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From the Department of Medicine,*
New York Medical
College, Valhalla, New York; and the Istituto di Ricerche
Farmacologiche Mario Negri,
Milano, Italy
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under ether anesthesia, MI was induced in 84 male Sprague-Dawley rats (Charles River Breeding Labs, North Wilmington, MA), weighing approximately 250 g.12,13,25 To reduce pain, buprenorphine hydrochloride was administered (0.65 mg/kg body weight, intramuscularly; Brufenex, Reckitt and Colman Pharmaceuticals, Richmond, VA). Thirty-seven infarcted rats died shortly after the operation; 47 infarcted rats were used. Thirty-one sham-operated (SO) rats were used as controls. Animals were killed 8 days after coronary occlusion or sham-operation. Before sacrifice, animals were anesthetized with chloral hydrate (300 mg/kg body weight, intraperitoneally), and measurements of left ventricular and right ventricular pressures and rate of pressure rise, + dP/dt, and decay, - dP/dt, were obtained.12-14,25 Protocols were approved by the New York Medical College.
Myocyte Isolation and Culture
Hearts were placed on a stainless steel cannula for retrograde
perfusion through the aorta and left ventricular myocytes were
enzymatically dissociated. The solutions were supplements of modified
commercial minimum essential medium (MEM) Joklik (Sigma Chemical
Co., St. Louis. MO).12-14,17,18,22,23,25
The cell
isolation procedure, used for SO and MI hearts, consisted of three main
steps: 1) calcium-free perfusion: blood wash-out and collagenase
(selected type II, Worthington Biochemical Corp., Freehold, NJ)
perfusion of the heart were carried out at 37°C with HEPES/MEM gassed
with 85% O2 and 15% N2.
2) Mechanical tissue dissociation: after the heart was removed from the
cannula, the left ventricle, inclusive of the septum, was separated
from the right ventricular free wall and minced. Pieces were
subsequently shaken in resuspension medium containing collagenase. 3)
Separation of intact cells: intact cells were enriched by
centrifugation at 30 x g for 3 minutes. Supernatant
was discarded. This procedure was repeated four times to remove
nonmyocytes, cellular debris, and residual collagenase. Myocytes were
resuspended in Percoll solution (final concentration 41%) and
centrifuged for 10 minutes at 34 x g. Intact cells
were recovered and washed. Smears were made to control the purity of
the preparation. Nonmyocytes constituted 1 to 2% of the
cells.12-14,25
The average number of myocytes obtained
from the left ventricle was 6 to 7 x 106
and 3.3 x 106
in SO and MI rats,
respectively. Myocytes were plated on a silicon rubber substrate
(Figure 1)
and stretched in an
equibiaxial strain device.17,18,26
Stretching effects on
cell injury were established by exposing cultures to ethidium monoazide
bromide (EMB; Molecular Probes, Eugene, CA). EMB binds to nucleic acids
only in cells with membrane breakage.27
Nonstretched and
stretched myocytes from noninfarcted and infarcted hearts were
evaluated at 3 and 12 hours. In some cultures, the
AT1 antagonist, losartan (Merck, Rahway,
NJ), and/or the AT2 antagonist, PD123319
(Parke-Davis, Ann Arbor, MI), were added at
10-8 mol/L 30 minutes before stretch, and kept
for the duration of the experiment. Cells were collected for
histochemical and molecular assays.17,18
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Myocyte dimensions were measured in freshly isolated binucleated myocytes with a computer system: 200 cells from each control and infarcted ventricle were analyzed to collect length, width, and area. Isolated myocytes flatten and the cross-sectional area resembles an ellipse. The minor-to-major axis ratio of the ellipse was obtained by examining 40 cells in each case by confocal microscopy. These values were used to compute myocyte volume.25
Terminal Deoxynucleotidyl Transferase and Taq Polymerase In Situ Ligation Assays
Cultures were incubated with 5 units of 2.5 mmol/L
CoCl2, 0.2 mol/L potassium cacodylate, 25 mmol/L
Tris-HCl, 0.25% bovine serum albumin, and 0.5 nmol/L
biotin-16-deoxyuridine triphosphate (dUTP). After exposure to
fluorescein-labeled Extravidin (Sigma), cytoplasm was stained by
-sarcomeric actin and nuclei by propidium iodide. Double-strand DNA
fragments for in situ ligation to 3' overhangs were prepared
by polymerase chain reaction using primers 5'-GTGGCCTGCCCAAGCTCTACCT-3'
and 5'-GGCTGGTCTGCCGTTTTCGACCCTG-3' complementary to pBluescript-bSDI1
plasmid.28,29
Digoxigenin-dUTP fragments were ligated to
DNA using T4 ligase.28,29
The number of myocytes labeled
by terminal deoxynucleotidyl transferase or Taq was
determined by examining 2000 to 3000 cells in each condition by
confocal microscopy.6,17,18,29
Western Blot
Myocytes were incubated with lysis buffer (50 mmol/L Tris/HCl, pH 7.5, 5 mmol/L EDTA, 250 mmol/L NaCl, 0.1% Triton X-100) containing protease inhibitors (0.2 mmol/L phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 5 mmol/L dithiothreitol, 1 mmol/L Na3VO4) on ice for 30 minutes. Equivalents of 50 µg of myocyte proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose and exposed to rabbit anti-human AT1 (306, Santa Cruz, Santa Cruz, CA) and goat anti-human AT2 (C-18, Santa Cruz) at a concentration of 1 µg/ml in Tris-buffered saline/Tween 20 (TBST). Bound antibodies were detected by peroxidase-conjugated anti-rabbit and anti-goat IgG. AT1 was detected as a 41-kd band and AT2 as a 44-kd band.17,18,30 Human cloned AT1 receptor (BioSignal, Montreal, Canada) was used as positive control.
Ang II
Ang II in conditioned medium was measured by enzyme-linked immunosorbent assay (Peninsula Laboratories, San Carlos, CA). Conditioned medium was treated with 10% trifluoroacetic acid and centrifuged. Supernatant was dried and dissolved in 0.1% trifluoroacetic acid. Ang II was purified using a C18 Sep-Pak column (Waters Associates, Milford, MA). Ang II fraction was eluted from the column with 30% acetonitrile in 5 ml of 0.1% trifluoroacetic acid, dried, and dissolved in 0.25 ml of TBST. Samples were analyzed in a microtiter plate using Ang II antibody (1:32,000) and a tracer, biotinylated Ang II. The microtiter plate was treated with streptavidin/horseradish peroxidase. Color reaction was developed with tetramethyl-benzidine substrate and terminated by 2 N HCl. Concentration was calculated from standard curves.18
Data Analysis
Results are presented as means ± SD. Autoradiograms were analyzed densitometrically by an image analyzer (Gel Doc 1000, Bio-Rad, Hercules, CA). Significance between two values, P < 0.05, was determined by Student’s t-test and among multiple preparations by the Bonferroni method.31 In the text and in figure legends n values for each determination are listed .
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Results |
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MI at 8 days was associated with a 6% and 7% reduction in body weight (SO: 305 ± 30 g, n = 10; MI: 286 ± 22 g, n = 25; NS) and heart weight which were not significant (SO: 914 ± 83 mg, n = 10; MI: 974 ± 90 mg, n = 25; NS). Infarcted hearts showed a threefold increase in left-ventricular end-diastolic pressure (SO: 7 ± 2.4 mmHg, n = 10; MI: 22 ± 4 mmHg, n = 25; P < 0.001) and an 18% decrease in left-ventricular systolic pressure (SO: 105 ± 8 mmHg, n = 10; MI: 86 ± 15 mmHg, n = 25; P < 0.001). Left ventricular +dP/dt was reduced 33% (SO: 9,976 ± 928 mmHg/second, n = 10; MI: 6,726 ± 902 mmHg/second, n = 25; P < 0.001) and -dP/dt 35% (SO: 8,840 ± 803 mmHg/second, n = 10; MI: 5,747 ± 934 mmHg/second, n = 25; P < 0.001). After infarction, right-ventricular end-diastolic pressure increased 2.4-fold (SO: 2.7 ± 1.8 mmHg, n = 10; MI: 6.5 ± 2.6 mmHg, n = 25; P < 0.001) and right ventricular systolic pressure 11% (SO: 27 ± 2.2 mmHg, n = 10; MI: 30 ± 3 mmHg, n = 25; P < 0.05). right ventricular +dP/dt (SO: 2,923 ± 399 mmHg/second, n = 10; MI: 2,675 ± 450 mmHg/second, n = 25; NS) and right ventricular -dP/dt (SO: 2,508 ± 258 mmHg/second, n = 10; MI: 2,330 ± 458 mmHg/second, n = 25; NS) did not change. Moreover, the volume of binucleated myocytes increased 45% (P < 0.001) with cardiac failure, from 25,300 ± 2,200 µm3 (n = 6) in controls to 36,800 ± 4,900 µm3 (n = 6) in infarcted hearts. These parameters, obtained by confocal microscopy, were the product of cross-sectional area (controls: 234 ± 19 µm2, n = 6; infarcts: 283 ± 37 µm2, n = 6; P < 0.05) and length (controls: 108 ± 6 µm, n = 6; infarcts: 130 ± 13 µm, n = 6; P < 0.005) measurements, evaluated separately in each animal.
Myocyte Stretch
A strain of 20% produced a 12% (P <
0.001) increase in sarcomere length in myocytes from SO animals, from
1.820 ± 0.022 µm to 2.037 ± 0.030 µm
(n = 61). Corresponding values in MI rats were
1.821 ± 0.024 µm and 2.040 ± 0.032 µm
(n = 61; P < 0.001). Sarcomere
length changes were constant in different preparations, but reflected
60% the extent of strain. Stretch did not increase the fraction of
myocytes with plasma membrane damage identified by EMB labeling of
nuclei (Figure 2
,A and B). At baseline,
ie, 24 hours after plating, and 3 and 12 hours later in the absence of
stretch, 0.58 ± 0.24% (n = 5), 0.68
± 0.35% (n = 5), and 0.73 ± 0.28%
(n = 5) control myocytes were EMB-positive.
Corresponding values in control stretched myocytes were 0.74 ±
0.41% (n = 5), 0.49 ± 0.33%
(n = 5), and 0.64 ± 0.39%
(n = 5). In postinfarcted nonstretched myocytes,
0.62 ± 0.32% (n = 5), 0.55 ± 37%
(n = 5), and 0.40 ± 0.22%
(n = 5) of cells were stained by EMB.
Percentages of EMB-labeled cells in stretched postinfarcted myocytes
were 0.56 ± 0.31% (n = 5), 0.66 ±
0.41% (n = 5), and 0.71 ± 0.29%
(n = 5).
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Figure 3
,A and B, illustrates the
detection of AT1 and AT2
receptor in myocytes before and after stretching. The densitometric
analysis of the changes in protein quantity is shown in Figure 3C
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AT1 protein was 1.8-fold
(P < 0.001) higher in freshly isolated myocytes
from infarcted hearts than in cells from control animals. Three hours
of stretch had no effect on the quantity of AT1
in myocytes from SO and MI rats but, at 12 hours, a 2.0-fold
(P < 0.001) and 2.4-fold
(P < 0.001) increase in
AT1 expression was detected in the two groups of
cells, respectively. Up-regulation of AT1 in
myocytes, induced in vitro by the mechanical stimulus, left
essentially intact the difference in AT1 between
SO and MI animals. Similarly, AT2 increased
1.7-fold (P < 0.001) in myocytes dissociated
from infarcted rats and stretch at 3 hours enhanced
AT2 quantity by 1.9-fold
(P < 0.001- P < 0.002) in
control and stressed cells; 12 hours of stretch resulted in a 2.2-fold
(P < 0.001) and 2.3-fold
(P < 0.001) accumulation of
AT2 in myocytes from SO and MI rats,
respectively.
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Figure 4A
illustrates that the
concentration of Ang II in conditioned medium was 42%
(P < 0.05) and 52% (P
< 0.05) higher at 3 and 12 hours in cultures of nonstretched myocytes
from MI animals than in culture of nonstretched myocytes from SO rats.
Stretch of control cells for 3 and 12 hours increased Ang II formation
66% (P < 0.005) and 91%
(P < 0.001), respectively. Corresponding
increases associated with stretch of postinfarcted myocytes were 62%
(P < 0.001) and 97% (P
< 0.001). The concentration of Ang II in conditioned medium of
stretched MI myocytes at 3 and 12 hours had values 39%
(P < 0.001) and 57% (P
< 0.001) greater than those in conditioned medium of stretched control
myocytes. Stretching of postinfarcted myocytes in the presence of
10-8 mol/L losartan, 10-8
mol/L PD123319 and losartan, or PD123319, had different consequences on
Ang II generation (Figure 4
, B and C). Losartan inhibited the synthesis
of Ang II produced by 3 and 12 hours of stretch of MI myocytes.
PD123319 did not interfere with Ang II secretion or enhance the impact
of losartan on stretch-mediated release of Ang II.
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Apoptosis was measured by confocal microscopy. Nuclei were
identified by the red fluorescence of propidium iodide, and
double-strand DNA cleavage with single-base 3' overhang was recognized
by the green fluorescence of Taq labeling. Myocytes were
distinguished by the red fluorescence of -sarcomeric actin staining.
Yellow fluorescence of nuclei reflected the combination of propidium
iodide and Taq labeling (Figure 5
,A–I). Alterations in
chromatin structure are apparent in the apoptotic myocytes shown in
Figure 5
, A–F, whereas nuclear morphology was preserved in the dying
cell illustrated in Figure 5
, G–I. The shape of the two first dying
myocytes was altered by cellular shrinkage (Figure 5
, C and F) and
partial loss of myofibrillar structures (Figure 5
C). In contrast, cell
morphology was primarily preserved in the myocyte shown in Figure 5
, G
and I. Similar observations were made when terminal deoxynucleotidyl
transferase was used.
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,A and B). However, values in MI
myocytes were 1.39-fold (P < 0.001) and
1.44-fold (P < 0.001) greater than in control
cells at 3 and 12 hours after stretch. In the absence of stretch,
baseline apoptosis was consistently higher in myocytes from MI rats.
Losartan prevented stretch-induced apoptosis in both groups of
myocytes. PD123319 had no effect on stretch-activated cell death
(Figures 6
, A and B). Because PD123319 had no impact on Ang II
formation and apoptosis, Ang II-mediated cell death most likely
occurred through ligand binding to the AT1
receptor subtype. This is consistent with results obtained by the
addition of nanomolar concentrations of Ang II to myocyte cultures
pretreated with losartan.23
Under this setting, apoptosis
was inhibited despite the presence of Ang II in the medium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AT1 and AT2 Receptors and Cardiac Myocyte Death
The role of AT1 and AT2 receptor subtypes in the activation of apoptotic death signals varies in different cell populations. Ligand binding to AT2 receptors in PC12W and R3T3 cells, which do not express AT1 receptors, leads to DNA fragmentation32 possibly through dephosphorylation of the survival factor mitogen-activated protein kinase. Mitogen-activated protein kinase phosphatase-1 dephosphorylates Bcl-2 inhibiting its antiapoptotic effect.24 AT2 receptor stimulation triggers apoptosis in vascular smooth muscle cells by antagonizing extracellular signal-regulated protein kinase (ERK) function, whereas AT1 receptor opposes the initiation of the endogenous cell death pathway.33-35 The AT1 receptor seems to prevent the impact of AT2 on ERK activity.34 Neonatal rat cardiac myocytes have been claimed to behave in a similar manner after exposure to Ang II in vitro.36,37 These observations are at variance with the results of the current study in which myocyte death mediated by Ang II occurred via AT1 receptor exclusively; AT2 receptor activation had no influence on cell viability of stretched myocytes.
Primary cultures of adult myocytes from SO and MI hearts showed a qualitatively identical response to stretch-induced synthesis and release of Ang II. Acute MI increased significantly the expression of AT1 and AT2 receptors on the remaining hypertrophied cells, but the AT2 receptor had no effect on the enhanced generation of ligand and level of myocyte apoptosis after the imposition of the mechanical stimulus. Pretreatment of myocytes from infarcted hearts with losartan blocked the formation of Ang II and the increase of cell death associated with 3 and 12 hours of stretch. Conversely, PD123319 alone did not interfere with the consequences of stretch on these cellular parameters. Moreover, the combination of losartan and PD123319 did not modify the results obtained by losartan only. Similar findings on the role of AT1 and AT2 in cell survival have previously been obtained in cultures of neonatal38 and adult20 myocytes exposed to Ang II. The discrepancy between our results and those indicated above36,37 is difficult to explain. However, our in vitro experimentation used Ang II at 10-9 mol/L,20,38 whereas concentrations 500,00036 and 50037 times higher were used in the other studies. In a comparable manner, doses of 10-7 to 10-8 mol/L of losartan and PD123319 were used by us which contrast the utilization of concentrations of these Ang II receptor antagonists 10,000,00036 and 1,00037 times greater. Our preparations of cardiac myocytes were all done in serum-free medium, but an initial 5% and a subsequent 0.5% serum, in combination with Ang II, was used in the cited work. Baseline apoptosis was markedly lower in our cultures, because the effects of changes in serum levels were avoided. Although these factors may account for some of the differences in the accumulated data, they still leave unexplained the contrasting results on the regulatory function of AT1 and AT2 receptors on myocyte death.
Myocardial Infarction, AT1 Receptor, Ang II Formation, and Myocyte Death
Myocytes surviving ischemic injury are characterized by hypertrophy1,8 and up-regulation of the local RAS which involves enhanced expression of angiotensinogen,39 renin,14 angiotensin converting enzyme40 and, as shown here in nonstretched cells, an increased formation of Ang II. Ang II binding sites in myocytes are also increased12-14 and this change in receptor density comprises both AT1 and AT2 subtypes. Additionally, these myocytes are more susceptible to apoptotic death signals; Bcl-2 quantity is reduced and Bax is increased,41 possibly through activation of p53 function.42,43 Forced induction of p53 in adult ventricular myocytes alters the Bcl-2-to-Bax protein ratio, up-regulates the local RAS, and potentiates apoptosis.22 Mechanical stretch mimics the effects of p53 overexpression on these cellular events.17
Postinfarcted hypertrophied myocytes are subjected to sarcomere elongation in vivo1,8 and this structural modification may be implicated in the changes in the local RAS detected in cultured myocytes in the absence of stretch. Moreover, these cells responded to stretch by enhancing the synthesis of Ang II and Ang II receptors. Although p53 function was not characterized in the current investigation, inhibition of the AT1 effector pathway prevented the formation and secretion of Ang II and myocyte apoptosis. This intervention may have interfered with protein kinase C phosphorylation of the C-terminal of p53,44 down-regulating the p53-dependent gene, angiotensinogen45 and the generation of Ang II. In vitro competition with mutated p53 abrogates stretch-mediated up-regulation of angiotensinogen, AT1 receptor, Ang II synthesis, and cell death despite an increased expression of renin, angiotensin converting enzyme, and AT2 receptor in myocytes.45 These observations are consistent with the notion that p53 is a major regulator of myocyte RAS and ligand-binding to AT1 receptors triggers apoptosis. Protein kinase C phosphorylation of L-type Ca2+ channels, elevation in cytosolic Ca2+, activation of Ca2+-dependent DNase I and, ultimately, DNA fragmentation have been implemented in the execution of the death process.17,20,38 Apoptosis was found here to consist of double-strand cleavage of the DNA with single-base 3' overhang which occurs only through the stimulation of Ca2+-dependent DNase I.28
The in vitro model of myocyte stretching and apoptosis requires some comments. The equibiaxial stretch apparatus leads to a stable condition of mechanical deformation of myocytes that does not apply to any in vivo state. It mimics only in part the diastolic stretch associated with increases in end-diastolic pressure and diastolic wall stress. Some similarities with the model may be found in the surviving myocardium after infarction in which lengthening of sarcomeres and a marked elevation in diastolic loading occur in the absence of alterations in the interstitial compartment two days after coronary artery occlusion.8,46 Myocyte apoptosis is the exclusive form of cell death found in this viable portion of the wall.41 Necrosis does not participate in the remodeling process of the acutely postinfarcted heart.41 However, the type of myocyte stretching used here cannot be assumed to reflect the increases in preload with anemia or dynamic exercise. Whether stretching of neonatal and adult ventricular myocytes produces comparable results is difficult to establish. Neonatal myocytes differ significantly from adult cells; a large portion of the cytoplasm is undifferentiated, DNA replication occurs in more than 10% of the cell population, and their volume is only 5% of adult cells.47
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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 January 14, 2000.
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,
difference from S-myocytes from SO hearts.