(Circulation. 1999;99:3071-3078.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Increased Cardiomyocyte Apoptosis and Changes in Proapoptotic and Antiapoptotic Genes bax and bcl-2 During Left Ventricular Adaptations to Chronic Pressure Overload in the Rat
From Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pa (G.C., R.R.); the Department of Experimental Medicine and Pathology, University "La Sapienza," Rome, Italy (G.C.); the Department of Cardiology, I.R.C.C.S., INM "Neuromed," Pozzilli (IS) (G.C., C.M., A.N., G. Sgaramella, A.d.R., B.T., G.L.); the Department of Internal Medicine, "Federico II" University, Naples, Italy (B.T., G.L.); Children's Hospital of Pittsburgh, Rangos Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pa (G. Stassi); and the Department of Surgical, Anatomical, and Oncological Sciences, Anatomy Section, University of Palermo, Italy (G. Stassi, F.F.).
Correspondence to Gianluigi Condorelli, MD, PhD, Kimmel Cancer Center (Room 1006), 233 S 10th St, Philadelphia PA, 19107. E-mail condore1{at}jeflin.tju.edu
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Abstract |
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Background—Left ventricular hypertrophy (LVH) represents both an adaptive response to increased cardiac work load and a precursor state of heart failure. Recent evidence linked cardiac myocyte death by apoptosis with LVH and heart failure. It remained unclear, however, whether apoptosis participated in the transition from LVH to left ventricular dysfunction (LVD).
Methods and Results—Cardiac myocyte apoptotic events and changes in apoptosis-specific genes were studied in a rat model of chronic pressure overload induced by transverse aortic constriction. The changes in left ventricular geometry and function were assessed by echocardiography. Transverse aortic constriction rats progressively developed "concentric" LVH and subsequently, LVD. A similar distribution of LVH and LVD was found 18 weeks after surgery. At this time point, we determined the occurrence of myocyte apoptosis by DNA laddering, in situ DNA TUNEL labeling, and light and electron microscopy. The monitoring of proapoptotic and antiapoptotic genes was determined by Western blot and immunohistochemistry. Our data demonstrated that cardiomyocyte apoptotic events increased from virtually undetectable (in sham-operated controls, SH) to 0.8/103 and 1.5/103 positive nuclei in LVH and LVD, respectively. Fibrosis also increased in the subendocardial and midwall regions of LVH and LVD rats compared with SH. Expression of the proapoptotic gene bax increased, whereas that of antiapoptotic gene bcl-2 decreased in LVH and LVD compared with SH.
Conclusions—These data suggest that in response to chronic pressure overload, cardiomyocyte-specific apoptosis contributed to the transition from LVH to LVD. LVH and LVD were accompanied by a dramatic cardiomyocyte upregulation of the proapoptotic gene bax and reduced bcl-2/bax ratio, predisposing cardiomyocytes to apoptosis.
Key Words: cells • heart failure • genes • apoptosis • hypertrophy
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Introduction |
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During heart failure (HF), the left ventricle (LV) is unable to pump blood at a rate commensurate with the metabolic requirements of peripheral tissues. An increase in imposed cardiac work load promotes an adaptive response consisting of cardiac hypertrophy. Although this response is initially compensatory, prolonged cardiac overload leads to a decreased global cardiac function and subsequently to HF. The mechanisms underlying the transition from compensatory hypertrophy to cardiac muscle dysfunction have not been completely elucidated yet. Among the several mechanisms involved, a critical role is played by loss of myocardial cells.1 Myocardial cell death may be accounted for by 2 independent mechanisms, necrosis and apoptosis (programmed cell death), which differ in several morphological and biochemical features.2 Necrosis is characterized by rapid cell swelling, plasma membrane breakdown, and a concomitant inflammatory response. It typically involves contiguous cells and is associated with tissue architectural disruption. In contrast, apoptosis generally affects scattered single cells and is identified by an enzymatic internucleosomal degradation of chromatin in the absence of membrane rupture and inflammatory response. Unlike necrosis, apoptosis is a cellular suicide, controlled by specific genetic programs, which can be activated by a wide range of physiological and pathophysiological events.2 Recent studies report that apoptosis is induced in cardiac myocytes in vivo and in vitro. Apoptosis may be a significant factor in the myocardial hypertrophy induced by pressure overload in rats,3 in spontaneously hypertensive rats,4 in the aging rat myocardium,5 in myocardial ischemia-reperfusion injury,6 and in acute myocardial ischemia in mice.7 Apoptosis is described to occur both in the early stage of LV hypertrophy3 and in overt human HF.8 9 However, little is known about the contribution of apoptosis to cardiac myocyte loss during the transition from LV hypertrophy to LV dysfunction after pressure overload.
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Methods |
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The experimental procedures performed in this study followed the guidelines of the American Physiological Society and were approved by the National Animal Care Committee.
Surgical, Echocardiographic, and Hemodynamic
Procedures
Male Wistar rats weighing 175 to 200 g (Charles River) were
anesthetized with a mixture of 120 mg/kg ketamine and
10 mg/kg xylazine, given by intraperitoneal
injection. Transverse aortic constriction (TAC) between the innominate
artery and the left carotid artery was performed.
Echocardiograms were performed on anesthetized animals (Sonos
100, equipped with a 7.5-MHz transducer, Hewlett-Packard).
Two-dimensional short-axis views of the LV were obtained. Whole studies
were recorded on half-inch S-VHS videotape (Panasonic AG7350).
Freeze-frames were printed on a color printer (UP 5000, Sony) and
analyzed with the use of the NIH image program. Anterior and
posterior end-diastolic wall thickness (AWT, PWT) and LV
diastolic and systolic internal dimensions (EDD,
ESD) were measured according to standard procedures as well as
calculations of LV mass (LVM), with the standard cube formula
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In our hands, the calculated LVM by echocardiography correlated significantly with postmortem LV weight (r=0.989, SEE=0.73, range=0.37 to 1.86 g, n=11; P<0.01). In addition, the intraobserver and interobserver coefficients of variation were 7% and 8%, respectively.
Two days after their final echocardiogram, rats were anesthetized, both right and left carotid arteries were then cannulated with a fluid-filled catheter pericardial effusion 50, catheters were connected to pressure transducers (Statham P23db), and hemodynamic data were recorded on a Gould recorder and then acquired in a computerized system by Gould's DASA. Analysis of the pressure curves was performed with the use of View II software (Gould Instruments Systems). Heart rate was obtained from the arterial pressure pulse. After arterial blood pressures were recorded, the catheter positioned in the right carotid artery was advanced into the LV under constant pressure monitoring to evaluate the LV systolic and end-diastolic pressures.
LV meridional peak systolic and end-diastolic wall stresses were estimated by use of the formula validated by Litwin et al.10 Additional information is available on request.
Tissue Histology Sections
Five animals per group were analyzed for
histological changes.
TUNEL Assay
LV were embedded in OCT (Miles Laboratories), frozen in liquid
nitrogen, and kept at -80°C until used. The LV was divided into 3
regions: base, middle and apex. Ten serial cryostat sections (4
µm) per region were then analyzed. Labeling of 3'-OH
fragmented DNA ends (TdT-mediated dUTP nick end labeling) was performed
by an in situ apoptosis detection kit, following manufacturer
instructions (In Situ Cell Death Detection, AP; Boehringer
Mannheim). Eosin was used as a counterstain. Photography was performed
with a Zeiss microscope.
Immunofluorescence
The TUNEL staining was performed with the use of
fluorescein-dUTP, omitting the AP-converting step. Sections
were then exposed to 1 µg/mL of phalloidin-TRITC label (Sigma) for 20
minutes at room temperature and washed in PBS twice for 3 minutes.
Sections were incubated with 10 µmol/L of a chromatin dye
(Hoechst 33342, Molecular Probes Inc) for 8 minutes to stain nuclei.
The slides were analyzed with the use of confocal laser
scanning microscopy (Fluoview+IX70, Olympus) under appropriate
wavelength.
Tissue Histology and Electron Microscopy
Ultrastructural analysis of morphological changes for
evidence of apoptosis and replacement and
interstitial fibrosis was performed on fixed specimens. The
embedding was performed in epoxy resin (Epon 812) and polymerized at
60°C overnight.11 Semithin sections (0.5 µm) cut
in an Ultracut-E (Reichert-Young) were mounted onto slides and stained
by warm methylene blue. Screening and photography were performed with
the use of Zeiss microscopy.
Ultrathin sections were mounted on nickel grids and counterstained with uranyl acetate for 10 minutes and lead acetate for 1 to 2 minutes. The specimens were analyzed with the use of a JEOL 1220 electron microscope. Necrosis was calculated as area of fibrosis, according to the point-counting method.12 The staining for the connective tissue was performed with the use of a modified basic trichrome technique on myocardial semithin sections embedded in epoxy resin.
Immunohistochemistry
The expressions of bcl-2, bax, and
bcl-xl were determined following manufacturer instructions
(Vectastain Quick Kit, Vector). Serial frozen heart sections were used
for this staining; 3-amino-9-ethylcarbazole was used as a
colorimetric substrate.
In Vitro DNA Extraction and Labeling
Extraction of DNA from 30 µm LV sections of 30 µm
was performed as described.7 Five microliters of each DNA
and 1 µg of 1 kb DNA marker were incubated with 10 µCi of
(
32P)dCTP (Amersham), a mix of cold dNTPs
(-dCTP), and 10 U of Klenow polymerase (Gibco) for 15 minutes at
30°C. The reactions were stopped with 10 mmol/L EDTA. Samples
were then run on 1.5% agarose gel, blotted onto a Magna nylon membrane
(Micron Separations), and exposed to Kodak X-OMAR x-ray film for 1 to 2
hours at room temperature.
Western Blots
Antibodies against rat bcl-2, bax, and
bcl-xl were purchased from Santa Cruz Biotechnology.
Anti-rat ANF was from Penynsula Inc and was used at the following
dilutions 1:500 ANF; 500 ng/mL for bcl-2, bcl-xl,
and bax.
LV was lysed and 30 µg/lane of proteins were loaded on gel. Western blots, done in triplicate, were developed with the ECL kit (Amersham). Additional information is available on request.
Statistical Analysis
Results are shown as mean±SEM. We identified 2 main
adaptive LV phenotypes on the basis of morphological and
functional parameters. LV hypertrophy (LVH) was
defined by an LVM increase of at least 2 SD of that recorded in
sham-operated controls (SH), with a preserved LV function (LVFS
comparable with that observed in SH). In contrast, LV dysfunction (LVD)
was characterized by a deterioration of LV function (LVFS decrease of
at least 2 SD of that observed in SH) independently by LVM value. All
tissue samples were analyzed in a blind fashion, and the LV
condition was unknown before statistical analysis was
performed. One-way ANOVA and 2-way ANOVA were performed to estimate the
changes of hemodynamic and histological
data. Post hoc simultaneous multiple comparisons were done
with Bonferroni analysis.
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Results |
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Hemodynamics
Two main adaptive phases are detectable during chronic pressure overload in the LV: The first is characterized by concentric LVH; the second is distinguished by LVD. To estimate the percentage of each of these 2 LV phenotypes throughout the study, we assessed the distribution of both LVH and LVD at each time point. There was a net prevalence of LVH (83%) at 12 weeks compared with LVD phenotype, whereas at 18 weeks a similar distribution of both phenotypes was observed (LVH 55% vs LVD 45%). Mostly LVD phenotype was detectable at 24 weeks (>90%).
Eighteen weeks after surgery, arterial blood pressure
recorded in the right carotid of LVH and LVD rats was comparable
and, as expected, markedly higher than that measured in SH rats
(Table
). Even LV
end-diastolic pressure was markedly increased in LVH and
LVD as compared with SH, and a significant difference was detectable
between the 2 groups of TAC rats. The analysis of the LV wall
stress revealed that LV diastolic wall stress was higher in
both groups of TAC rats compared with SH. In contrast, LV
systolic wall stress was significantly elevated in LVD compared
with both SH and LVH rats.
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Evaluation of Interstitial and Replacement
Fibrosis
The extent of fibrosis was determined in the subendocardium,
midwall, and subepicardium of SH, LVH, and LVD animals. Whereas
interstitial and replacement fibrosis were virtually absent
in SH animals, interstitial fibrosis was present in the
subendocardial region in LVH and represented 0.125 of a
0.33 mm2 observational area. Replacement
fibrosis represented 0.030 of a 0.33
mm2 observed area in the same region. In the
midwall region of LVH, interstitial fibrosis
represented an area of 0.05 of a 0.33
mm2, whereas replacement fibrosis was absent. The
epicardial region showed no fibrotic process. In LVD, although the
epicardial region was spared, the endocardial region was affected by
the fibrotic process to 0.033 of 0.33 mm2,
whereas interstitial fibrosis represented
20% of the fibrotic process. Interstitial edema also
was significant in the subendocardial region in LVD. In LVD as opposed
to LVH, areas of recent necrosis, recognized as pale with methylene
blue staining, also were present (Figure 1).
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In both LVH and LVD, the LV free wall was more affected then the septal region, which was almost completely spared from the fibrotic process.
DNA Laddering
Analysis of genomic DNA obtained by agarose gel
demonstrated DNA degradation in both LVH and LVD compared with the
control SH group. In addition, there was a marked increase of
low-molecular-weight DNA fragments in LVH and LVD compared with SH
(Figure 2). The density of the DNA ladder
from LVD hearts was higher than that measured in LVH. In addition, the
pattern of DNA laddering supports both an apoptotic
(internucleosomal) and nonspecific, necrotic-type DNA
degradation.13
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Evaluation of Cardiac Myocyte Apoptosis
Analysis of the number of cardiomyocyte
nuclei with DNA fragmentation detected by TUNEL increased progressively
from SH to LVD. In particular, the number of TUNEL-positive nuclei
increased in LVH and LVD compared with SH. Cardiac myocyte nuclei can
be recognized because they have an elliptical shape on longitudinal
sections and are included within myofibers (Figure 3, white arrowheads). We calculated that
the number of myocytes undergoing apoptosis increased from
approximately undetectable to 0.06±0.01 to 0.11±0.03 nuclei/0.33
mm2, respectively (P>0.05). Because
there are 72±11 cardiomyocyte/0.33
mm2, we calculated that
0.8/103 and 1.5/103 cells
scored positive for TUNEL. Nuclear damage in cardiac myocytes
undergoing apoptosis was confirmed by electron microscopy
analysis, which showed nuclear collapse and the presence of
apoptotic bodies within the nucleus in LVH and LVD but not in
SH (Figure 4). The number of nuclei of
nonmyocytes stained for TUNEL were 0.15±0.02/0.33
mm2 in LVH and 0.15±0.02/0.33
mm2 in LVD, whereas in SH animals the number of
nuclei of nonmyocyte was almost undetectable.
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A triple fluorescence staining was also performed in which
nuclei were identified by Hoechst and
immunofluorescence; the cytoplasm of
cardiomyocytes was recognized by typical staining with
phalloidin, which reacts with actin fibers (Figure 5). These results confirmed the number of
cardiomyocytes considered positive on TUNEL.
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Changes in Expression of Proapoptotic Gene
bax and Antiapoptotic Gene bcl-2
in LVH and LVD
bcl-2 was the first gene to be identified of a family
that includes molecules sharing sequence homology and with
proapoptotic and antiapoptotic
activity.14 bcl-2 gene expression in
cardiomyocytes has been shown to decrease in
physiopathological states associated with increased stretching of
myocardial cells in vitro15 or in ischemic
myocardium in vivo.16 Under similar
conditions, the expression of bax was
upregulated.15 16 Therefore the ratio between
bcl-2 and bax expression has been proposed as an
important marker of myocardial cell survival probability. To determine
whether the increase in cardiomyocyte apoptosis
rate in LVH and LVD was accompanied by a change of the ratio between
bcl-2 and bax, Western blot and
immunohistochemical analyses were conducted. Results show a
relative decrease in bcl-2 and an increase in bax
expression in LVH and LVD compared with SH. In contrast,
bcl-xl levels did not change significantly. ANF protein
levels, induced in ventriculocytes subject to pressure overload, were
upregulated in LVH and LVD, as expected (Figure 6).
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Because changes in cardiac expression of the apoptotic genes
determined by Western blot from whole tissue cardiac extracts might not
reflect changes in cardiomyocyte specific genes, expression
of bcl-2, bax, and bcl-xl was also
determined by immunohistochemistry. The results confirmed the data
obtained by Western blot, because the number of bax-positive
cells was markedly increased in LVH and LVD and peaked in LVD, whereas
bcl-2/bax ratio was reduced in LVH and LVD
compared with SH (Figure 7). Also, the
number of bcl-xl–positive cells was equal in the 3
conditions (not shown). These experiments demonstrated that the
bcl-2/bax ratio was decreased in LVH and LVD
compared with SH, and bax expression was upregulated in
almost all cardiocytes under these conditions. LVD hearts
showed maximal levels of bax expression.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This model of cardiac pressure overload allows determination of an initial compensatory phase characterized by concentric LVH followed by an enlargement of cardiac chambers associated with a further deterioration of LV function. Twelve weeks after TAC, we detected LVH almost exclusively, whereas at 24 weeks there was a net prevalence of LVD. Similar distribution of the 2 types of LV response to pressure overload was observed 18 weeks after TAC, the time interval selected for histopathological analysis. TUNEL analysis showed a moderate increase of cardiomyocyte apoptosis in LVH. In LVD, the rate of apoptotic events was further increased compared with either LVH or SH. Triple fluorescent staining confirmed TUNEL data. In this model of HF, apoptosis is localized in the subendocardial-midwall region in LVH and LVD. In addition, cardiomyocyte apoptotic figures are mainly present in areas in which interstitial and replacement fibrosis are more abundant. In both LVH and LVD, apoptosis was confined within the free wall of the LV, whereas the septal region was almost spared. Our model cannot discriminate between the single contributions of apoptosis and necrosis in determining HF. It does suggest, however, that these 2 phenomena are tightly linked. Moreover, we did not determine the rate of apoptosis throughout the course of the study but at 1 point only (18 weeks). Therefore the contribution of cardiomyocyte apoptosis to the pathogenesis of HF might be underestimated.
Our data share similarities with the pathological analysis conducted on spontaneously hypertensive rats during the LVD phase.4 LVD was accompanied by increased cardiac apoptosis, which was more frequent in the free wall than in the septal region of the LV and was associated with subendocardial fibrosis. The number of apoptotic cells reported by these investigators is within the range described in the current study. A different myocardial wall stress in our rats could account for the small difference in the number of apoptotic cells between the 2 studies.
Quantitation of the expression of genes involved in the apoptotic pathway might represent a good index of the probability for a cell to undergo apoptosis. The proapoptotic bax gene shares structural similarities with bcl-2 and is thought to inactivate bcl-2 by binding to it.14 The number of bax-expressing cells in our study dramatically increases in LVH and LVD, whereas a tendency toward a decrease in bcl-2 expression is also revealed. Interestingly, bcl-xl (antiapoptotic) levels did not change. Although the threshold of bcl-2/bax ratio at which apoptosis is triggered in the cardiac cell is not known, our data, together with others,15 16 suggest that a decreased bcl-2/bax ratio increases the probability for a myocardial cell to undergo apoptosis in LVH and LVD. The role of other proapoptotic and antiapoptotic genes needs further investigation.
Recent studies indicate that trophic factors or cytokines might
influence the rate of cardiomyocyte survival and could be
an important determinant in cardiomyocyte
apoptosis. In fact, transgenic mice overexpressing insulin-like
growth factor-1 (IGF-1) show a better survival rate after
coronary occlusion trials.17 The beneficial
effects of IGF-1 in enhancing cardiac function during the onset of
cardiac failure also was demonstrated in rats.18 Other
reports demonstrated the hypertrophic19 or
proapoptotic20 21 effect of tumor necrosis
factor- on cardiomyocytes in rodents.
Possibly, pressure overload activates specific signal
transduction pathways in an autocrine-paracrine way, which triggers the
apoptotic cascade. In fact, it has been shown that Gq, a key
postreceptor signal transduction molecule activated by AT1,
ET1, and 1-adrenergic receptors in cardiomyocytes, is
able to trigger the apoptotic program in
cardiocytes.22 Moreover, cardiac-selective
overexpression of Gq in transgenic mice induces
hypertrophy23 and HF.22 Results
of these studies suggest a scenario in which the hypertrophic cell is
more prone to apoptosis and that similar signal transduction
pathways are involved in cardiomyocyte
hypertrophy and apoptosis
In conclusion, our results support the hypothesis that apoptosis is involved in the transition from compensated LVH to LVD. Whether or not apoptosis is the primary event leading to LVD during chronic pressure overload cannot be answered by our data. Whatever the initial event, our data point out the possibility that apoptosis in LVH may further contribute to the progression toward HF.
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Acknowledgments |
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This work was supported by grants from Fondo 1%, Ministero della Sanitá (Italy), Progetto Terapia Genica-ISS, AIRC (to Dr Condorelli), and funds of Thomas Jefferson University. Dr Condorelli would like to thank Carlo M. Croce for his support. Dr Condorelli dedicates this work to his mother.
Received November 12, 1998; revision received March 9, 1999; accepted March 9, 1999.
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cardiomyocyte apoptotic events and the changes of
antiapoptotic and proapoptotic genes
bcl-2 and bax in a rat model of left
ventricular pressure overload–induced heart failure. In
particular, cardiomyocyte apoptosis and changes in
apoptotic genes were evaluated during the transition from left
ventricular hypertrophy (LVH) to left
ventricular dysfunction (LVD). Our data show that
apoptosis is correlated to the extent of necrosis, evaluated as
fibrosis, in LVH and LVD. Although virtually absent in sham animals,
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overload–induced heart failure.
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G. Yaniv, M. Shilkrut, R. Lotan, G. Berke, S. Larisch, and O. Binah Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas (CD95/Apo-1) receptor: Fas activation and apoptosis in hypoxic myocytes Cardiovasc Res, June 1, 2002; 54(3): 611 - 623. [Abstract] [Full Text] [PDF]
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G. CONDORELLI, J. K. AYCOCK, G. FRATI, and C. NAPOLI Mutated p21/WAF/CIP transgene overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidation-sensitive mechanisms, and restenosis in hypercholesterolemic apolipoprotein E knockout mice FASEB J, October 1, 2001; 15(12): 2162 - 2170. [Abstract] [Full Text] [PDF]
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S. Adachi, H. Ito, M. Tamamori-Adachi, Y. Ono, T. Nozato, S. Abe, M.-a. Ikeda, F. Marumo, and M. Hiroe Cyclin A/cdk2 Activation Is Involved in Hypoxia-Induced Apoptosis in Cardiomyocytes Circ. Res., March 2, 2001; 88(4): 408 - 414. [Abstract] [Full Text] [PDF]
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S. Gupta, A. J.C Prahash, and I. S Anand Myocyte contractile function is intact in the post-infarct remodeled rat heart despite molecular alterations Cardiovasc Res, October 1, 2000; 48(1): 77 - 88. [Abstract] [Full Text] [PDF]
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P. M. Kang and S. Izumo Apoptosis and Heart Failure : A Critical Review of the Literature Circ. Res., June 9, 2000; 86(11): 1107 - 1113. [Full Text] [PDF]
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H. J. Oskarsson, L. Coppey, R. M. Weiss, and W.-G. Li Antioxidants attenuate myocyte apoptosis in the remote non-infarcted myocardium following large myocardial infarction Cardiovasc Res, February 1, 2000; 45(3): 679 - 687. [Abstract] [Full Text] [PDF]
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