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1Center for Perinatal Biology, Department of Physiology and Pharmacology, and 2Department of Microbiology/Molecular Genetics, Loma Linda University School of Medicine, Loma Linda, California 92350
Submitted 3 January 2003 ; accepted in final form 13 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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protein in fetal hearts. Chronic hypoxia
significantly increased the percentage and size of binucleated myocytes and
increased apoptotic cells from 1.4 ± 0.14% to 2.7 ± 0.3% in the
fetal heart. In addition, the active cleaved form of caspase 3 was
significantly increased in the hypoxic heart, which was associated with an
increase in caspase 3 activity. There was a significant increase in Fas
protein levels in the hypoxic heart. Chronic hypoxia did not change Bax
protein levels but significantly decreased Bcl-2 proteins. In addition,
chronic hypoxia significantly suppressed expression of heat shock protein 70.
However, chronic hypoxia significantly increased expression of the
anti-apoptotic protein 14–3-3 
, among other 14–3-3
isoforms. Chronic hypoxia differentially regulated 
-adrenoreceptor
(-AR) subtypes with an increase in 1-AR levels but no
changes in 2-AR. The results demonstrate that maternal
hypoxia increases apoptosis in fetal rat heart, which may be mediated by an
increase in Fas and a decrease in Bcl-2 proteins. Chronic hypoxia-mediated
increase in 1-AR and decrease in heat shock proteins may also
play an important role in apoptosis in the fetal heart.
fetus
Apoptosis is a highly selective process controlled and regulated by
intracellular signal transduction that involves the activation of cysteine
proteases known as caspases, resulting in protein cleavage and breakdown of
DNA molecules. Overexpression of caspase 3 in the mouse heart increased
myocardial cell death and depressed cardiac function
(6). On the other hand,
inhibition of caspase 3 reduced myocyte cell death
(49). Although it is likely
that multiple mechanisms are involved in the regulation of apoptosis, several
studies (15,
41,
48) have suggested that
hypoxia-induced apoptosis in cultured neonatal rat cardiomyocytes is mediated
by the Fas death receptor pathway. In addition, hypoxia-induced apoptosis in
the rat heart was associated with a significant change in the Bcl-2/Bax ratio
(16), and overexpression of
Bcl-2 protected against hypoxia-induced cell death
(12,
37). Although it is well known
that the adult heart may protect itself against physiological stresses,
including hypoxia, by upregulating heat shock proteins (HSP), such as HSP70,
which protects the heart from apoptosis
(38), it is unknown whether
the fetal heart has the similar response. In addition, little is known about
the effect of hypoxia on -adrenoceptors (-AR) in the fetal heart,
although it has been shown that 2-AR protects cardiomyocytes
from hypoxia-induced apoptosis
(4,
51).
In the present study, we tested the hypothesis that prenatal chronic hypoxia increased apoptotic cell death in the fetal heart. The aims of this study were the following: 1) to determine whether in vivo maternal chronic hypoxic exposure during pregnancy increases apoptosis in the hearts of near-term fetal rats, and 2) investigate whether prenatal chronic hypoxia alters the expression of pro- and anti-apoptotic proteins in the fetal rat heart.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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40 mm/min) was adjusted to bring the percentage of oxygen in
each hypoxic chamber to 10.5, which was maintained and monitored continuously
with an oxygen analyzer (model OM-14, Sensormedics; Anaheim, CA). Experiments
from our laboratory have shown that an ambient O2 level of 10.5%
lowers maternal arterial PO2 to 50 mmHg
(34). The normoxic group was
housed identically with just air flowing through the chambers. Food and water
were provided as desired. Pregnant dams were euthanized by cervical
dislocation on day 21 (term = day 22) of gestation, and
fetal hearts were isolated. For tissue slide preparation, the fetal hearts
were fixed in 10% buffered formalin and embedded in paraffin. For the other
studies, fresh tissues were used. All procedures and protocols used in the present study were approved by the Institutional Animal Care and Use Committee of Loma Linda University and followed the guidelines put forward in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.
Myocyte measurements. Cardiomyocytes were isolated from 21-day-old
fetal rat hearts as described previously
(46). Cells were cultured at
37°C in 95% air-5% CO2 incubator for 24 h. Over 95% of the
cells manifested spontaneous contractions. Cells were then fixed with 4%
paraformaldehyde solution for 30 min at room temperature, washed with PBS, and
incubated with permeabilization solution (0.1% Triton X-100 and 0.01% sodium
citrate) for 7 min at room temperature. Cardiomyocytes were double stained
with the use of -cardiac sarcomeric actin monoclonal antibody labeled
with FITC-conjugated secondary antibody and Hoechst 33258 for staining nuclei.
To measure the myocyte size, cells were viewed and photographed by microscope
with the SPOT digital camera, and cell sizes were measured with computerized
planimetry (Image-Pro Plus) in a double-blind manner. To count and measure
sizes of binucleated and mononucleated cells, a total of 1,319 cells from 12
control fetal hearts and a total of 1,134 cells from 12 hypoxic fetal hearts
were analyzed.
Quantitative analysis of apoptotic cells. Fluorescent DNA binding
dyes were used to define nuclear chromatin morphology as a quantitative index
of apoptosis, as described previously
(47). The fetal heart was
sectioned (4 µm thick) horizontally at two positions: apical and middle.
Six sections from each heart were analyzed for the presence of apoptosis. The
tissue sections were deparaffinized with xylene and rehydrated with graded
dilutions of ethanol in water. The tissue sections were then stained with
Hoechst 33258 (Sigma; St. Louis, MO) at 8 µg/ml for 10 min. To confirm the
myocyte location of apoptosis, a combination of nuclear Hoechst 33258 staining
and -cardiac sarcomeric actin staining with the monoclonal antibody was
used in the same tissue sections. The nuclei with DNA fragmentation stained
blue amid the surrounding green color of actin staining developed by
FITC-conjugated second antibody, and nuclei without DNA fragmentation had
clear nuclear regions. The nuclear morphology was examined by fluorescence
microscopy. Individual nuclei were visualized at x400, and cells were
scored as apoptotic if they exhibited unequivocal nuclear chromatin
condensation and/or fragmentation. Sample identity was concealed during
scoring. To quantify apoptosis, a total of 2,000 nuclei from each heart were
analyzed, and apoptotic cell counts were expressed as a percentage of the
total number of nuclei counted.
DNA fragmentation determination by ELISA. Apoptosis in the heart was also determined as DNA fragmentation, quantified by specific determination of cytosolic mononucleosomes and oligonucleosomes with the use of an ELISA kit (Boehringer Mannheim) as described previously (33). Briefly, tissue samples were put into the 500-µl lysis buffer supplied in the kit, homogenized in a tissue grinder, and incubated for 30 min at room temperature. After centrifugation at 200 g for 10 min, the supernatant (cytosolic fraction) was further diluted 40-fold in PBS, and used as the antigen source in the sandwich ELISA. The absorbance was measured at 405/490 nm, and the background value of the immunoassay was subtracted.
Western blotting analysis. The tissues were cut into pieces and
homogenized in a glass-glass tissue grinder in 5 vol of cold lysis buffer
composed of 20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM
EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml
aprotinin, and 10 µg/ml leupeptin, followed by incubation on ice for 30
min. The homogenates were ultrasonicated, followed by centrifugation
(Eppendorf model 5417R) at 14,000 revolutions/min (20,200 g) for 30
min at 4°C. Proteins were quantified in the supernatant with the Bio-Rad
Protein Assay Kit II (Bio-Rad; Hercules, CA). Samples with equal protein were
loaded on SDS-polyacrylamide gel (from 7.5 to 12%) and were separated by
electrophoresis at 100 V for 1 h. Proteins were then transferred onto
Immobilon-P membrane and were probed with the primary antibodies that
recognize hypoxia-inducible factor-1 (HIF-1; Novus Biological;
Littleton, CO), 1-AR, 2-AR, Fas, caspase-3,
14–3-3 isoforms, Bcl-2, Bax, and HSP70 (Santa Cruz Biotechnology; Santa
Cruz, CA), respectively. After being washed, the membranes were incubated for
1 h with the horseradish peroxidase-conjugated secondary antibodies (Amersham;
Arlington Heights, IL), and proteins were then visualized with an enhanced
chemiluminescence detection system. Results were quantified with Kodak
Electrophoresis Documentation and Analysis System and Kodak 1D Image Analysis
Software.
Caspase activity assay. The activities of caspase-3 and -8 were determined with the use of the corresponding caspase activity detection kits (R&D Systems) as described previously (47). Briefly, 100-µg proteins isolated from the fetal hearts were added to the 50-µl reaction buffer and 5-µl substrates of Acetyl-Asp-Glu-Val-Asp-p-nitroanilide (for caspase-3) and Ile-Glu-Thr-Asp-p-nitroanilide (for caspase-8), respectively. Samples were incubated at 37°C for 8 h, and the enzyme-catalyzed release of p-nitroanilide was measured at 405 nm with the use of a microtiter plate reader. The values of hypoxic samples were normalized to the controls, allowing determination of the fold increase in caspase activity.
Statistical analysis. Data were expressed as means ± SE and were analyzed by Student's t-test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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protein expression in the fetal heart. As shown in
Fig. 1, HIF-1 was
detected in the fetal hearts of maternal hypoxia but not in the control
hearts. Figure 2 shows examples
of freshly isolated fetal myocytes stained with anti--cardiac
sarcomeric actin antibody conjugated to FITC and the Hoechst 33258 stain for
nuclei. Freshly isolated myocytes were predominately (95%) mononucleated cells
with minimum (5.3 ± 0.4%) binucleated cells. Chronic hypoxia
significantly increased binucleated cells to 7.5 ± 0.6%
(Fig. 2). As expected,
binucleated cells were 65% larger than mononucleated cells. Chronic
hypoxia did not affect the sizes of mononucleated cells but significantly
increased the sizes of binucleated cells by 13%
(Fig. 2).
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Effect of chronic hypoxia on fetal heart apoptosis. Assessment of nuclear chromatin morphology by the Hoechst 33258 staining using fluorescence microscopy indicated that chronic hypoxia significantly increased condensed, coalesced, and segmented apoptotic nuclei in the fetal heart (Fig. 3A). Quantification of the hypoxia-induced apoptotic nuclei defined by the fluorescent DNA-binding dye Hoechst 33258 demonstrated a significant increase in apoptotic cell death from 1.4% in the control heart to 2.7% in the hypoxic heart. Apoptosis was further assessed by quantitative determination of fragmented DNA into mononucleosomes and oligonucleosomes determined by an ELISA specific for cytosolic histone-bound DNA, as previously reported in rat hearts. Consistent with the results from the Hoechst 33258 staining, chronic hypoxia significantly increased DNA fragmentation in the fetal heart (Fig. 3B). Hypoxia-induced apoptosis was further demonstrated by Western blot analysis, showing a significant increase in the active, cleaved form (10 kDa) of caspase 3 in the hypoxic heart compared with the control heart (Fig. 4). This was associated with a significant increase in the activity of caspase 3 in the hypoxic hearts (Fig. 5). In addition, there was a significant increase in caspase 8 activity in the hypoxic hearts, compared with the control hearts (Fig. 5).
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Effect of chronic hypoxia on apoptotic-associated proteins. To
elucidate the potential mechanisms underlying the hypoxia-induced apoptosis in
the fetal heart, we determined the effect of chronic hypoxia on Fas protein
expression in the fetal heart. Previous studies in cultured neonatal rat
cardiomyocytes suggested an involvement of Fas death receptor pathway in
hypoxia-induced apoptosis. As shown in Fig.
6, Fas was undetectable in the control heart of near-term fetal
rats. Chronic hypoxia markedly increased Fas expression in the fetal heart
(Fig. 6). Given that Bcl-2
family proteins are important modulators of cardiac myocyte apoptosis, and the
relative concentrations of pro-apoptotic (e.g., Bax) and anti-apoptotic (e.g.,
Bcl-2) proteins act as a rheostat for the cell death program, we measured Bax
and Bcl-2 protein levels in the control and hypoxic fetal hearts. Both Bax and
Bcl-2 proteins were expressed in near-term fetal rat hearts, with a
significantly higher level of Bcl-2 than that of Bax in the control hearts
(Fig. 7). Chronic hypoxia had
no effect on Bax protein levels but significantly decreased Bcl-2 protein
levels (Fig. 7). As a
consequence, chronic hypoxia increased the Bax/Bcl-2 ratio in near-term fetal
rat hearts. In addition, chronic hypoxia differentially regulated
anti-apoptotic protein 14–3-3 isoforms in the fetal heart. Among the
four isoforms examined, 
, 
, , 
, chronic hypoxia
selectively increased 14–3-3 protein levels in near-term fetal
rat hearts (Fig. 8).
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Effect of chronic hypoxia on HSP70 and -AR. As
shown in Fig. 9, there was a
constitutive expression of HSP70 in near-term fetal rat hearts. Chronic
hypoxia significantly decreased protein levels of HSP70 in fetal hearts
(Fig. 9). In addition, chronic
hypoxia differentially regulated -AR subtypes in the fetal heart. There
was no difference in 2-AR protein levels between the control
and hypoxic hearts (Fig. 10).
In contrast, 1-AR protein levels were significantly increased
in the hypoxic heart, compared with that in the control heart
(Fig. 10).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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, suggesting the
hypoxic myocardium in fetal rats. Given the finding that hypoxia induced
apoptosis in cultured neonatal rat cardiomyocytes
(24,
41,
48), it is likely that there
is a connection between myocardial hypoxia and cardiomyocyte apoptosis in
fetal rat hearts in the present study. Apoptosis in the fetal heart was clearly demonstrated by morphological changes as condensed and fragmented apoptotic nuclei and the biochemical hallmark of DNA fragmentation. Further support for hypoxia-induced apoptosis came from the finding that hypoxia increased the active, cleaved form of caspase 3 and its activity in the fetal heart, which is a unique feature of apoptotic cell death. Apoptosis in fetal rat and mouse hearts has been demonstrated with the TdT-mediated dUTP nick end labeling (TUNEL) method, which stains in situ DNA breaks in individual nuclei in tissue sections (1, 40, 42). However, the TUNEL method suffers from the lack of selectivity for apoptotic over nonapoptotic and caspase-independent cell death. In addition, active gene transcription can also result in TUNEL positive (19). In our recent study (47), we developed a method by using the fluorescent DNA-binding dye Hoechst 33258 to stain tissue sections, which allowed us to distinguish the normal uniform nuclear pattern from the characteristically condensed and/or fragmented chromatin pattern of apoptotic cells. With this method, the present study demonstrated 1.4% apoptotic cells in fetal rat heart of 21 days of gestation, which is in agreement with our previous findings (47). Given the possibility that some of the apoptotic cells had already been cleared by the time the heart sections were obtained, hypoxia-induced apoptosis in the fetal heart may be underestimated in the present study.
The present finding of decreased fetal body weight and increased fetal
heart-to-body weight ratio is consistent with our previous studies that 2-day
chronic hypoxic exposure during gestation caused fetal asymmetric growth
restriction (44). In
chronically anemic fetal sheep, an increase in HIF-1 in the heart and
an increase in fetal heart-to-body weight ratio has been shown
(25). Similar findings of
decreased fetal body weight were observed in maternal cocaine administration
(47). However, cocaine did not
affect fetal heart-to-body weight ratio, suggesting that cocaine may have
direct effects on fetal heart, other than causing fetal hypoxia. Given that
hypoxia increased cell death in the fetal heart, it is speculated that the
hypoxia-induced asymmetric enlargement of the fetal heart is due to cardiac
hypertrophy in the present study. It has been shown that the maturation
process of cardiomyocytes in rodents is marked by binucleation over postnatal
days 4–12
(22,
39). In the present study,
almost all myocytes are mononucleated in term fetal rats. Although we cannot
exclude cardiomyocyte hyperplasia in response to chronic hypoxia, we found
significant increases in the percentage and size of binucleated myocytes in
hypoxic fetal hearts, suggesting a premature exit of the cell cycle of
cardiomyocytes and myocyte hypertrophy. We have demonstrated that chronic
hypoxia increases the level of cytochrome c, a mitochondrial marker
protein, in the fetal heart, which is likely to be a metabolic adaptation in
cardiac muscle during asymmetrical enlargement of the heart
(44). It has been demonstrated
in adult animals that during the early stages of cardiac hypertrophy there are
disproportionate accumulation of mitochondria with respect to other cellular
components and specific increases in the synthesis of mitochondrial
cytochromes, leading to a stimulation of mitochondrial biogenesis
(32,
50).
It is likely that multiple mechanisms may be involved in hypoxia-induced apoptosis in the fetal heart. Although it has been well known that Fas is a widely expressed cell surface receptor that can initiate apoptosis when activated by its ligand (FasL), the expression and regulation of Fas in the fetal heart are less clear. In the present study, we found that Fas levels were very low or undetectable in control near-term fetal rat heart. Chronic hypoxia markedly increased Fas levels in the fetal heart. Hypoxia-induced increase in Fas mRNA and protein levels has been demonstrated in neonatal rat cardiomyocytes (41, 48). By upregulating Fas expression, hypoxia predisposed cardiomyocytes to Fas-induced apoptosis (48). Recent studies (20) have provided direct evidence that activation of Fas by overexpression of FasL can induce apoptosis both in cultured myocytes and in the myocardium of intact animals but not in lymphoproliferative animals that lack functional Fas. These results suggest that the upregulation of Fas plays an important role in chronic hypoxia-induced apoptosis in the fetal heart. This is further supported with the finding that the activity of caspase 8 was significantly increased in the hypoxic hearts in the present study. Caspase 8 has been involved in Fas-mediated apoptosis (2).
The finding that both Bcl-2 and Bax were expressed in fetal rat heart with higher levels of Bcl-2 is in agreement with the previous results in human, mouse, and rat embryonic hearts (1, 23, 47). It has been demonstrated that Bcl-2 mRNA levels are high in fetal rat heart, decrease markedly at 1 and 5 days postnatally, and progressively increase at 11 and 21 days, which is inversely related to apoptosis in the heart (17). In the present study, chronic hypoxia significantly decreased Bcl-2 protein levels, which may play an important role in the hypoxia-induced apoptosis in the fetal heart. Our previous studies (47) demonstrated that cocaine-induced apoptosis in the fetal rat heart was associated with a decrease in Bcl-2 and an increase in Bax. Similar findings were obtained from the adult rat heart, in which the induction of apoptosis in chronic hypoxic hearts correlated with a significant decrease of Bcl-2 protein levels and an increase of Bax protein expression (16). It has been shown that overexpression of Bcl-2 protects cardiac myocytes from apoptosis (18). In contrast, overexpression of Bax in the ventricles of spontaneously hypertensive rat hearts may contribute to apoptosis (7). The present finding of no change in Bax protein levels does not necessarily preclude the potential role for Bax in hypoxia-induced apoptosis in the fetal heart. It has been documented that one of the crucial steps before Bax can exert its proapoptotic activity is its translocation from the cytosol to the mitochondria and induction of cytochrome c release, and hypoxia and Fas induces Bax mitochondrial translocation (28, 36).
The finding that chronic hypoxia selectively upregulated the
isoform of 14–3-3 proteins in the fetal heart is intriguing and suggests
a compensatory protection mechanism for the fetal heart on hypoxic insult.
Among other functions, 14–3-3 proteins exert anti-apoptotic effects by
binding to proapoptotic proteins such as Bad and Bax, and prevent their
translocation to mitochondria
(8,
30). There are multiple
isoforms of 14–3-3 proteins, but there is a lack of isoform-specific
interactions with their targets. Instead, the interaction of 14–3-3 with
target proteins is regulated by fluctuation of total 14–3-3 pool levels
via unique transcriptional controls for each isoform
(8). Isoform-specific
expression of 14–3-3 proteins appears to be a normal part of cellular
response to insults. It is likely that upregulation of 14–3-3
isoform represents a myocardial adaptive mechanism to hypoxia-induced
apoptosis in the fetal heart.
In the present study, we found that chronic hypoxia decreased HSP70 protein
levels in fetal heart. It has been well known that the adult heart may protect
itself against physiological stresses, including hypoxia, by upregulating the
heat shock the protein HSP70
(38). To the best of our
knowledge, the present study is the first study to demonstrate that chronic
hypoxia decreases HSP expressions in the heart. Given the findings that HSP70
antisense molecules inhibited HSP70 synthesis and decreased tolerance of
cardiomyocytes to hypoxic stress
(29), and heart-targeted
overexpression of HSP70 protected myocardial cell from apoptosis
(11,
14), it is likely that
hypoxia-mediated reduction in the heat shock proteins results in a decrease in
the protective mechanism of myocyte cell death, which may contribute to
hypoxia-induced apoptosis in the fetal heart. The present study also
demonstrated that chronic hypoxia differentially regulated -AR subtypes
in the fetal rat heart, with an increase in 1-AR, but no
changes in 2-AR. This is in agreement with the previous
finding that prenatal chronic hypoxia increased -AR density in neonatal
rat heart (35). Both in vivo
and in vitro studies have shown that enhanced 1-AR signaling
induces cardiac myocyte apoptosis via a stimulatory G protein-mediated,
protein kinase A-dependent mechanism
(45,
51). The role of increased
1-AR in hypoxic-induced apoptosis in the fetal heart remains
to be elucidated.
In summary, we have demonstrated for the first time, to our knowledge, in a
rat model, that maternal chronic hypoxic exposure in vivo increased apoptotic
cell death in the fetal heart. The increased cell death may lead to cardiac
hypertrophy, resulting in an asymmetric enlargement of the fetal heart in
hypoxic animals. Although the mechanisms underlying hypoxiainduced apoptosis
in the fetal heart are not clear at present, and are likely to be multiplex,
the present study demonstrated that the hypoxia-induced apoptosis was
associated with an increase in Fas receptors and a decrease in Bcl-2 proteins.
Chronic hypoxia differentially regulated endogenous protective mechanisms in
the fetal heart by downregulating HSP70 but upregulating the isoform
of 14–3-3 proteins. The physiological and pathophysiological
consequences of selective increase in 1-AR in the hypoxic
hearts are not entirely clear at present and present an intriguing avenue for
future investigation.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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2001.This article has been cited by other articles:
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