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Regular Articles |
From the Department of Medicine,*
New York Medical
College, Valhalla, New York; and the Department of Molecular and
Cellular Pharmacology,
University of Miami,
Miami, Florida
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Introduction |
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Materials and Methods |
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Myocytes were isolated from 3-month-old Sprague-Dawley
rats.2,9,11,14
From each left ventricle, 6.0 x
106
myocytes were obtained. Cells were plated at
a density of 2 x 104
per
cm2
in a device with a silicon substrate coated
with 0.5 µg/cm2
laminin.2,9
The
entire study included 44 animals and a total of 
300 stretch devices.
Myocyte Infection
Cells were infected with a replication-deficient adenovirus, containing human mutated p53, Adp53m, under the control of the human cytomegalovirus immediate early gene promoter.15,16 p53mutations consisted of substitution of cysteine in position 135 with serine, and proline in position 72 with arginine.15 A second vector, expressing ß-galactosidase, AdLacZ, was used as control. Myocytes were infected 2 hours after plating and maintained in serum-free medium (SFM) for 12 hours; infection was terminated by removing the medium and adding fresh SFM. This phase lasted 36 hours. Myocytes were infected for 48 hours with 100, 300, 500, and 1,000 pfu/cell of AdLacZ; infection efficiency was 60 ± 9% (n = 3), 87 ± 5% (n = 5), 88 ± 3% (n = 5), and 91 ± 3% (n = 5), respectively. In all studies, infection with 300 pfu/cell was used.
Myocyte Stretch
After 48 hours of infection, a 20% strain was applied to a stretch device for 12 hours.2,9 In each preparation, sarcomere length was measured in 150 myocytes before and after stretch. To assess whether stretch injured cells, ethidium monoazide bromide (EMB; Molecular Probes, Eugene, OR), 5 µg/ml, was used. EMB binds to nucleic acids only in cells with membrane breakage.17 After incubation with EMB and exposure to light, cells were fixed in 1% paraformaldehyde. EMB-labeled cells were measured by confocal microscopy.
Gel Retardation Assay
Two synthetic oligonucleotides, 5'-AGCCTCTGTACAGAGTAGCC-TGGGAATAGATCCATCTTC-3' and 5'-GAAGATGGATCTATTCCCAGGCTACTCTGTAC-AGAGGCT-3', corresponding to two half-sites of the p53 motif in the rat angiotensinogen (Aogen) promoter were used. Two synthetic oligonucleotides, 5'-GCTGAGCTTGGATCTGGAAGGCGACACTGGG-3' and 5'-CCCAGTGTCGCCTTCCAGATCCAAGCTCAGC-3', corresponding to two half-sites of the p53 motif in the rat AT1 promoter were used. Two synthetic oligonucleotides, 5'-AGCTTGCTCACAAGTTAGAGACAAGCCTGGGCGTGGCTATATTGA-3' and 5'-AGCTTCAATATAGCCCACGCCCAGGCTTGTCTCTAACTTGTGAGCA-3', corresponding to two half-sites of the p53 motif in the human bax promoter were used. Two additional sites of the p53 motif are located in the 3'-region of the perfect p53 element at 0 and 6 bp where they partially overlap.7 Nuclear extracts were obtained by incubating myocytes in hypotonic buffer and in high-salt buffer2,4,9,10 ; 30 µg of proteins were diluted in binding buffer with 1 to 2 µl of labeled probe. Nuclear extracts were exposed to p53 antibodies consisting of 0.5 µg pAb 240 and DO-1 (Santa Cruz, Santa Cruz, CA), or 0.5 µg pAb 246 (Ab-4, Calbiochem, San Diego, CA). Controls included unlabeled Aogen, AT1, and bax probes as competitors and unlabeled mutated Aogen (5'-AGCCTCTATAAAGAGTAGCCTGGGAATAGATCCATCTTC-3'), AT1 (5'-GCTGAGATTAGATCTGGAAGGCGACACTGGG3'), and bax (5'-AAGTTAGAGATAATGCTGGGCGAG-3') as noncompetitors.
Western Blot of p53, Bax, Bcl-2, Aogen, Renin, ACE, and AT1 and AT2 Receptors
Myocytes were lysed in the presence of protease inhibitors.
Samples were incubated on ice, centrifuged, and 50 µg proteins were
separated by 8 to 12% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Proteins were transferred to nitrocellulose and
exposed to mouse monoclonal anti-human p53 (pAb 240, Santa Cruz),
rabbit polyclonal anti-human Bax (P19, Santa Cruz), rabbit polyclonal
anti-human Bcl-2 (
C21, Santa Cruz), mouse anti-rat Aogen (Swant,
Bellinzona, Switzerland), mouse anti-rat renin (Swant), mouse anti-rat
ACE (gift from Dr. David E. Dostal), rabbit polyclonal
anti-human AT1 (306, Santa Cruz), and goat
polyclonal anti-human AT2 (C-18, Santa Cruz)
antibodies. Bound antibodies were identified by horseradish
peroxidase-conjugated anti-mouse, anti-rabbit, anti-goat IgG, or
anti-mouse IgM and recognized by a peroxidase chemiluminescent
detection reagent.2,4,9,10
Immunoprecipitation and Western Blot of Bax and Bcl-2
Bcl-2 Bound to Bax
Two hundred µg of proteins were incubated with 3 µg of rabbit
polyclonal anti-human Bax antibody (P19, Santa Cruz) and 250 µl of
HNTG buffer (20 mmol/L Hepes, pH 7.5, 150 mmol/L NaCl, 0.1% Triton
X-100, 10% glycerol), containing protease inhibitors at 4°C
overnight. Fifty µl of protein A-agarose was added. Proteins were
separated by 12% polyacrylamide gel electrophoresis. Proteins were
transferred to nitrocellulose membranes and exposed to rabbit
polyclonal anti-human Bcl-2 (C21, Santa Cruz) at a concentration of
1 µg/ml Tris-buffered saline/Tween 20 (TBST).
Bax Bound to Bcl-2
An identical procedure was followed, with the exception that
rabbit polyclonal anti-human Bcl-2 antibody (C21, Santa Cruz) was
used to immunoprecipitate myocyte lysates9,10
and that
membranes were exposed to rabbit polyclonal anti-human Bcl-2 and rabbit
polyclonal anti-human Bax.
p53 Localization
Cells were fixed in methanol and acetone (2:1) at -20°C and were incubated with two p53 antibodies: DO-1 (Santa Cruz) which recognizes human p53 and pAb 246 (Ab-4; Calbiochem, San Diego, CA) which binds to rodent wild-type p53. The fraction of p53-positive myocyte nuclei was evaluated by confocal microscopy;4 500 cells were examined in each case.
Terminal-Deoxynucleotidil Transferase Assay Assay
Cultures were incubated with 50 µl of solution, containing 5 U
of TdT, 2.5 mmol/L CoCl2, and 0.5 nmol/L
biotin-16-dUTP. Myocytes were stained with 5 µg/ml of fluorescein
isothiocyanate-extravidin in 4x standard saline citrate buffer. Cells
were treated with 
-sarcomeric actin antibody and with
rhodamine-labeled anti-mouse IgG. Nuclei were labeled by propidium
iodide (PI).2,4,9,18
In Situ Ligation of Hairpin Oligonucleotide
Myocytes were stained with 50 mmol/L Tris/HCl, pH 7.8, 10 mmol/L MgCl2, 10 mmol/L dithiothreitol, 1 mmol/L ATP, 15% polyethylene glycol 8,000, 1 U/µl T4 ligase, and 35 ng/µl hairpin probe with single-base 3' overhang.19 Cells were exposed to fluorescein isothiocyanate-extravidin in bicarbonate buffer and stained for confocal microscopy.2,18
Ang II Amount
Ang II in conditioned medium was measured by enzyme-linked immunosorbent assay (Peninsula, Belmont, CA). Conditioned medium was treated with 10% trifluoroacetic acid and centrifuged. Supernatant was dried, residue dissolved in 0.1% trifluoroacetic acid, and purified in a C-18 Sep-Pak column. Ang II fraction was eluted with 30% acetonitrile in 5 ml of 0.1% trifluoroacetic acid, dried, and dissolved in 0.25 ml of TBST. Samples, 50 µl, were analyzed in a microtiter plate using Ang II antibody (1:32,000) and a tracer, biotinylated Ang II. Color reaction was developed with tetramethyl-benzidine. Absorbance was recorded at 450 nm and concentration calculated from standard curves.9,10
Data Analysis
Results are presented as mean ± SD. Autoradiograms were assessed by an image analyzer. Significance between two measurements, P < 0.05, was determined by Student’s t-test. Significance among several preparations was established by the Bonferroni method.20
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pAb 240 p53 antibody recognized both wild-type and mutated p53; small amounts of p53 were detectable in control AdLacZ-infected myocytes. In Adp53m-infected cells, p53 increased from 24 to 48 hours when 100 and 300 pfu/cell were added. In comparison with AdLacZ-infected cells for 48 hours, infection of myocytes with 100 and 300 pfu/cell of Adp53m resulted in a 71-fold (P < 0.001) and 107-fold (P < 0.001) increase in p53 (data not shown). The 1.5-fold difference in p53 quantity between these doses of Adp53m was significant (P < 0.001).
Stretch and p53 DNA Binding
Stretching of noninfected and infected myocytes produced a 14%
(P < 0.001) increase in sarcomere length, from
1.85 ± 0.02 µm (n = 90) to 2.11 ±
0.03 µm (n = 90). Sarcomere length was
constant among experiments.2,9
Cell membrane was
occasionally disrupted. EMB-positive myocytes were 0.4 to 0.6% in
cultures of noninfected and infected cells and in the absence and
presence of stretch; the 420 molecular weight molecule was mostly
detected in the nucleus (Figure 1)
. The
minimal level of myocyte damage indicates that the utilization of 300
pfu/cell of Adp53m as an infection protocol did not alter cell
viability. Similar values have been obtained with mechanical
deformation only.2
Additionally, this high dose was used
because the medium added to the stretch devices was 5 ml. This quantity
of medium was necessary for the large size of the devices; when more
than 1 ml of medium has to be used, the diffusion factor is decreased
and larger titers of virus have to be applied.21
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. The p53-shifted complex was also
stronger in stretched than in nonstretched noninfected myocytes.
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Similarly, p53 complex in stretched noninfected myocytes was slightly
higher than in nonstretched myocytes. The promoter of bax contains two copies of the 10-bp element of the p53 motif which are separated by a single nucleotide: 5'-TCACAAGTTA-3' and 5'-AGACAAGCCT-3'. They correspond to the human bax sequence, from -484 bp to -465 bp, located 70 bp 5' from the TATA box. The first half-site has three mismatched nucleotides and the second is perfect. Two additional sites for p53 are present in the 3' region of the perfect p53 motif at 0 and 6 bp: 5'-GGGCGTGGGC-3' and 5'-GGGCTATATT-3'. They have seven of 10 and eight of 10 matches with the consensus sequence of p53.7 Thus, a 46-bp oligonucleotide was labeled and used.
p53 binding to the bax promoter was evaluated to confirm that Adp53m
functions as a dominant-negative despite the fact that the 135 mutation
was located in the DNA-binding domain. A similar behavior of p53
binding to the well-known sequences present in the bax promoter would
support the observations with Aogen and AT1
receptor probes. Figure 2C
documents that stretching of myocytes
increased p53 binding to bax under control condition and after
infection of myocytes with Adp53m. To confirm the specificity of the
assay, nuclear extracts from Adp53m-infected myocytes were exposed to
the anti-human p53 antibody DO-1 because the adenoviral vector carried
a human mutated form of p53; this interaction opposed the appearance of
a p53-shifted complex. Conversely, the addition of the anti-rat p53
antibody pAb 246 did not interfere with the p53 bands. The slight
decrease in optical density of the bands most likely reflects the
semiquantitative nature of this assay and not an actual difference in
DNA binding. A similar phenomenon was noted when nuclear extracts were
treated with an unlabeled mutated form of bax that left the p53 bands
essentially intact (Figure 2D)
. Nuclear proteins from noninfected
myocytes were not exposed to DO-1 antibody, because DO-1 does not react
with rat p53. Consistency in protein loading, lack of protein
degradation, and uniformity in the relative purity of nuclear extracts
are shown in Figure 2E
.
Increased binding of mutated p53 to the Aogen,
AT1, and bax promoter with stretch (Figure 2)
suggests that translocation to the nucleus of this inactive p53 form
may require phosphorylation, in a manner comparable to wild-type p53.
This possibility is consistent with the higher fraction of myocyte
nuclei labeled by mutated p53 after sarcomere elongation. These
results, obtained by immunolabeling and confocal microscopy, are
described below.
Stretch and p53
To establish whether translocation of wild-type p53 was impaired
in myocytes overexpressing Adp53m, the effects of 12 hours of stretch
on the nuclear localization of mutated and wild-type p53 was assessed
by confocal microscopy in Adp53m-infected myocytes and compared with
the distribution of wild-type p53 in AdLacZ-infected cells. Endogenous
wild-type p53, detected by anti-rat p53 pAb 246 antibody, was seen in
1.3 ± 1.0% (n = 6) of nuclei of
nonstretched myocytes infected with AdLacZ (Figure 3, A–C)
and in 20 ±
7% (n = 6) of nuclei of the same cells after stretch (Figure 3, D–F)
. This latter change was consistent with activation and
translocation to the nucleus of functioning p53. Mutated p53 was not
detectable in the nucleus of nonstretched Adp53m-infected myocytes by
anti-human p53 DO-1 antibody (Figure 3, G–I)
, but was observed with
the same antibody in 83 ± 5% (n = 6) of
nuclei of Adp53m-infected cells after stretch (Figure 3, J–L)
. In
Adp53m-infected myocytes, endogenous wild-type p53, identified by
anti-rat p53 pAb 246 antibody, was found in 0.4 ± 0.2%
(n = 6) of nuclei of nonstretched and in
5.4 ± 1.1% (n = 6) of nuclei of stretched
cells. The presence of mutated p53 in the majority of myocyte nuclei
after stretch suggests that activation of the mutated protein occurred
with mechanical deformation. This contention was supported by the
characteristics of DNA binding shown in Figure 2
. Therefore, it is
possible that the mutated p53 may have acted as a dominant-negative by
competing with wild-type p53 for the translocation to the myocyte
nucleus, and, subsequently, at the level of the nucleus, for the
binding to specific DNA sequences. Interaction between mutated p53 and
target DNA did not result in transactivation of p53-regulated genes
(see Figures 5, A and B, and 6
, A and B). Infection with Adp53m reduced
by 75% (5% versus 20%) the translocation of endogenous
wild-type p53 to the nucleus as a result of stretching of sarcomeres.
The impact of 12 hours of stretch on p53 quantity was measured by
Western blot in Adp53m-infected cells and in control noninfected and
AdLacZ-infected myocytes. Stretch increased p53 protein 4.3-fold
(P < 0.001) and 4.8-fold
(P < 0.001) in noninfected and
AdLacZ-infected myocytes (Figure 4)
.
In contrast, the high p53 level in nonstretched Adp53m-infected
cells remained constant after stretch (+15%, NS). There was no
apparent difference in the electrophoretic mobility of wild-type and
mutated p53 proteins.
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Bax and Bcl-2 were measured in nonstretched and stretched,
noninfected and AdLacZ- and Adp53m-infected myocytes. Stretch for 12
hours increased Bax in noninfected and AdLacZ-infected myocytes
2.3-fold (P < 0.001) and 2.7-fold
(P < 0.001), respectively (Figure 5A)
. This difference was not significant,
but a larger variability was noted in AdLacZ-infected myocytes.
Infection with Adp53m not only prevented stretch-mediated up-regulation
of Bax in myocytes, but also reduced its quantity by 97%
(P < 0.001). Adp53m had an opposite effect on
Bcl-2 (Figure 5B)
. Bcl-2 increased 2.0-fold (P
< 0.001) in stretched Adp53m-infected myocytes, whereas it decreased
94% (P < 0.001) and 96%
(P < 0.001) in stretched noninfected and
AdLacZ-infected myocytes. The changes in Bax and Bcl-2 in stretched
Adp53m-infected myocytes were consistent with the localization of
mutated p53 in 83% of nuclei. This may have resulted in inhibition of
wild-type p53 function below baseline.
As stated, stretch essentially abolished the expression of Bcl-2 and
more than doubled Bax quantity in control-noninfected and -infected
myocytes. Conversely, stretch of Adp53m-infected myocytes resulted in
opposite changes in the amount of Bcl-2 and Bax in the cells. To
confirm these findings and evaluate the relationship between these two
antiapoptotic and proapoptotic proteins, the interaction of Bax with
Bcl-2 was examined by immunoprecipitation. Myocyte lysates were
incubated with Bax antibody and immunoprecipitated proteins were then
exposed to Bcl-2 antibody to obtain the fraction of Bcl-2 bound to Bax
(Figure 5C)
. The decrease of Bax in stretched Adp53m-infected myocytes
(Figure 5A)
resulted in an almost undetectable amount of Bcl-2 linked
to Bax. The higher levels of Bax in stretched noninfected and
AdLacZ-infected myocytes (Figure 5A)
were coupled with minimal
quantities of Bcl-2 associated protein, reflecting the down-regulation
of Bcl-2 in these cells with stretch (Figure 5, A and B)
. Moreover,
total Bcl-2 and the fraction of Bax linked to Bcl-2 were identified by
immunoprecipitation of myocyte lysates with Bcl-2 antibody and
subsequent exposure of the blots to Bax and Bcl-2 antibodies (Figure 5D)
. The large quantity of total Bcl-2 in stretched Adp53m-infected
myocytes was not coupled with Bax because this latter protein almost
disappeared in these cells after stretch. On the other hand, the lack
of Bax associated with Bcl-2 in noninfected and AdLacZ-infected
myocytes was because of severe reduction of Bcl-2 in these cells after
stretch.
Stretch and Local RAS
Aogen and AT1 are p53-regulated
genes.2,9-11
Stretch increased Aogen by twofold
(P < 0.001) in noninfected and
AdLacZ-infected myocytes. However, Aogen decreased 45%
(P < 0.01) in stretched Adp53m-infectedmyocytes
(Figure 6A)
. Similarly,
AT1 increased 90% (P <
0.001) and 120% (P < 0.001) in stretched
noninfected and AdLacZ-infected myocytes, but decreased 60%
(P < 0.01) in stretched Adp53m-infected cells
(Figure 6B)
. Infection with Adp53m had no effect on the p53-independent
genes, renin, ACE, and AT2 receptor in myocytes.
Stretch increased renin 198% (P < 0.001),
220% (P < 0.001) and 232%
(P < 0.001) in noninfected, and in AdLacZ- and
Adp53m-infected myocytes (Figure 6C)
. ACE increased a ninefold average
(P < 0.001) with stretch in the three groups of
myocytes (Figure 6D)
. AT2 was also enhanced by
stretch, 68 to 74% (P < 0.05-P
< 0.005), in all cells (Figure 6E)
.
Stretch and Ang II Formation
Ang II was measured by enzyme-linked immunosorbent assay in conditioned medium of nonstretched and stretched AdLacZ- and Adp53m-infected myocytes. Ang II increased 2.4-fold (P < 0.001) in stretched AdLacZ-infected myocytes, from a baseline value of 454 ± 93 pg/hour/106 cells (n = 5) to a value of 1,104 ± 115 pg/hour/106 cells (n = 5) at 12 hours after stretch. Conversely, the 10% increase noted with stretch in Adp53m-infected cells was not statistically significant: nonstretched, 493 ± 88 pg/hour/106 cells, n = 4; stretched, 540 ± 122 pg/hour/106 cells, n = 7; P = 0.98.
Stretch and Apoptosis
Myocyte apoptosis was measured by TdT assay14
and
in situ ligation of a hairpin probe with single-base 3'
overhang.19
This latter method identifies DNA damage which
is present only with apoptosis.2,18,19,26
Labeled nuclei
were assessed by confocal microscopy that allowed detection of
structural features of apoptosis (Figure 7)
. With both techniques, apoptosis
increased an average 6.2-fold (P < 0.001) in
stretched AdLacZ-infected myocytes (Figure 8)
. However, the 63% increase in cell
death in stretched Adp53m-infected myocytes was not significant
(P = 0.67–0.61). Apoptosis in stretched
Adp53m-infected myocytes was 70% (P < 0.001)
lower than in stretched AdLacZ-infected myocytes.
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Adp53m and p53 Transcriptional Activity
The p53 mutant used in this study lacks transcriptional activation function because of the cysteine to serine substitution at amino acid position 135.15,16,27 DNA binding to p53 consensus sites is retained, and the mutated p53 acts as a dominant-negative by competing with the wild-type protein for binding to target genes. The p53 motif consists of 10 nucleotides22 and high-affinity binding requires two copies of this sequence.23,25 They were previously found in the promoter of bax,7 and here in the promoter of Aogen and AT1 receptor genes. Sarcomere elongation in noninfected myocytes was characterized by enhanced p53 binding to the promoter of Aogen, AT1 receptor, and bax that resulted in equivalent increases in the expression of Aogen, AT1 receptor, and Bax proteins. In contrast, opposite findings were obtained in stretched Adp53m-infected myocytes; increases in p53-shifted complexes were not coupled with corresponding accumulations of Aogen, AT1 receptor, and Bax proteins in the cells.
The system used, which depressed p53 function after sarcomere elongation,2,9 inhibited stretch-induced up-regulation of Bax and down-regulation of Bcl-2. Moreover, expression of Aogen and AT1 receptor was markedly attenuated after stretch, documenting that p53 plays a relevant role in the modulation of these two components of the cellular RAS. Decreased availability of Aogen may reduce Ang II production.28 Additionally, ligand binding to AT1 receptors is critical for initiation of myocyte growth29,30 and apoptosis,2,10,11 and reduction in Ang II binding sites may affect cell hypertrophy and cell death. Ang II formation, myocyte apoptosis, and reactive myocyte growth are enhanced in pathological states.30-32 Lack of interference with p53 transactivation2,9 and potentiation of p53 activity11 increase Ang II-mediated myocyte death2,9,11 and possibly myocyte growth.
Observations indicate that mechanical deformation is necessary for p53 translocation to the nucleus and activation of p53-dependent genes and local RAS in myocytes. However, we have previously shown that infection with an adenoviral vector containing wild-type human p53 led to similar responses in myocytes in the absence of sarcomere stretching.11 These results suggest that high level of expression of wild-type human p53 by viral infection was characterized by translocation and activation of exogenous p53 at the level of the nucleus, independently from the application of physical forces. Such a notion is consistent with enhanced p53 DNA binding to the promoter of bax, Aogen, and AT1 receptor, and increased transcription of Aogen and AT1 receptor mRNAs.11 Additionally, up-regulation of p53 binding activity was accompanied by a supershift for bax, Aogen, and AT1 when the anti-human p53 DO-1 antibody was used.11 This excludes activation of endogenous p53 in myocytes as a secondary event promoted by infection of wild-type human p53. However, these findings do not prove unequivocally that p53 translocation to the nucleus is required for the induction of apoptotic cell death.
Adp53m and Myocyte RAS
Myocytes possess the various components of RAS29,33,34 and generate Ang II.2,9-11,29 Conditions of overload in vivo up-regulate this local system34 and Ang II blockade improves the outcome of ventricular dysfunction and failure of ischemic and nonischemic origin.31,32,35-37 We have identified previously that Aogen and AT1 belong to the group of p53-inducible genes.9-11 However, myocyte stretching was used and this in vitro manipulation activates multiple factors38,39 that could have stimulated RAS independently from p53. Conversely, overexpression of p53 in myocytes, in the absence of stretch, typically showed enhanced p53 binding to the promoter of Aogen and AT1 receptor, increased expression of these two genes, and synthesis and secretion of Ang II.11 The current investigation strengthens these findings by documenting two additional points: 1) inhibition of p53 function after stretch, by previous infection of myocytes with Adp53m, decreased below baseline the expression of Aogen and AT1 receptor; and 2) down-regulation of Aogen prevented the increase in Ang II generation in mechanically loaded myocytes, despite an increase in renin and ACE proteins. Aogen played a key role in the synthesis of the peptide; availability of Aogen as a substrate catalyzed by renin seemed to be the limiting factor in Ang II formation.
Stretching increased the quantity of Aogen, renin, ACE, and AT1 and AT2 receptors in noninfected and AdLacZ-infected myocytes, and this adaptation of the entire RAS was associated with a nearly 2.5-fold increase in the generation of Ang II. Similar increases in renin, ACE, and AT2 receptor were noted in stretched Adp53m-infected myocytes, demonstrating that p53 dominant-negative had no influence on p53-independent genes. Enhanced expression of renin and ACE in stretched Adp53m-infected myocytes may have compensated for the attenuation of Aogen, contributing to maintain cellular Ang II to levels comparable to those in nonstretched AdLacZ-infected myocytes. Additionally, this may suggest a greater conversion of Aogen in Ang I. The reduction in AT1 receptors below controls in stretched cells expressing dominant-negative p53 may be accounted for by impaired receptor cycling mediated by p53 with time.
AT2 receptors have not been previously identified in adult myocytes. AT2 receptors are numerous in neonatal cardiac myocytes, but their number decreases rapidly postnatally and adult ventricular myocytes have been claimed to possess exclusively AT1.40,41 The possibility was raised that AT2 is re-expressed in the overloaded heart in both myocytes42 and fibroblasts,43 but the myocyte localization remained unclear. In this study, AT2 was detected in nonstretched myocytes and its expression increased with the mechanical stimulus. The functional significance of AT2 receptor in differentiated myocytes has not been defined. They oppose the growth-promoting effect of AT1 receptors in neonatal myocytes,44 but a similar response was not observed in adult myocytes.30 In aortic smooth muscle cells, AT2 receptors inhibit cell growth and trigger apoptosis, by interfering with the AT1 effector pathway.45,46
Adp53m and Myocyte Apoptosis
The proapoptotic effects of p53 have been shown in numerous
systems,47-49
including myocyte stretching,2
hypoxia of transformed cells,50
and DNA
damage.51
p53 up-regulates Bax and down-regulates Bcl-2,
increasing the susceptibility of cells to undergo
apoptosis.7
However, changes in Bax and/or Bcl-2 alone are
not sufficient to trigger cell death.7
Overexpression of
wild-type p53 in myocytes increases Bax and decreases Bcl-2, but cell
death is conditioned by the synthesis and secretion of Ang
II.11
Interference with ligand binding to surface
AT1 receptors abrogates myocyte
apoptosis.2,9,11
Infection of myocytes with p53
dominant-negative impacts at least three steps in the stretch-induced
apoptotic pathway: first, it increases the Bcl-2:Bax protein ratio;
second, it inhibits the formation of Ang II; and third, it decreases
surface AT1 receptor. Several pathways of
apoptosis have also been described that are not regulated by
p53.48,52
The activity of p53 is highly dose-, cell type-,
and cell context-dependent: p53 overexpression does not by itself
induce apoptosis in vascular smooth muscle cells53,54
;
overexpression of p53-dependent p21 protects skeletal muscle from
apoptosis55
; and activation of p53 by 
-radiation may
induce apoptosis or cell cycle withdrawal.51
Infection of stretched myocytes with Adp53m reduced the formation of
Ang II and AT1 receptor, both responsible for the
initiation of events resulting in DNA fragmentation; DNA damage was
characterized by staggered ends with single-base 3'
overhang.19,26
DNase I activation is critically influenced
by elevated intracellular Ca2+, proposed here to
be mediated by AT1 receptor stimulation and
downstream translocation of protein kinase C 
and
.56
Contrary to what has been found in smooth muscle
cells,57
AT2 plays no role in this
model of myocyte apoptosis. The death promoting effects of Ang II on
myocytes are inhibited by AT1 blockers in
vitro2,11,56
and in
vivo.31,32
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Footnotes |
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Supported by National Institutes of Health Grants HL-38132, HL-39902, HL-43023, and AG-15756.
Accepted for publication May 25, 2000.
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V. Grishko, V. Pastukh, V. Solodushko, M. Gillespie, J. Azuma, and S. Schaffer Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2364 - H2372. [Abstract] [Full Text] [PDF]
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 A. Gonzalez, M. A Fortuno, R. Querejeta, S. Ravassa, B. Lopez, N. Lopez, and J. Diez Cardiomyocyte apoptosis in hypertensive cardiomyopathy Cardiovasc Res, September 1, 2003; 59(3): 549 - 562. [Abstract] [Full Text] [PDF]
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M. A. Fortuno, A. Gonzalez, S. Ravassa, B. Lopez, and J. Diez Clinical implications of apoptosis in hypertensive heart disease Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1495 - H1506. [Full Text] [PDF]
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S. Jesmin, Y. Hattori, I. Sakuma, C. N. Mowa, and A. Kitabatake Role of ANG II in coronary capillary angiogenesis at the insulin-resistant stage of a NIDDM rat model Am J Physiol Heart Circ Physiol, October 1, 2002; 283 (4): H1387 - H1397. [Abstract] [Full Text] [PDF]
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M. Sotoudeh, Y.-S. Li, N. Yajima, C.-C. Chang, T.-C. Tsou, Y. Wang, S. Usami, A. Ratcliffe, S. Chien, and J. Y.-J. Shyy Induction of apoptosis in vascular smooth muscle cells by mechanical stretch Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1709 - H1716. [Abstract] [Full Text] [PDF]
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 F. Fiordaliso, A. Leri, D. Cesselli, F. Limana, B. Safai, B. Nadal-Ginard, P. Anversa, and J. Kajstura Hyperglycemia Activates p53 and p53-Regulated Genes Leading to Myocyte Cell Death Diabetes, October 1, 2001; 50(10): 2363 - 2375. [Abstract] [Full Text]
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J. Kajstura, F. Fiordaliso, A. M. Andreoli, B. Li, S. Chimenti, M. S. Medow, F. Limana, B. Nadal-Ginard, A. Leri, and P. Anversa IGF-1 Overexpression Inhibits the Development of Diabetic Cardiomyopathy and Angiotensin II-Mediated Oxidative Stress Diabetes, June 1, 2001; 50(6): 1414 - 1424. [Abstract] [Full Text]
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 L. Barlucchi, A. Leri, D. E. Dostal, F. Fiordaliso, H. Tada, T. H. Hintze, J. Kajstura, B. Nadal-Ginard, and P. Anversa Canine Ventricular Myocytes Possess a Renin-Angiotensin System That Is Upregulated With Heart Failure Circ. Res., February 16, 2001; 88(3): 298 - 304. [Abstract] [Full Text] [PDF]
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A. Frustaci, J. Kajstura, C. Chimenti, I. Jakoniuk, A. Leri, A. Maseri, B. Nadal-Ginard, and P. Anversa Myocardial Cell Death in Human Diabetes Circ. Res., December 8, 2000; 87(12): 1123 - 1132. [Abstract] [Full Text] [PDF]
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