© 1999 American Heart Association, Inc.
Original Contribution |
Apoptosis of Cardiac Myocytes in Gs
Transgenic Mice
From the Cardiovascular and Pulmonary Research Institute (Y-J.G., Y.I., D.E.V., S.F.V.), Allegheny University of the Health Sciences, Pittsburgh, Pa; Departments of Molecular and Cellular Biology and Oncology Research (T.E.W.), The Greenville Hospital System, Greenville, SC; Department of Microbiology and Molecular Medicine (T.E.W.), Clemson University, Clemson, SC; Department of Pathology (S.P.B.), University of Alabama at Birmingham; and COR Therapeutics, Inc (C.J.H.), South San Francisco, Calif.
Correspondence to Stephen F. Vatner, MD, George J. Magovern Professor and Director, Cardiovascular Research Institute, Allegheny University of Health Sciences, 320 East North Avenue, Pittsburgh, PA 15212.
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Abstract |
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Abstract—The stimulatory GTP-binding protein Gs
transmits signals from
catecholamine receptors to activate adenylyl
cyclase and thereby initiate a cascade leading to cardiac chronotropy
and inotropy. Transgenic mice overexpressing the Gs subunit (Gs)
selectively in their hearts exhibit increased cardiac
contractility in response to ß-adrenergic receptor
stimulation. However, with aging, these mice develop a
cardiomyopathy. This study sought morphological and
biochemical evidence that overexpression of Gs is associated with
increased myocyte apoptosis in the older animals and to
determine whether such overexpression can promote apoptosis of
isolated neonatal cardiac myocytes exposed to ß-adrenergic receptor
agonists. In the hearts of 15- to 18-month-old Gs transgenic mice,
histochemistry and electron microscopy illustrated the existence of
numerous myocytes with abnormal nuclei embedded in collagen-rich
connective tissue. Terminal deoxyribonucleotide
transferase-mediated dUTP nick-end labeling (TUNEL, for in situ
labeling of DNA breaks) demonstrated that 
0.6% of myocyte
nuclei contained fragmented DNA. Agarose gel electrophoresis provided
further biochemical evidence of apoptosis by showing
internucleosomal DNA fragmentation. Cultured cardiac myocytes from
newborn Gs transgenic mice showed increased TUNEL staining and
internucleosomal DNA fragmentation compared with wild-type controls
when treated with the ß-agonist isoproterenol. Thus, enhanced
activation of ß-adrenergic signaling by overexpression of Gs in
the hearts of transgenic mice induces apoptosis of cardiac
myocytes. This represents a potential mechanism that may
contribute to the development of cardiomyopathy in
this model.
Key Words: adenylyl cyclase • ß-adrenergic receptor • catecholamine • programmed cell death • cardiomyopathy
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The heterotrimeric GTP-binding proteins (G proteins) composed of
, ß, and 
subunits mediate signal transduction in a
broad diversity of cell types.1 2 The stimulatory G
protein Gs transmits signals from ß-adrenergic receptors (ßARs)
to adenylyl cyclase (AC), leading to activation of this enzyme,
production of cAMP, and enhancement of cardiac
contractility.1 2 Alterations in this
signaling pathway (ßAR-Gs-AC), particularly catecholamine
desensitization, are a hallmark of heart failure in both human disease
and animal models.2 3 4 However, it remains controversial
whether chronic ßAR stimulation or the reverse, desensitization, is
adaptive or maladaptive in the pathogenesis of heart failure. Several
lines of evidence show that acutely5 or
chronically6 7 8 enhanced stimulation with
catecholamines can result in myocyte death and cardiac
fibrosis. We previously established a transgenic mouse model by
selectively overexpressing a Gs transgene in the
heart,7 8 9 which enhances inotropic and chronotropic
responses of the heart to sympathetic stimulation.7
Furthermore, with aging the transgenic heart develops morphological
alterations characteristic of cardiomyopathy,
including myocyte hypertrophy and fibrosis,7 8
suggesting that persistent ßAR activation may be deleterious over the
long term. However, other studies favor the opposing view that
enhancement of ß-adrenergic signaling may be beneficial, eg,
overexpressing a ß-adrenergic signaling component may be a treatment
to reverse the cardiac depression that occurs in heart
failure.10 11
We hypothesize that overdriving the ßAR/Gs/AC signaling pathway may
induce apoptosis as well as necrosis. A key observation is that
the older animals overexpressing cardiac Gs develop significant
myocyte hypertrophy but have only a modest increase in
heart weight/body weight,7 suggesting the potential
for myocyte deletion. Apoptosis or genetically programmed cell
death plays an important role in determination of tissue cellularity
during embryonic development and adult tissue turnover.12
We examined the hearts of mice overexpressing the Gs transgene in a
cardiac-selective manner for the presence of programmed myocyte death.
We also tested the hypothesis that cardiac myocytes cultured in a
controlled environment undergo apoptosis, when the Gs-coupled
ßAR-AC pathway is stimulated vigorously for a short period in
vitro. This study demonstrates that increased myocyte nuclear
degeneration and DNA fragmentation occur in the hearts of transgenic
mice overexpressing Gs. Our results suggest that intrinsic,
long-term overactivation of the ßAR/Gs/AC pathway may accelerate
apoptosis of cardiac myocytes and thereby play a role in the
development and/or progression of cardiomyopathy
and heart failure.
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Materials and Methods |
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Gs
Transgenic MiceTransgenic mice selectively overexpressing Gs
in the heart
were generated by microinjection of a Gs minigene construct into the
eggs of C57BL/6J mice as previously described.9 Briefly,
exons 1 to 12 from a canine Gs cDNA within a 1.3-kb
MulI-BamHI fragment were conjugated to a
human Gs genomic fragment containing intron 12, exon 13, and the
polyadenylation signal. This chimeric minigene construct was then
positioned downstream of a rat -myosin heavy chain promoter
contained within a 0.9-kb EcoRI-XbaI gene
fragment. This construction was then inserted into the plasmid pGEM-7Z
(Promega) for propagation, and a 3.5-kb
KpnI-SacI fragment was isolated for
microinjection. Positive founders carrying the transgene were bred to
normal adult C57BL/6XC3H (B6C3) F(1) hybrid females to establish
independent germlines. The successful establishment and expression of
the transgene in the mice were confirmed by Southern blotting
analysis for the transgene DNA, Northern blotting for Gs
transcripts, and Western blotting for the Gs
protein.9 There was 3- to 5-fold overexpression of
the protein for Gs in the heart.9 13
Tissue Preparation and Histochemical Examination
After deep anesthesia with an
intraperitoneal injection of sodium pentobarbital,
the hearts were removed from adult mice at either 4 to 7 months or 15
to 18 months of age and then fixed by immersion in 10%
phosphate-buffered formalin or by in situ perfusion fixation with
formalin through the left ventricular apex. The fixed
tissues were dehydrated, embedded in paraffin, and sectioned at 6-µm
thickness. Histological examination was carried out by
staining with hematoxylin and eosin and Gomori aldehyde fuchsin
trichrome. For silver staining of the glycocalyx and other matrix
substances, 1.0-µm-thick sections embedded in glycol methacrylate
were prepared for staining using a microwave procedure
(Acustain, Sigma). Myocytes were judged to be cut normal to
their long axis by the nearly round shape of perfused capillaries in
the region.
Electron Microscopy
Transmission electron microscopy was performed on a separate
series of animals in which the myocardium was perfusion
fixed with phosphate-buffered 2% glutaraldehyde, en
bloc stained with osmium tetroxide, and embedded in Spurr epoxy resin,
and thin sections were stained with lead citrate and uranyl
acetate.
Culture of Cardiomyocytes
Cardiomyocytes were isolated from the hearts of newborn Gs
and wild-type control mice by proteolytic digestion with trypsin and
collagenases and cultured in DMEM supplemented with
vitamins and antibiotics. The cells, at a density of
5x103 cells/mL, were grown on a glass coverslip
in 6-well culture plates or in 8-chamber slides. They were then
stimulated with isoproterenol (5 to 20 µmol/L) for 48 hours.
Cellular morphology was analyzed by phase-contrast and
fluorescent microscopy, and DNA integrity was determined by
enzymatic labeling and agarose gel electrophoresis (see below).
Cell Viability
The viability of the isolated myocytes was determined by
staining with the nucleic acid–binding fluorochromes acridine orange
and ethidium bromide.14 15 At the end of incubation with
isoproterenol, the chamber slides were incubated with the DNA-binding
dyes, acridine orange and ethidium bromide, at 10 µg/mL each for 2
minutes on ice. Coverslips were applied to the slides, and the sections
were observed under a Nikon E800 fluorescent microscope with a
triple filter. Viable cells exclude ethidium bromide but not acridine
orange, which stains nuclei, yielding a green fluorescence.
When cells die, they become permeable to both acridine orange and
ethidium bromide, which bind cellular nucleic acid, producing a red or
orange fluorescence. For each sample, at least 200 cells were
counted using the x40 objective on the microscope. The percentage of
viable cells was determined by the following formula: % cell
viability=number of viable cells/total number of cellsx100.
In Situ Labeling of DNA Fragments (TUNEL)
DNA fragmentation was detected in situ by using terminal
deoxyribonucleotide transferase (TdT)–mediated dUTP
nick-end labeling (TUNEL) in tissues16 17 from
Gs-overexpressed transgenic and wild-type mice and in cultured
neonatal myocytes. Briefly, the paraffin sections were deparaffinized
by immersing in xylene; rehydrated through 100%, 95%, 75%, and 0%
ethanol; and incubated in PBS with 2%
H202 to
inactivate endogenous peroxidases. After
incubation with proteinase K (20 µg/mL), the sections were washed in
PBS. DNA fragments in the sections were labeled with 2 nmol/L
biotin-conjugated dUTP and 0.1 U/µL TdT for 1 hour at 37°C. The
incorporation of biotin-16-dUTP into DNA was determined by incubating
the sections with FITC-ExtrAvidin (1:200, Sigma) at room temperature
for 30 minutes. After the TUNEL procedure, the slides were washed in
PBS and mounted in a Vector DAPI medium for fluorescent
microscopic observation. Using left ventricular regions
where myocytes were cut in cross section, the mean number of myocyte
nuclei per x40-objective field was determined by manual counting of
DAPI-stained nuclei using UV excitation. At the same magnification, a
minimum of 20 fields with myocytes cut in cross section from the left
ventricle of each heart was examined to count TUNEL-positive myocytes.
For TUNEL staining of myocytes treated with or without isoproterenol,
freshly isolated neonatal myocytes were cultured on a glass coverslip
in 6-well plates. After treatment with isoproterenol, cells were fixed
in 10% formalin in PBS for 10 minutes, washed in cold PBS 3 times, and
then incubated in 0.1% saponin in PBS with 1 µmol/L EGTA for 20
minutes at room temperature. DNA 3' end labeling was performed using
the biotin-16-dUTP/TdT system followed by FITC-ExtrAvidin staining. All
morphometric measurements were performed in a blinded manner.
DNA Isolation and Electrophoresis
Hearts from Gs transgenic or control mice were removed
from deeply anesthetized animals, snap frozen, and crushed in
liquid nitrogen. The tissue was then mixed with 1 mL of DNA extraction
solution containing 20 µmol/L Tris-HCl, pH 7.4, 0.1 mol/L NaCl,
5 µmol/L EDTA, and 0.5% SDS. For isolation of DNA from cultured
myocytes, 1 mL of DNA extraction buffer was directly added into the
culture flask after removing the culture medium. The cell lysates were
incubated with 100 µg/mL proteinase K at 37°C for 16 hours. After
incubation, 1 mL of phenol/chloroform (1:1) was mixed with the
enzyme-digested cell lysates and then centrifuged at
20 000g for 20 minutes; DNA in the upper (aqueous) phase
was incubated with 5 µg/mL DNase-free RNase A at 37°C for 1 hour
and extracted with phenol/chloroform again. DNA was collected by
precipitation with 1 mL of isopropanol and 0.1 mL of 5 mol/L NaCl at
–20°C overnight. After centrifugation, the resulting
DNA pellets were washed with 75% ethanol and air dried. DNA was
dissolved in 10 µmol/L Tris-HCl buffer with 1 µmol/L
EDTA, and its concentration was determined at 260 nm by
spectrophotometry. DNA electrophoresis was carried out in 1.5% agarose
gels containing 1 µg/mL ethidium bromide, and DNA bands were
visualized under UV light.
Immunoblotting Assay
Total membrane proteins were extracted from cardiac cells of
Gs or wild-type mice. Protein (30 µg/lane) was loaded onto a 10%
SDS-PAGE gel. After electrophoresis, protein bands were transblotted to
a membrane, which was then blocked and immunostained with
anti-Gs antibody (1:5000). Anti-rabbit IgG conjugated with
peroxidase was used as the second antibody. The blots were developed by
enhanced chemiluminescence (Amersham).
Statistical Analysis
Data are reported as mean±SD. The difference between means was
evaluated using Student's t test. For statistical
analysis of data from multiple groups, ANOVA was used.
Significance levels were established at P<0.05.
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Results |
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Histopathological Evaluation of the Myocardium
Histopathological evaluation confirmed our previous observations of absence of lesions in young adult (4 to 7 months old) animals and extensive multifocal areas of fibrosis in 16-month-old Gs
transgenic
mice (Figure 1
).7 In
addition to the multifocal fibrosis, cellular degeneration was
apparent, including pale-staining cells due to loss of myofibrils,
cytoplasmic vacuolation, nuclear aberrations, including irregular size,
condensed chromation, and vacuoles, and hypertrophy and
atrophy of myocytes. The individual variation and increased size
in myocyte cross-sectional area was shown by staining 1-µm-thick
methacrylate sections with a silver stain to outline the glycocalyx
surrounding each cell (Figure 2).7
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Nuclear Morphology and in Situ DNA 3' End Labeling
The myocytes of the transgenic hearts also displayed distinct
morphological alterations in their nuclei. Light microscopy showed
alterations in the nuclear morphology of transgenic myocytes
characterized by prominent chromatin condensation and vacuole formation
(Figure 1
). By transmission electron microscopy, we observed
marked irregularity of the nuclear membrane with invaginations and
vacuole formation (Figure 3b). Hence,
both light and electron microscopy demonstrated nuclear morphological
alterations in transgenic myocytes, but no such changes were found in
age-matched, wild-type controls (Figures 1a and 3a). In
most cells, these nuclear morphological alterations occurred with
little cytoplasmic abnormality (Figure 3b). However, in other
cells, nuclear changes were accompanied by disorganization or loss of
the striated myofibrils and other degenerative changes. Most of the
myocytes with cytoplasmic and/or nuclear degenerative changes
maintained an intact plasma membrane.
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Evaluation of tissue sections stained by the TUNEL technique revealed
only small numbers of TUNEL-positive cells in the control hearts. The
myocytes of normal hearts were of regular shape, and counterstaining of
their nuclei showed a homogeneous blue fluorescent
stain with DAPI. The transgenic 15- to 18-month-old mice had a
significant increase in the number of TUNEL-positive myocyte nuclei
compared with wild-type controls, (0.6±0.1% of myocytes versus
0.1±0.04% in control). The 4- to 7-month-old animals had only rare
TUNEL-positive cells, and there was no significant difference between
transgenic and wild-type animals (Figure 4). An example of a TUNEL-positive
myocyte is shown in Figure 5.
Nonmyocyte interstitial cells were also labeled by
the TUNEL procedure and were more frequently present in the older
transgenic mice than in wild-type animals (data not shown). Limiting
the counting of total myocyte nuclei and the TUNEL-positive nuclei to
areas with true cross sections of myocytes made it possible to
selectively count only those nuclei that clearly were within a myocyte
(Figure 5).
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Internucleosomal DNA Fragmentation in Hearts Overexpressing
Gs
Internucleosomal DNA fragmentation biochemically characterizes
apoptosis. Agarose gel electrophoresis of DNA isolated from the
myocardium of 15- to 18-month-old mice showed bands of DNA
fragments at 180 to 200 bp or multiples with the laddering appearance
typical of apoptosis (Figure 6a, lanes 1 and 2). In contrast, little or no DNA laddering was observed in
the age-matched wild-type animals (Figure 6a, lanes 3 and
4).
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To confirm overexpression of Gs in the hearts exhibiting
internucleosomal DNA fragmentation, we examined Gs protein content
by immunoblotting with rabbit polyclonal anti-Gs
antibody. As shown in Figure 6b, much stronger Gs bands
(short isoform encoded by the transgene) were observed in the blots
prepared from the transgenic animals (lanes 1 and 2) than from
wild-type controls (lanes 3 and 4), indicating the presence of
overexpressed Gs protein in the transgenic hearts with increased DNA
fragmentation.
In Vitro Apoptosis of Myocytes With Overexpressed Gs in
Response to ß-Adrenergic Stimulation
To determine whether apoptosis of myocytes might occur as
a direct consequence of overactivation of the ß-adrenergic pathway,
short-term vigorous stimulation of this signaling pathway in cardiac
myocytes was carried out in vitro. Cardiac myocytes isolated from the
hearts of newborn Gs transgenic or wild-type mice were exposed to
isoproterenol under identical culture conditions. After 2 days in
culture, no difference in cell viability (>95%) was observed between
untreated Gs and wild-type cells. After exposure for 2 days to the
ßAR agonist isoproterenol (5 to 20 µmol/L), many myocytes were
contracted and fragmented (Figure 7c). By
fluorescent microscopy, nuclear aberrations were also seen
(Figure 7d).
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We also analyzed nuclear DNA integrity by the TUNEL technique
in neonatal myocytes after exposure to isoproterenol (5 to 20
µmol/L) for 48 hours. There was a concentration-dependent increase in
the number of TUNEL-positive nuclei in the isoproterenol-treated Gs
neonatal myocyte cultures when compared with the wild-type controls
(Figure 8). To determine the sizes of DNA
fragments, we analyzed nuclear DNA from both control and
isoproterenol-treated cells by agarose gel electrophoresis. Under the
baseline condition of primary culture, no internucleosomal DNA
fragmentation occurred in myocytes from the hearts of either Gs
transgenic or wild-type mice (Figure 9, lanes 4 and 5). Treatment with isoproterenol for 2 days markedly
increased internucleosomal DNA fragmentation in myocytes isolated from
Gs transgenic hearts (Figure 9, lane 2) but not in wild-type
myocytes (Figure 9, lane 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present investigation, we have demonstrated apoptosis, a form of genetically programmed cell death, as a potential mechanism mediating myocyte death in the heart. We used a combination of techniques to obtain morphological and biochemical evidence for apoptosis, including histopathological evaluation with diverse staining techniques, electron microscopy, in situ nuclear DNA-end labeling, and DNA agarose gel electrophoresis. Although other forms of cell death might have occurred, our data suggest that apoptosis contributes to the loss of myocytes in the heart with overexpressed Gs
. ßARs, via the stimulatory G protein Gs, transduce the signal from norepinephrine to activate AC, thereby catalyzing the synthesis of cAMP. This signaling pathway is important for enhancement of cardiac contractility and heart rate, particularly when there is an increase in demand for cardiac output.1 2 18 Rapid changes in cardiac output, occurring in a time frame measured in seconds, are required of the heart to meet a varied set of demands. Thus, the ßAR signaling pathway is being continuously activated and deactivated as the sympathetic nerves adjust their activity in response to the body's greater or lesser demand for blood supply. In contrast, in pathophysiological states such as heart failure, it is thought that the sympathetic nerves are continuously activated, as the organism senses an inadequate cardiac output and attempts to correct the situation. What remains unclear is whether this state of persistent sympathetic nerve stimulation is, in fact, deleterious to the heart over the long term.
Transgenic mice with overexpression of cardiac Gs respond to a
sympathetic stimulus with enhanced
contractility.7 13 The augmented response
is not due to a heightened state of sympathetic nerve activity but
rather to an accentuated postsynaptic response pathway. Thus, we
believe that this mouse model mimics the state of heightened
sympathetic nerve activity that occurs in heart failure, albeit by a
different mechanism and in the absence of a preceding cardiac insult.
It also offers a unique perspective on the consequences of chronically
enhanced ßAR stimulation, occurring over the life of the animals.
Initially, these Gs transgenic mice exhibit an enhanced cardiac
response to catecholamines. As they age, these mice develop
cardiac dilatation with decreased ejection fraction but do not develop
myocardial desensitization to
catecholamines.13 Consequently, myocyte
hypertrophy, cellular dropout, and fibrosis occur,
mimicking the human syndrome of
cardiomyopathy.7 8
We observed that in the hearts of older Gs transgenic mice, an
increased number of cardiac cells showed changes characteristic of
apoptosis. The observation that a significant difference in
TUNEL staining existed between the transgenic and wild-type control
hearts in the older mice (15 to 18 months old) but not in the younger
animals (4 to 7 months of age) suggests an age dependency of
accelerated apoptosis in the transgenic hearts. Therefore, the
Gs transgene itself appears to exert no direct cytotoxic effect on
myocytes. Apoptosis in aging tissues has also been reported to
mediate cell loss.19 However, the aging process itself
cannot account for the myocyte degeneration and apoptosis in
Gs transgenic mice, since age-matched wild-type mice do not display
the histopathological alterations found in the transgenic mice. As
a consequence of Gs overexpression and chronically enhanced
ß-adrenergic signaling, the hearts of the transgenic animals may
express proapoptotic factors and become sensitive to
apoptotic stimuli from a variety of environmental sources.
Alternatively, it is possible that the apoptotic program is
only initiated after cardiac dysfunction develops (ie, that
environmental factors created by the cardiomyopathy
in turn initiate signals that activate the apoptotic
program, namely, an altered neurohormonal milieu).
We performed in vitro experiments to test the above possibilities. In
particular, we examined whether apoptosis could be induced in
the Gs-overexpressing myocytes in vitro by assessing the effects of
short-term potent ß-adrenergic stimulation on myocyte death in
culture. The results from our in vitro experiments demonstrate that
stimulation with the ß-adrenergic agonist and G-protein
activator isoproterenol promotes myocyte apoptosis
in Gs myocytes but not in myocytes from wild-type controls. Hence,
overexpression of Gs appears to increase the apoptotic
response of myocytes to isoproterenol. This finding extends a previous
report by Mann et al5 demonstrating that
catecholamine stimulation is deleterious and can induce
death of cardiac myocytes in culture.
Results from recent studies on cAMP-mediated cell death by
apoptosis are controversial. cAMP reportedly induces
apoptosis of cultured Schwann cells20 and
contributes to apoptosis of human thymocytes,21
but it has also been shown to prevent T cell receptor–mediated
apoptosis22 and reduce apoptosis mediated
by atrial natriuretic peptide in myocytes.23
Thus, the role of AC and cAMP in regulation of myocyte
apoptosis appears controversial and potentially not
inconsistent with our findings of Gs-mediated
apoptosis, since isoproterenol mediates a significant increase
in L-type Ca2+ channel activity and in the
Ca2+ transient in Gs-overexpressing myocytes
as compared with wild-type controls (Reference 2424 and S.-J. Kim et al,
unpublished data, 1998).
A variety of factors have been reported to trigger cell death via
apoptosis in the heart, including reperfusion injury after
ischemia, myocardial infarction,5 25 26 pressure
overload,27 and mechanical stretch.28 A
recent report has shown that transgenic overexpression of an
activated form of the G protein, Gq, can induce
cardiomyopathy and myocyte
apoptosis.29 The present study provides
evidence that acceleration of myocyte apoptosis also occurs
secondary to overexpression of Gs, a protein that promotes
ß-adrenergic signaling and thereby hyperresponsiveness to
catecholamines. Our finding that mature myocytes exhibit
apoptosis provides a potential explanation to the paradox of
myocyte hypertrophy without an increase in heart weight
that occurs in the older Gs transgenic mice. It is thus plausible
that the cellular hypertrophy was offset by a decrease in
myocyte number caused by apoptosis, explaining the lack of an
overall increase in heart weight in the transgenic mice, in spite of
increased myocyte size. This consequence of cardiac Gs
overexpression is particularly interesting in light of our present
understanding of heart failure and the factors that contribute to its
progression. Two opposing views currently exist with regard to the
activity of the ßAR-Gs-AC signaling pathway in both the development
and progression of heart failure. One holds that
catecholamine stimulation may be useful for the treatment
of heart failure.10 Another view suggests that chronic
sympathetic nerve stimulation is disadvantageous and that inhibition of
ßAR activity with antagonists has beneficial effects over
the long term.30 31 Our findings in the Gs transgenic
mice favor the latter possibility and support the hypothesis that
chronic, heightened ß-adrenergic signaling can result in
apoptosis in the heart, leading to myocyte loss and potentially
contributing to cardiac decompensation. Enhanced apoptosis
occurs not only in this murine model of
cardiomyopathy but also in human idiopathic dilated
cardiomyopathy32 33 and chronic heart
failure,33 34 35 thus implicating apoptosis in the
development of or progression of human
cardiomyopathy and heart failure.
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-33107, HL-59139, HL-33065, and AG14121, and by an American Heart Association Grant-in-Aid Affiliate (Pennsylvania) Award. We thank Dr Peter Libby, Brigham and Women's Hospital, Harvard Medical School, for his comments and support for this study.
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Footnotes |
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This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received January 6, 1998; accepted October 26, 1998.
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