytes
was 2310±607 and 6285±659 in control and paced dogs, respectively.
The 2.7-fold greater value with pacing was significant
(P<0.001). The distribution of
Ang II–positive dots in labeled cells and the fraction of negative
myocytes are shown in
Figure 8. Ang II was increased in all cell categories of
paced dogs. Whether the increase in Ang II correlated with an increase
in myocyte size with the progression of pacing remains to be
investigated.
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Discussion |
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The results of the present study indicate that canine ventricular myocytes expressed the various molecular components of RAS and were capable of forming Ang II. Cathepsin D and chymase were detectable in these cells, pointing to the possibility of the conversion of Aogen to Ang I and Ang I to Ang II through pathways independent from renin and ACE. Surface AT1 and AT2 receptors were identified in myocytes. Cardiac failure was characterized by an upregulation of Aogen, renin, cathepsin D, ACE, and Ang II, whereas chymase was not increased; AT1 receptor protein increased, but the quantity of AT2 was not significantly altered. p53 function was enhanced in myocytes, leading to activation of the p53-regulated genes Aogen and AT1 receptor. These observations support the notion that the local synthesis of Ang II may be implicated in myocyte death and cellular hypertrophy in experimental dilated cardiomyopathy.
Ventricular Pacing and Generation of Ang II
in Myocytes
Analysis of the local RAS in the dog and baboon heart
has been restricted to myocardial
samples18 19 20
or examination of interstitial
fluid.24 25 These
investigations have provided significant information concerning the
role of the chymase system in the generation of Ang II in the normal
and overloaded
heart.18 19 20 24 25
Similar findings have been reported in humans, strengthening the
contribution of chymase in the formation of Ang II in the nonfailing
and failing
heart.21 22
Although not all studies are in
agreement,23 the major
source of chymase was linked to interstitial cells, and ACE was
localized to the endothelial cell membrane
only.23 On this basis,
myocytes were not identified as a cell population expressing chymase or
ACE activity and thereby were assumed of limited importance in the
synthesis and secretion of Ang II in the myocardium of large
mammals.18 19 20 21 22 23 24 25
Results in this study document that canine myocytes contained Ang II. Contrary to expectation, chymase and ACE were detected in these cells, and the upregulation of the myocyte RAS with cardiac failure was associated with increased ACE without changes in chymase. This does not exclude that both enzymes were involved in the generation of Ang II in the overloaded myocytes. Renin mRNA has been previously identified in rat cardiac myocytes,14 and its expression increases with heart failure.16 The present data confirm these observations in canine myocytes. Renin can be synthesized directly in cardiac muscle cells, and its formation is enhanced in the decompensated dog heart. Sarcomere stretching in vitro, which mimics diastolic overload in vivo, results in an upregulation of renin32 and other components of RAS, leading to the production and release of Ang II from the cells.6 7 Cathepsin D, which is ubiquitously present in the lysosomes of all cells,33 was found in myocytes, and a 3-fold increase was measured after ventricular pacing. This aspartyl protease34 may be implicated in combination with renin in the synthesis of Ang I with pacing. In summary, dog myocytes are most likely implicated in the regulation of the myocardial RAS under normal and pathological states.
Ventricular Pacing and Ang II–Receptor
Subtypes
Two isoforms of Ang II receptors,
AT1 and AT2, have been
identified in neonatal
myocytes.34 With maturation,
AT2 progressively decreases and
AT1
predominates.35 36 37
AT2 receptors are upregulated in the myocardium
after pressure overload or
infarction,17 38 39
but their role remains unclear. In neonatal myocytes, Ang II–mediated
hypertrophy is potentiated when AT2 blocking
agents are used.40
Similarly, deletion of the AT2 gene leads to an
abnormal elevation in arterial blood pressure, coupled with alterations
in resistance vessels and smooth muscle cell
hyperplasia.41 By
autoradiography of the human heart, AT2
receptors have been found in fibroblasts; AT2
expression is increased with heart failure, and this may oppose
fibroblast proliferation induced by
AT1–receptor
activation.37 Myocytes show
minimal amount of AT2 at baseline, and this
quantity is not affected by cardiac
decompensation.37
In this study, AT1 and AT2 were detected in pure preparations of myocytes from nonpaced and paced hearts. The documentation of AT2 in adult canine myocytes has no precedent. Cardiac failure was characterized by an increase in AT1, but the changes in AT2 were not significant. This differential adaptation suggests that AT1 receptor activation is implicated in the cellular responses that condition remodeling of the paced ventricle: myocyte apoptosis5 26 and cellular enlargement.4 Increases in Ang II and AT1 receptors with tachycardia may promote hypertrophy8 or trigger death in myocytes susceptible to growth or apoptotic signals.6 7
Ventricular Pacing and p53
Although stretching and the secretion of Ang II from
myocytes may be the immediate consequence of the elevation in left
ventricular end-diastolic pressure with
pacing,42 it is more
difficult to understand the mechanism by which the local RAS is
activated, maintaining the formation of Ang II. p53 binding to the
promoter of Aogen and AT1 receptor was enhanced
in the failing heart, resulting in an upregulation of Aogen and
AT1 proteins in myocytes. Because renin and
cathepsin D, as well as ACE and chymase, are normally present in canine
myocytes, the formation of Aogen may be the limiting factor in the
generation of Ang II. Overexpression of p53 in myocytes in vitro
enhances transcription of Aogen and AT1
receptor, leading to the production and release of Ang
II.43 Similarly, sarcomere
elongation increases the quantity and functional activity of p53, which
are coupled with the induction of multiple p53-regulated genes,
including Aogen and AT1
receptor.6 7 p53
binding to the bax promoter is enhanced, and the amount of Bax protein
in myocytes increases several
fold.6 Conversely, Bcl-2 is
decreased. These cellular responses are inhibited by infection of
myocytes with an inactive form of
p53.28 Thus, upregulation of
p53 function with pacing may play a critical role in the chronic
synthesis of Ang II and Ang II–mediated myocyte hypertrophy and death
in this
model.4 26
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-38132, HL-39902, HL-43023, AG-15756, HL-65577, HL-69923, AG-17042 and by grant JDFI 1-2000-62.
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Footnotes |
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Original received October 2, 2000; revision received November 27, 2000; accepted December 13, 2000.
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References |
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