(Circulation. 1999;100:305-311.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
- and ß-Adrenergic Pathways Differentially Regulate Cell Type–Specific Apoptosis in Rat Cardiac Myocytes
From the Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Correspondence to Koji Hasegawa, MD, PhD, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail koj{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Background—The apoptosis of cardiac myocytes may play a role in the development of heart failure. Norepinephrine is one of the factors activated in heart failure and can induce myocardial cell apoptosis in culture. However, it is unknown if
- and
ß-adrenergic pathways coordinately or differentially regulate
apoptosis and if this apoptotic pathway uses common or
cell type–specific apoptotic signals.
Methods and Results—We stimulated cultured neonatal rat cardiac
myocytes with an 1-adrenergic agonist (PE,
phenylephrine), a ß-adrenergic agonist (isoproterenol
[Iso]) or a membrane-permeable cAMP analogue (8-Br-cAMP) in
serum-free conditions for 48 hours. Iso and 8-Br-cAMP markedly
increased the number of TUNEL-positive cells (%TUNEL-positive nuclei
>40%) compared with saline stimulation (<10%). DNA fragmentation
was also confirmed by ladder formation in agarose gels.
Apoptotic myocytes were characterized by cell shrinkage and
nuclear condensation, consistent with morphological features of
apoptosis. The Iso-induced apoptosis was almost
completely inhibited by the protein kinase A–specific
inhibitor KT5720. In contrast, PE inhibited
8-Br-cAMP–induced myocardial cell apoptosis. The
apoptosis-inhibitory effect by PE was negated by
the 1-adrenergic receptor antagonist
prazosin and the MEK-1–specific inhibitor PD098059.
Interestingly, although 8-Br-cAMP markedly induced apoptosis in
cardiac myocytes, it completely blocked serum depletion–induced
apoptosis in PC12 cells, a rat pheochromocytoma cell line.
Conclusions—These findings indicate that - and ß-adrenergic
pathways differentially regulate myocardial cell apoptosis. The
results also suggest that a cAMP– protein kinase A pathway is
necessary and sufficient for ß-adrenergic agonist–induced
apoptosis and that this apoptotic pathway is not
functional in other cell types, for example, PC12 cells.
Key Words: apoptosis • myocytes • heart failure
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Introduction |
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Apoptosis, or programmed cell death, is a central feature of normal tissue development in the fetus and of cell replacement in certain adult tissues (eg, thymus).1 2 3 4 5 In contrast to the classic swelling and membrane rupture associated with necrosis, apoptotic cells shrink and maintain their membrane integrity. The hallmarks of apoptosis include cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptotic cells are phagocytosed by neighboring cells, effectively removing unwanted cells without an inflammatory response. Apoptosis is most often associated with cells that are progressing through the cell cycle. Although adult cardiac myocytes are terminally differentiated and have lost their ability to divide, accumulating evidence suggests that these cells can undergo apoptosis in vivo in various animal models of heart failure including models of rapid ventricular pacing6 7 and pressure overload caused by aortic constriction8 and aged spontaneously hypertensive rats.9 Since the functional aspects of the failing myocardium are impaired in proportion to the reduction in the myocyte fractional area, the apoptosis of myocytes may play a significant role in the deterioration of cardiac function. As such, the identification of the signaling pathways that mediate cardiac myocyte cell death and survival is critical to the ultimate elucidation of the molecular basis of cardiac muscle failure.
Despite an increasing body of evidence concerning myocardial cell
apoptosis in vivo, little is known regarding the relevant
physiological stimuli. The control of programmed
cell death is dependent on a balance between inhibitors and
inducers of apoptosis. Since a number of neurohormonal factors
are activated in congestive heart failure,10 11
they may play positive and negative roles in regulating myocardial cell
apoptosis. Norepinephrine is one such factor, the
elevation of which in plasma closely correlates with the severity and
poor prognosis of heart failure.11 It was recently shown
that norepinephrine can induce apoptosis in cardiac
myocytes in vitro.12 Norepinephrine exerts its
effect on cardiac myocytes through both - and ß-adrenergic
receptor pathways. However, it is not known whether - and
ß-adrenergic pathways regulate apoptosis in a coordinated or
differential manner.
Apoptosis in other cell types has been extensively studied.
Several reports suggest that a subset of apoptosis inducers is
common in cardiac myocytes. For example, reactive oxygen species and
tumor necrosis factor-, both of which are potent apoptosis
inducers in most cell types,12 13 14 15 16 17 have also been
implicated in myocardial cell apoptosis.18 19 It
was recently reported that p53, a well-known trigger of
apoptosis in a variety of cell types, is sufficient to trigger
apoptosis by itself in cardiac myocytes.20 21
However, cardiac muscle cells are distinct from other cell types in
many biological aspects. Because the signaling pathways leading to
apoptosis differ among cell types, it is likely that in
addition to the conserved pathways, cardiac myocytes possess their own
apoptotic pathways. It is unknown, however, whether
norepinephrine-induced apoptosis occurs in a cell
type–specific manner or uses common apoptotic signals.
The present study investigated the effects of - and
ß-adrenergic stimulation on myocardial cell apoptosis. In
this study, we show that a cAMP– protein kinase A (PKA) pathway is
necessary and sufficient for ß-adrenergic stimulation–induced
apoptosis, whereas MEK-1 appears to be involved in an
inhibitory effect by 1-adrenergic
stimulation. Interestingly, in contrast to cardiac myocytes, cAMP
almost completely inhibited apoptosis in PC12 cells, a
pheochromocytoma cell line, suggesting that this cAMP-PKA
apoptotic pathway might use some cardiac-specific
machinery.
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Methods |
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Cell Culture
Primary ventricular cardiac myocytes were prepared as previously described.22 Briefly, hearts from 1- to 2-day-old Sprague-Dawley rats were removed, the ventricles were pooled, and the ventricular cells were dispersed by digestion with pancreatin (Life Technologies). The cells were preplated for 1 hour to enrich the culture with myocytes (90% to 95% of cells after this step). Cells were plated at a high density (1000 cells/mm2) onto 60-mm tissue culture dishes (Primaria, Falcon; Becton Dickinson & Co) and cultured in media consisting of Hanks' salts plus minimal essential medium (MEM) vitamin stock, MEM amino acids, MEM nonessential amino acids, 2 mmol/L L-glutamine, 0.67 mmol/L glycine, 0.92 mmol/L hypoxanthine, 19.6 mmol/L NaHCO3 (pH 7.1 to 7.2), penicillin, streptomycin, and 10% (vol/vol) fetal bovine serum (all from GIBCO BRL) at 37°C, 5% CO2.
PC12 cells, a rat pheochromocytoma cell line, were obtained from a health science research resources bank (JCRB No. 0733) and kept in the RPMI 1640 medium with 10% horse serum and 5% fetal bovine serum.
Nucleosomal Ladder Assay
Forty-eight hours after plating, the neonatal rat cardiac
myocytes were washed twice with serum-free media and cultured in
serum-free medium in the presence or absence of
phenylephrine (PE), isoproterenol (Iso) or 8-B-cAMP for 48
hours. These agents were obtained from Sigma and were of the highest
purity available. The cells were then harvested by scraping into the
media. After centrifugation at 500g for 5
minutes at 4°C, the cells were lysed in lysis buffer and subjected to
a nucleosomal ladder assay with the use of a commercial kit (Takara
Biomedicals) according to the manufacturer's recommendations. The
presence of characteristic 180- to 200-bp multiple oligonucleosomal
fragmentation was examined on 1.5% agarose gels stained with SYBR
Green I (Takara Biomedicals).
In Situ Labeling of Apoptotic Cells and Quantitative
Analysis
Terminal deoxynucleotidyl transfer–mediated
end-labeling of fragmented nuclei (TUNEL assay) was performed on
cardiomyocytes that had been plated on flask-style glass
slides (Nalgen Nunc). The in situ TUNEL assay was then performed in
accordance with the manufacturer's protocol for cultured cells (Takara
Biomedicals) after fixing the cells in 10% neutral buffered formalin
for 10 minutes at room temperature. Individual nuclei were visualized
at a magnification of x400 for quantitative analysis. An
average of 400 to 500 nuclei from random fields were analyzed
in each slide. The apoptotic index (percentage of
apoptotic nuclei) was calculated as (apoptotic
nuclei/total nuclei)x100%. Sample indicates were concealed
during scoring, and samples from at least 3 independent experiments
were scored per group.
Immunocytochemistry
To identify cardiac myocytes, immunocytochemical staining was
performed as described23 24 with the use of a monoclonal
antibody against muscle-specific -actin (HHF35) at a dilution of
1:100. Signals were detected with the use of an alkaline
phosphatase–conjugated Fab fragment of the secondary antibody (a
dilution of 1:600, Jackson Immunoresearch Laboratories) and nitroblue
tetrazolium dye as the substrate.
Statistical Analysis
Data are presented as mean±SE. Statistical comparisons
were performed with the use of unpaired 2-tailed Student's
t tests or ANOVA with Scheffé's test when
appropriate, with a probability value of <0.05 taken to indicate
significance.
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Results |
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ß-Adrenergic Stimulation Activates an Apoptotic Program in Cardiac Myocytes
For the investigation of the possible contribution of a ß-adrenergic pathway to the development of myocardial cell apoptosis, neonatal rat cardiac myocytes were treated with a ß-adrenergic agonist (Iso) or saline as a control in the serum-free condition. In our experimental conditions in which cells were plated at a high density, serum deprivation alone did not increase the number of TUNEL-positive nuclei (<10%) (Figure 1A
). As shown in Figure 1B, however, the stimulation with Iso markedly increased the number of
TUNEL-positive cells (>40%). These positive cells may specifically
indicate the presence of internucleosomal DNA fragmentation, since no
positive cells were found when we omitted the terminal deoxytransferase
treatment (Figure 1C). The cells stimulated with Iso displayed
small condensed nuclei, cell shrinkage, and nuclear fragmentation,
consistent with the morphological features of apoptosis
(Figures 1C and 1D). These cells were derived from cardiac
myocytes, as evidenced by the positive immunostaining
for HHF35, which reacts with the -actin of cardiac myocytes but not
that of fibroblasts (Figure 1D). Figure 2 and Figure 4 show the
Iso-induced typical ladder formation of fragmented internucleosomal DNA
in agarose gels, a hallmark of apoptosis. The Iso-induced
apoptosis occurred in a dose-dependent manner, as shown in
Figure 3. Apoptosis was evident
later than 36 hours but not at 24 hours after Iso stimulation (data not
shown). The Iso-induced apoptosis was almost completely blocked
by a ß-adrenergic receptor antagonist,
propranolol (10-4 mol/L) (Figure 2, lane 3 and Figure 5, lane 5), whereas this
concentration of propranolol alone did not increase the
number of TUNEL-positive cells (Figure 5, lane 2). To further
test the specificity of propranolol, we examined the effect
of this agent on 8-Br-cAMP–induced apoptosis. We found that
10-4 mol/L propranolol did not
affect the 8-Br-cAMP–stimulated increase of TUNEL-positive cells
(compare Figure 5, lanes 7 and 8). These findings provide
evidence that a ß-adrenergic receptor–dependent pathway
activates an apoptotic program in cardiac myocytes.
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cAMP/PKA Pathway Mediates Iso-Induced Apoptosis
Stimulation of the ß-adrenergic receptor activates
adenylate cyclase, which increases intracellular cAMP and
activates cAMP-dependent PKA. To determine whether the
activation of PKA is required for Iso-induced apoptosis, we
examined the effect of KT5720, a highly selective inhibitor
of PKA.25 As shown in Figure 4
, 10-6 mol/L of
KT5720 was able to completely inhibit the Iso-induced internucleosomal
cleavage of genomic DNA. The quantitative analysis by TUNEL
staining also revealed that KT5720 (10-6 mol/L)
almost completely inhibited the Iso-stimulated increase of
TUNEL-positive cells (Figure 5, lane 6),
whereas the same concentration of this agent alone did not induce
apoptosis (Figure 5, lane 3). In accord with these
results, the administration of a cell-permeable cAMP analogue,
8-Br-cAMP (30 mmol/L), also induced apoptosis in cardiac
myocytes to an extent similar to that shown by Iso (Figure 5, lane 7 and Figure 6A, lane 2). Taken
together, these results demonstrate that cAMP-PKA is necessary and
sufficient for Iso-induced apoptosis.
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1-Adrenergic Pathway Inhibits cAMP-Induced
Cardiac Apoptosis
To determine the effects of 1-adrenergic
stimulation on myocardial cell apoptosis, neonatal rat cardiac
myocytes were treated with an 1-adrenergic
agonist (PE). In contrast to Iso, stimulation with PE did not increase
the number of TUNEL-positive cells, even at a high concentration
(10-4 mol/L) (Figure 3). As shown in
Figure 6A, lane 3, PE inhibited the internucleosomal cleavage of
genomic DNA in 8-Br-cAMP–stimulated cardiac myocytes. The
antiapoptotic effect of PE was further revealed by TUNEL
staining (Figure 6B). Fewer myocardial cells treated with PE in
addition to 8-Br-cAMP were positive for internucleosomal cleavage by
TUNEL staining (Figure 6B, lane 5) compared with the cells
treated with 8-Br cAMP alone (Figure 6B, lane 4). These results
provide evidence that PE has an antiapoptotic effect in
cultured cardiac myocytes. To evaluate whether the inhibition of
myocardial apoptosis by PE is mediated through an
1-adrenergic receptor pathway, we used
prazosin, an 1-adrenergic receptor
antagonist. Prazosin negated the PE-mediated inhibition of
apoptosis (Figure 6B, lane 6), whereas the same
concentration of this agent did not increase the number of
TUNEL-positive cells (Figure 6B, lane 2) compared with saline
stimulation (Figure 6B, lane 1).
1-Adrenergic stimulation has been shown to
activate a MAP kinase cascade in cardiac myocytes. To determine
whether the activation of MAP kinase is required for the PE inhibition
of apoptosis in cardiac myocytes, we used PD098059, a specific
MEK inhibitor that selectively inhibits MEK-1
activity.26 27 A previous study confirmed that 10 µM of
PD098059 completely inhibits the PE-stimulated activation of ERK1 and
ERK2 in cardiac myocytes.28 As shown in Figure 6A, lane 4, and Figure 6B, lane 7, 10 µM of PD098059 negated the
inhibitory effects of PE on myocardial cell
apoptosis. To exclude the possibility of a nonspecific
cytotoxic effect of PD098059, we tested whether it was capable of
inducing cell death in the serum-free condition. We found that 10 µM
or 50 µM of PD098059 alone did not induce apoptosis (Figure 6B, lane 3) compared with saline stimulation (Figure 6B, lane 1). This result suggested that PD098059 might block the downstream
signaling pathway by which PE prevents apoptosis. Taken
together, these results provide evidence that PE has an
antiapoptotic effect in cultured cardiac myocytes and that a
MAP kinase pathway appears to be involved in this process.
cAMP-Mediated Apoptotic Pathway Is Not Functional in
PC12 Cells
In cardiac myocytes, as shown in Figure 7A, serum
deprivation alone barely induced internucleosomal cleavage
of genomic DNA compared with the condition of 10% fetal bovine serum.
Cell-permeable 8-Br-cAMP induced marked apoptosis in the
serum-free condition (Figure 7A, lane 3).
To determine whether cAMP-induced apoptosis is a conserved
phenomenon among different cell types or a phenomenon unique to cardiac
myocytes, we examined the effect of 8-Br-cAMP on apoptosis in
different cell lines. For this purpose, we used PC12 cells, rat
pheochromocytoma cells that are dependent on the presence of growth
factors such as nerve growth factor and insulin growth factor-1 in the
medium and die by apoptosis after serum
deprivation.29 30 In these cells, 12 hours
after serum deprivation, marked internucleosomal cleavage
of genomic DNA was observed. Notably, as shown in Figure 7B, the
administration of 30 mmol/L of 8-Br-cAMP completely inhibited the
serum deprivation–induced apoptosis in PC12 cells.
Antiapoptotic effects of 8-Br-cAMP were also observed in other
mitotic cell lines such as mouse fibroblast cells (NIH3T3 cells) (data
not shown). These findings indicate that a cAMP pathway is involved in
promoting cell survival in these cell lines. Thus 8-Br-cAMP displayed
opposite effects on apoptosis in cardiac myocytes and PC12
cells.
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Discussion |
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Adult cardiac muscle cells are terminally differentiated and have lost their proliferative capacity. As a result, the maintenance of cardiac muscle cell survival is critical for the maintenance of normal cardiac function. The present study demonstrated that a ß-adrenergic agonist induced apoptosis in cardiac myocytes and that this apoptosis was mediated through a cAMP-PKA pathway. In contrast, an
1-adrenergic agonist
antagonized cAMP-induced apoptosis. Interestingly, cAMP had
opposite effects on apoptosis in cardiac myocytes and PC12
cells, suggesting that cAMP involves cardiac-specific signal
transduction mechanisms.
Accumulating evidence suggests that myocyte apoptosis occurs in
failing hearts,6 7 8 9 suggesting that apoptosis
contributes to progressive myocardial dysfunction. Nevertheless, little
is known about the stimuli that initiate the program of
apoptosis or the molecular and cellular events that mediate the
ensuing cell death. A number of neurohormonal and autocrine substances,
including A-type and B-type natriuretic peptides,
endothelin-1, and norepinephrine, are present at high
levels in patients with heart failure.10 11 The elevated
plasma levels of norepinephrine, an activator
of both - and ß-adrenergic pathways, is closely associated with
the severity and poor prognosis of heart failure.10 11
With the use of 3 independent criteria, that is, TUNEL staining, DNA
ladder formation, and nuclear condensation, the present study has
documented that ß-adrenergic stimulation can induce apoptosis
in cultured neonatal rat cardiac myocytes. In addition, a highly
selective inhibitor of PKA, KT5720, completely blocked
ß-adrenergic–induced cardiac apoptosis, suggesting a
PKA-dependent effect. Consistent with these results, the
membrane-permeable cAMP analogue (8-Br-cAMP) also induced
apoptosis in these cells. These findings demonstrate that a
cAMP-PKA pathway mediates ß-adrenergic–induced apoptosis.
Although small numbers of fibroblasts are present in every culture
of neonatal myocytes, the apoptosis that resulted from either
ß-adrenergic agonist or 8-Br-cAMP was confined to myocytes, as shown
by muscle-specific immunostaining with HHF35. Cardiac
specificity was also confirmed by the findings that the cellular
composition of these cultures was >90% myocytes and that these
stimuli did not induce apoptosis in other cell lines such as
NIH3T3 or PC12 cells.
We used cultured neonatal cardiac myocytes, which retain some ability
to undergo DNA synthesis.31 32 It should be further
investigated whether ß-adrenergic stimulation can induce
apoptosis in terminal differentiated adult cardiac myocytes in
vivo. However, the results of our study are compatible with 2 recent
reports: (1) norepinephrine can induce apoptosis in
adult cardiac myocytes in culture through a ß-adrenergic
pathway12 ; and (2) the overexpression of
Gs results in the accelerated
apoptosis of adult cardiac myocytes in transgenic
mice.33 Thus the induction of cardiomyocyte
apoptosis through ß-adrenergic receptor–dependent signaling
pathways is not confined to in vitro assays in neonatal cells but may
also occur in adult cardiac myocytes in vivo.
The present study also demonstrated that
1-adrenergic stimulation inhibited
cAMP-induced apoptosis. 1-Adrenergic
stimulation triggers downstream signaling pathways through
Gq and subsequently activates protein
kinase C. It is also becoming clear that an
1-adrenergic pathway cross-talks with
Ras and MAP kinase cascades. The present treatment of
cultured neonatal cardiac myocytes with 10 mmol/L of a
MEK-specific inhibitor, PD098059, which has been shown to
completely inhibit the PE-stimulated activation of ERK1 and ERK2 at
this concentration,28 negated the
apoptosis-inhibitory effect of
1-adrenergic agonist. These findings
demonstrate that MAP kinase–dependent pathways are required for the
inhibition of cardiac myocyte apoptosis. Our data do not rule
out a possible role of MAP kinase–independent pathways in the
apoptosis inhibition. However, the nearly complete blockade of
the 1-adrenergic–mediated inhibition of
apoptosis by PD098059 suggests the essential role for this
pathway in the cell survival function. MAP kinase pathways have been
found to be necessary for the effects of nerve growth factor and
insulin growth factor-1 on the promotion of the survival of neuronal
cell types (PC12), whereas the inhibition of MAP kinase has been shown
to be critical for the induction of apoptosis in these
cells.29 30 These studies provide further evidence that
MAP kinase–dependent pathways play a particularly important role in
promoting the survival of terminally differentiated cell types as
well.
Another interesting feature of our results is the opposing effects of cAMP on apoptosis in 2 different cell types. In contrast to the data in primary cardiac myocytes in culture, the administration of 8-Br-cAMP completely inhibited serum deprivation–induced apoptosis in PC12 pheochromocytoma cells. Thus cAMP-mediated apoptosis in cardiac myocytes might involve some cell type–specific signal transduction mechanisms. At present, the precise molecular effectors and targets of cAMP-induced cardiac apoptosis are unclear. Regarding apoptosis in terminally differentiated cells, one of the candidate molecules is the adenovirus E1A-associated cellular protein p300, which appears to play a role in myocardial cell survival.32 33 p300 is a homologue of CBP, a protein that is associated with and coactivates the transcription factor CREB, mediating the induction of cAMP-responsive promoters.34 35 The possible involvement of the p300/CBP family in cAMP-mediated apoptosis in cardiac myocytes requires further investigation.
Several lines of evidence suggest that the activation of the
sympathetic nervous system exerts a direct deleterious effect on the
heart that is independent of the hemodynamic actions of
these endogenous mechanisms. Therapeutic interventions by
ß-adrenergic receptor blockers favorably alter the natural history of
heart failure, and such benefits cannot be explained by the effect of
these agents on cardiac contractility and ejection
fraction. The present study demonstrated that a ß-adrenergic
pathway but not the 1-adrenergic pathway
induced cell type–specific apoptosis in cardiac myocytes.
These findings might indicate a mechanism of the beneficial effects of
ß-adrenergic receptor blockers in patients with heart failure. It
would be of particular interest to test whether ß-adrenergic receptor
blockers can inhibit myocyte apoptosis in experimental animal
models of heart failure. In addition, the elucidation of precise
signaling pathways leading to myocardial cell apoptosis may
contribute to novel strategies for heart failure therapy in humans.
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Acknowledgments |
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This work was supported in part by grants (to Dr Hasegawa) from the Japanese Heart Foundation, the Japan Cardiovascular Research Foundation, Yamanouchi Foundation for Research on Metabolic Disorders, Uehara Memorial Foundation, The Tokyo Biochemical Research Foundation, and the Ministry of Education, Science, and Culture of Japan.
Received November 18, 1998; revision received March 4, 1999; accepted March 31, 1999.
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