© 2000 American Heart Association, Inc.
Integrative Physiology |
Calcineurin-Mediated Hypertrophy Protects Cardiomyocytes From Apoptosis In Vitro and In Vivo
An Apoptosis-Independent Model of Dilated Heart Failure
From the Departments of Pediatrics (L.J.D.W., H.W.L., T.T., J.D.M.) and Cardiology (G.W.D.), University of Cincinnati, and Division of Molecular Cardiovascular Biology (L.J.D.W., H.W.L., T.T., J.D.M.), Children’s Hospital Medical Center, Cincinnati, Ohio; Departments of Medicine and Cell Biology (D.W., R.N.K.), Albert Einstein College of Medicine, The Bronx, NY; and Kimmel Cancer Center and Department of Microbiology and Immunology (G.C.), Thomas Jefferson University, Philadelphia, Pa.
Correspondence to Jeffery D. Molkentin, Department of Pediatrics, Division of Molecular Cardiovascular Biology, Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail molkj0{at}chmcc.org
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Abstract |
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Abstract—We have previously shown that the calcium-calmodulin–regulated phosphatase calcineurin (PP2B) is sufficient to induce cardiac hypertrophy that transitions to heart failure in transgenic mice. Given the rapid onset of heart failure in these mice, we hypothesized that calcineurin signaling would stimulate myocardial cell apoptosis. However, utilizing multiple approaches, we determined that calcineurin-mediated hypertrophy protected cardiac myocytes from apoptosis, suggesting a model of heart failure that is independent of apoptosis. Adenovirally mediated gene transfer of a constitutively active calcineurin cDNA (AdCnA) was performed in cultured neonatal rat cardiomyocytes to elucidate the mechanism whereby calcineurin affected myocardial cell viability. AdCnA infection, which induced myocyte hypertrophy and atrial natriuretic factor expression, protected against apoptosis induced by 2-deoxyglucose or staurosporine, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) labeling, caspase-3 activation, DNA laddering, and cellular morphology. The level of protection conferred by AdCnA was similar to that of adenoviral Bcl-xL gene transfer or hypertrophy induced by phenylephrine. In vivo, failing hearts from calcineurin-transgenic mice did not demonstrate increased TUNEL labeling and, in fact, demonstrated a resistance to ischemia/reperfusion–induced apoptosis. We determined that the mechanism whereby calcineurin afforded protection from apoptosis was partially mediated by nuclear factor of activated T cells (NFAT3) signaling and partially by Akt/protein kinase B (PKB) signaling. Although calcineurin activation protected myocytes from apoptosis, inhibition of calcineurin with cyclosporine was not sufficient to induce TUNEL labeling in Gq
-transgenic mice or in cultured cardiomyocytes.
Collectively, these data identify a calcineurin-dependent mouse model
of dilated heart failure that is independent of apoptosis.
Key Words: calcineurin • apoptosis • cardiac hypertrophy • phenylephrine • caspase-3
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Introduction |
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Heart failure can result from a wide range of pathological conditions such as infarction, chronic left ventricular hypertrophy, ischemic cardiomyopathy, idiopathic dilated cardiomyopathy, hypertrophic cardiomyopathy, valvular heart disease, and viral infection. In response to these pathologic conditions, cardiomyocytes may undergo apoptosis, or programmed cell death, which has been postulated to contribute to the progressive pathology associated with heart failure.1 2 3 4 Apoptosis is an energy-requiring process that results in cellular shrinkage, nuclear condensation, and DNA fragmentation in response to developmental cues or to activation/inhibition of specific reactive signaling cascades (reviewed in References 5 and 65 6 ).
Increased hypertrophic signaling via either of the heterotrimeric G
proteins, Gq or Gs, in transgenic mouse hearts was shown to
promote cardiomyocyte apoptosis and
cardiomyopathy, suggesting that overstimulation of
G protein–coupled receptors might promote cell death.7 8
Consistent with this interpretation, pharmacological
stimulation of ß-adrenergic receptors in vivo caused
apoptosis independent of left ventricular
hypertrophy or tachycardia.9 In
vitro studies in cultured primary cardiomyocytes also
implicate certain signaling pathways in apoptosis.
Stretch-mediated release of angiotensin II is associated
with p53 activation and cardiomyocyte
apoptosis.10 11 Similarly, atrial
natriuretic factor (ANF) and tumor necrosis factor-
likely promote cardiomyocyte
apoptosis.12 13 Mitogen-activated protein
kinase (MAPK) activation is also thought to be a regulator of
apoptosis. Specifically, activation of p38 may promote
cardiomyocyte apoptosis.14 15
In contrast, numerous studies have demonstrated protection from apoptosis through activation of certain intracellular signaling pathways. Cardiotrophin-1 signaling through the gp130 receptor was shown to protect cardiomyocytes from apoptosis.16 Induction of cardiac hypertrophy through MKK6 and P38ß has also been associated with protection from apoptosis.14 15 Insulin-like growth factor-1 (IGF-1) stimulation protects cardiomyocytes from apoptosis through phosphatidylinositol 3-kinase and MAPK-dependent signaling pathways.17 18 19 Hypertrophic agonists such as phenylephrine (PE) and endothelin-1 are also associated with protection from apoptosis, suggesting that hypertrophy itself may confer protection.20
We recently described a novel mediator of cardiac hypertrophy through the calcium-calmodulin–regulated intracellular phosphatase calcineurin (PP2B) and the transcriptional regulator nuclear factor of activated T cells (NFAT3).21 Expression of a constitutively active form of calcineurin in transgenic mouse hearts resulted in profound cardiac hypertrophy that progressed to dilated heart failure within 8 to 12 weeks.21 It was of interest to determine whether hypertrophic signaling mediated by calcineurin induced apoptosis, which might explain the rapid progression to heart failure in these mice. However, we determined that cardiomyocyte terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling (TUNEL) in calcineurin-transgenic hearts is not significantly different from that in wild-type hearts. In fact, calcineurin-transgenic hearts were significantly less susceptible to ischemia/reperfusion–induced apoptosis than were wild-type hearts. To define the mechanism whereby calcineurin signaling might protect cardiac myocytes from apoptosis, we generated a calcineurin adenovirus (AdCnA) to infect cultured rat neonatal cardiomyocytes. AdCnA infection induced a hypertrophic response that was protective against staurosporine- and 2-deoxyglucose–induced apoptosis. The likely mechanism whereby calcineurin protects myocytes from apoptosis is associated with NFAT3 and Akt/protein kinase B (PKB) activation.
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Materials and Methods |
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The mouse model of ischemia/reperfusion by ligation of the left descending coronary artery was performed as described previously.22 Primary cultures of neonatal rat cardiomyocytes were obtained as described previously.23 E1a-deleted, replication-deficient adenovirus expressing a constitutively activated form of mouse calcineurin A
amino acids 1 to 398 (AdCnA), full-length human
Bcl-xL (AdBcl-xL), or a
constitutively nuclear form of NFAT3 (AdNFAT3) was initially
constructed in pACCMV-pLpA and cotransfected into HEK293 cells with
pJM17 as described before.24 For the generation of the
adenovirus expressing a calcineurin-inhibitory peptide
(Adcain), a 582-bp fragment corresponding to amino acids 1989 to 2182
of cain25 26 was generated by polymerase chain reaction
and subcloned with a flag epitope as a HindIII fragment into
pACCMVpLpA. All initial recombinants were plaque-purified, expanded,
and titered by duplicate plaque assays in monolayers of HEK293 using
the agarose gel overlay method.27 Adenoviral
infection of cardiomyocytes was performed at a multiplicity
of infection of 100 plaque-forming units in 2 mL (6-cm culture dishes)
of DMEM supplemented with 2% FBS for 2 hours at 37°C in a
humidified, 5% CO2 incubator. Under these
infection conditions, 
99% of the cells were positive for protein
expression by immunocytochemistry or stained ß-galactosidase positive
after 24 hours. TUNEL labeling of cultured cardiomyocytes
or tissue sections was performed with the CardioTACS kit (Trevigen) as
recommended by the manufacturer. Cardiomyocytes were prepared for
immunocytochemistry as described previously.21 Data are
expressed as mean±SEM, and differences were evaluated for significance
using the Student t test for unpaired data or 1-way ANOVA
followed by the Bonferroni post test when appropriate. An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Calcineurin Adenovirus Drives Neonatal Cardiomyocyte Hypertrophy In Vitro
To investigate the importance of calcineurin signaling in vitro, we generated a replication-deficient adenovirus expressing the constitutively active form of mouse calcineurin A
(AdCnA) (amino
acids 1 to 398) to perform gene transfer in cultured cardiac myocytes.
Consistent with the effect of activated calcineurin
expression in vivo, AdCnA induced morphological
hypertrophy, whereas control infection with a
ß-galactosidase adenovirus (Adßgal) had no effect (Figure 1A
through 1H). The hypertrophic
response was independent of cellular density and produced a
quantitative increase in cell surface area 96 hours after infection
(Figure 1I). AdCnA-infected cardiomyocytes also
demonstrated greater sarcomeric organization (Figure 1E and 1F),
rhythmic beating 24 hours after infection, and ANF protein expression
(Figure 1K). Quantification of ANF expression in a
representative experiment revealed that among
cardiomyocytes, 9.2% (±2.4%) of those infected with
Adßgal, 39% (±2.5%) of those infected with AdCnA, and 54%
(±2.7%) of those treated with PE were positive (P<0.05).
These data indicate that expression of activated calcineurin is
sufficient to drive the hypertrophic program in cultured neonatal
cardiomyocytes.
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Calcineurin Adenoviral Infection Protects Cardiomyocytes From
Apoptosis
Activation of certain intracellular signaling pathways in
cardiomyocytes has been shown to profoundly affect
myocardial cell viability. It was of interest to determine whether
constitutive calcineurin activation would promote or protect
cardiomyocytes from cell death. To this end, we determined
that AdCnA infection of cultured neonatal cardiomyocytes
did not induce apoptosis over any time period compared with
Adßgal infection or uninfected cells (data not shown). In contrast,
AdCnA infection was found to protect cultured
cardiomyocytes from apoptosis induced by 2
different pharmacological agents. Cells were cultured for 24 hours
after adenoviral infection and treated with either
staurosporine (1.0 µmol/L) or 2-deoxyglucose (2
mmol/L) in glucose- and serum-free medium. Staurosporine
was previously shown to induce cardiomyocyte
apoptosis in a caspase-dependent manner.28
Similarly, metabolic inhibition with 2-deoxyglucose was
also shown to induce apoptosis in cultured
cardiomyocytes.29 30 The data show that AdCnA
infection protected cardiomyocytes from morphological
features associated with cell death, whereas Adßgal infection had no
protective effect (Figure 2C, 2D, 2G, and 2H). As a positive control for these assays, we generated an
adenovirus expressing the antiapoptotic gene
Bcl-xL. AdBcl-xL infection
also protected cardiomyocytes from both
staurosporine- and 2-deoxyglucose–mediated effects (Figure 2E and 2F). As a further control, the
calcineurin-inhibitory drug cyclosporine was
added to AdCnA-infected cardiomyocytes, resulting in a
reversal of protection. However, cyclosporine alone did not
induce apoptosis in untreated cardiomyocytes in
serum-free medium (data not shown).
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TUNEL assays were performed to quantify the induction of cell death by
staurosporine and 2-deoxyglucose. Only 2% to 4% of
nontreated cardiomyocytes were TUNEL-positive, whereas
staurosporine or 2-deoxyglucose treatment induced 17% and
26% TUNEL-positive staining, respectively (P<0.05) (Figure 3). Consistent with cellular
morphology shown above, AdCnA and AdBcl-xL
infection significantly reduced staurosporine- and
2-deoxyglucose–induced cell death, whereas control Adßgal infection
had no protective effect (P<0.05) (Figure 3).
Adenoviral delivery routinely resulted in 98% to 99%
cardiomyocyte infection, so that uninfected cells did not
appreciably influence quantification. The protective effect of AdCnA
was only partially reversed by cyclosporine
(P<0.05), likely because this agent is not a complete
inhibitor of calcineurin. This interpretation is
consistent with the known profile of only partial immune
suppression in vivo, even at a high dosage of
cyclosporine.31 AdCnA infection also
significantly protected cardiomyocytes from
staurosporine- or 2-deoxyglucose–induced caspase-3
activation, poly(ADP-ribose) polymerase and protein kinase C
degradation, and DNA laddering compared with Adßgal infection (data
not shown). We and others have previously characterized that
2-deoxyglucose or staurosporine treatment of cultured
cardiomyocytes results in caspase activation and cytochrome
c release.28 29 30
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P<0.05 vs Adßgal,
*P<0.05 vs control, #P<0.05 vs
AdCnA.
Activation of Endogenous Calcineurin Protects Myocytes
From Apoptosis
It was previously reported that -adrenergic stimulation (PE)
protected cultured cardiomyocytes from
apoptosis.20 We have determined that PE
stimulation leads to calcineurin activation in cultured
cardiomyocytes.31a To investigate the
potential involvement of calcineurin in PE-mediated protection from
apoptosis, we treated cardiomyocytes with
cyclosporine or a calcineurin-inhibitory
adenovirus. As previously reported, we also found that PE significantly
protected cardiomyocytes from staurosporine-
and 2-deoxyglucose–induced cell death (Figure 3
). Remarkably,
inhibition of calcineurin with cyclosporine partially
reversed the antiapoptotic effects of PE to 2-deoxyglucose
(Figure 4). Because
cyclosporine is known to affect proteins other than
calcineurin, we also specifically targeted calcineurin by adenoviral
expression of a 194–amino acid inhibitor peptide from the
cain protein.25 26 Adcain infection significantly reversed
the antiapoptotic effects of PE hypertrophy to
2-deoxyglucose (Figure 4), whereas Adßgal had no effect.
Adcain infection alone in serum-free medium did not induce cell death
(data not shown). These data indicate that calcineurin is a necessary
component of PE-mediated protection from cardiomyocyte cell
death in vitro. These data also indicate that
physiological activation of endogenous
calcineurin affords protection from cell death.
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It was also of interest to determine whether MAPK signaling factors
played a role in PE-mediated protection from apoptosis.
Inhibition of p38-reactive signaling with SB202190 (20 µmol/L)
did not reverse the protective effect of PE, whereas inhibition of
extracellular signal–regulated kinase (ERK) signaling with the
mitogen-activated protein/ERK (MEK)–1 inhibitor
PD98059 (25 µmol/L) partially reversed the protective effect of
PE (Figure 4). These data indicate that ERKs, but not p38, are
components of PE-mediated protection from cardiomyocyte
apoptosis. However, the hierarchy between calcineurin and MAPK
signaling pathways in regulating the hypertrophic program or protection
from apoptosis is currently unknown.
Because NFAT3 acts downstream of calcineurin in mediating cardiac
hypertrophy,21 we investigated the ability of
NFAT3 to protect cardiomyocytes from apoptosis
induced by 2-deoxyglucose using an adenovirus expressing the same
truncated NFAT3 cDNA that promoted cardiac hypertrophy in
transgenic mice.21 AdNFAT3 infection partially protected
cardiomyocytes from 2-deoxyglucose–induced
apoptosis (Figure 5). These data
suggest that NFAT3 is partially responsible for mediating the
antiapoptotic effects of calcineurin activation in
cardiomyocytes. However, AdNFAT3 infection was not as
protective as AdCnA infection, suggesting that calcineurin provides
protection by additional mechanisms.
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To this end, we examined the levels or activation state of multiple
proteins known to promote cell survival. Although the
phosphorylation state of BAD and the protein amounts of
Bcl-2, Bcl-xL, and Bax proteins were unchanged,
the Akt/PKB phosphorylation state was found to be
enhanced. Akt was previously shown to directly promote cell viability
through multiple mechanisms.32 33 34 We observed that PE
stimulation for 3 hours was sufficient to induce Akt
phosphorylation, but that inhibition of calcineurin
with cyclosporine blocked Akt
phosphorylation (Figure 6A). Augmentation of intracellular
calcium levels with the ionophore A23187, a known inducer of
calcineurin, stimulated Akt phosphorylation after 6
hours (Figure 6B). This induction of Akt after 6 hours by A23187
was blocked by cyclosporine, indicating that calcineurin
was partially regulating Akt activation. Finally, transgenic mice
expressing the activated calcineurin cDNA were analyzed
for Akt activation. We observed a significant increase in Akt
phosphorylation in calcineurin-transgenic hearts at 8
weeks, but not 8 days (Figure 6C). The increase in Akt
phosphorylation at 8 weeks is associated with the onset
of heart failure in these mice, whereas day 8 is prehypertrophic.
Collectively, these data indicate that Akt is partially responsible for
mediating the antiapoptotic effects of calcineurin in vitro and
in vivo.
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Calcineurin-Transgenic Mice Show Dilated Heart Failure
We have previously reported that transgenic mice expressing the
constitutively active calcineurin cDNA in the heart develop profound
hypertrophy characterized by a 2- to 3-fold increase in
heart-to-body-weight ratio, which rapidly progresses to dilated heart
failure.21 To analyze this effect in more detail,
we performed echocardiography on 8-week-old
transgenic mice (mouse line 37). A
representative M-mode tracing demonstrates the severity
of heart failure in these mice (Figure 7). Fractional shortening was
consistently reduced by 70% to 80% by 8 weeks of age compared
with nontransgenic littermate controls (S.A. Witt, T.R. Kimball, and
J.D. Molkentin, unpublished results, 1998).
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Absence of Increased Cardiomyocyte TUNEL Staining in
Calcineurin-Transgenic Hearts
Because dilated heart failure is associated with increased
cardiomyocyte apoptosis, we reasoned that failing
calcineurin-transgenic hearts would show enhanced cell death. However,
an exhaustive histologic analysis of TUNEL-stained 8-week-old
hearts failed to reveal a significant difference in
cardiomyocyte TUNEL reactivity between transgenic and
wild-type mice (Figure 8A). In contrast,
calcineurin-transgenic hearts showed a statistically significant
increase in nonmyocyte TUNEL staining, likely because of the
increase in interstitial space associated with mild edema,
immune cell infiltration, and heart failure (P<0.05)
(Figure 8B). A typical TUNEL-stained
histological section from a calcineurin-transgenic and
a nontransgenic heart demonstrates predominantly
interstitial cell reactivity (blue) (Figures 8C and 8D).
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Calcineurin Protects Against Apoptosis Occurring After
Ischemia/Reperfusion
To test the hypothesis that activated calcineurin might
protect cardiomyocytes from apoptosis in vivo,
calcineurin-transgenic and wild-type hearts were subjected to
ischemia/reperfusion–induced cell death. Temporary occlusion
of the left coronary artery followed by reperfusion is known to
potently induce cardiomyocyte
apoptosis.17 35 Cardiac ischemia followed
by reperfusion in nontransgenic control mice produced substantial DNA
laddering, indicating activation of apoptosis (Figure 9). However, ischemia/reperfusion
of calcineurin-transgenic hearts demonstrated substantial protection
against typical DNA fragmentation, indicating that calcineurin-mediated
hypertrophy protects cardiomyocytes from
apoptotic stimuli in vivo.
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Cyclosporine Administration Does Not Induce
Apoptosis in Gq-transgenic Mice
The observation that calcineurin protects
cardiomyocytes from apoptosis in vivo might suggest
that cyclosporine would be proapoptotic. To
investigate this possibility in vivo, we treated Gq-transgenic mice
with cyclosporine for 8 weeks. Gq-transgenic mice were
chosen because they are uniquely sensitized to apoptotic
stimulation such that increased transgene number or the postpartum
period induces substantial apoptosis.7 However,
hearts from cyclosporine-treated Gq-transgenic mice,
or wild-type hearts, did not demonstrate increased TUNEL labeling
(Figure 10). The quantification
of TUNEL labeling was performed in a blinded manner. As a positive
control for increased TUNEL labeling, reactivity was quantified in a
single Gq heart from an aortic-banded animal. Aortic banding is a
potent stimulus for apoptosis in Gq-transgenic mice (Figure 10) (J. Ross and G.W. Dorn II, unpublished data, 1999).
Collectively, these data indicate that cyclosporine is not
sufficient to induce apoptosis in vivo or in vitro. This
suggests that, whereas calcineurin activation protects myocytes from
cell death, its inhibition is not sufficient to induce
apoptosis in vivo.
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Discussion |
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In this study, we provide multiple lines of evidence that calcineurin protects cardiomyocytes from apoptosis both in vivo and in vitro. First, apoptosis induced by ischemia/reperfusion injury was largely rescued in calcineurin-transgenic hearts compared with nontransgenic hearts. Second, AdCnA infection protected cardiomyocytes in culture from TUNEL labeling, caspase activation, and DNA laddering. Third, direct inhibition of endogenous calcineurin either with cyclosporine or with a noncompetitive peptide inhibitor reversed the antiapoptotic effects of PE stimulation. We demonstrate calcineurin-dependent protection from apoptosis both in vivo and in vitro either through expression of an activated calcineurin mutant or by activation of endogenous calcineurin. Taken together, these data establish a role for calcineurin signaling in protecting myocardial cells from apoptotic insults. These results also establish a mouse model of dilated heart failure that is independent of apoptosis.
Inspection of the literature suggests that humans in dilated heart
failure and many animal models that mimic human disease have increased
numbers of apoptotic myocytes (reviewed in Reference 3636 ). These
cumulative studies have suggested a causal relationship between the
onset of apoptosis and progression of heart failure.
Calcineurin-transgenic mice develop dramatic concentric
hypertrophy (3-fold increase in heart size) that
transitions to dilated heart failure by 8 weeks of life. Despite this
phenotype, dilated and failing calcineurin-transgenic hearts do
not show increased TUNEL labeling, but instead show resistance to
ischemia/reperfusion–induced DNA laddering. This predicts that
calcineurin-transgenic mice are a nonapoptotic model of heart
failure. However, the causality of heart failure in
calcineurin-transgenic mice is likely linked to other pathologies.
First, the extreme degree of cardiac hypertrophy in
calcineurin-transgenic mice is predicted to negatively impact function
based on geometric constraints. Second, the profound
hypertrophy in calcineurin mice is also associated with
bradycardia and arrhythmia (Figure 7, data not shown).
Third, calcineurin-transgenic hearts show considerable
interstitial cell fibrosis.21 Finally, the
default response of the myocardium when unable to
hypertrophy further may simply be to dilate. It is likely
that these factors, in the absence of apoptosis, are sufficient
to induce heart failure.
The relationship between hypertrophic signaling pathways and the
regulation of apoptosis is rather complex. Whereas some studies
have shown antiapoptotic effects associated with various
hypertrophic stimuli, other studies have shown induction of
apoptosis by certain reactive signaling pathways. Among these
proapoptotic pathways are the heterotrimeric GTP-binding
proteins Gq and Gs.7 8 Similarly,
cardiomyocyte apoptosis has been shown to be
augmented by p38, ß-adrenergic receptors, NO synthase activity,
atrial natriuretic peptide, tumor necrosis factor-,
angiotensin II, overexpression of the transcription factor
Id, and p53.9 10 11 12 13 15 37 38 39 Taken together, it appears
that the regulation of cardiac myocyte apoptosis in the
hypertrophied myocardium is complex and depends on the
relative activation pattern of distinct signaling pathways, with Gq,
Gs, p53, and p38 stimulation promoting cardiomyocyte
death and calcineurin, NFAT3, p38ß, IGF-1/phosphatidylinositol
3-kinase/Akt, and ERK activation promoting
cardiomyocyte survival.
It is difficult to dissect the direct mechanisms whereby calcineurin
protects myocytes from apoptosis, because calcineurin
activation also induces significant hypertrophy, which
itself may afford protection. Previous studies have demonstrated that
certain hypertrophic agonists or activation of certain reactive
signaling pathways provides protection from
apoptosis.14 15 16 17 18 19 20 Although PE, endothelin-1,
cardiotrophin-1 (gp130), IGF-1, ERK activation, and p38ß activation
have all been shown to be protective, a correlation with cell
death–regulatory pathways (caspases or death receptors) or with
proteins that regulate mitochondrial integrity (Bcl-2) has not been
described.14 15 16 17 18 19 20 The direct mechanisms whereby certain
reactive signaling pathways protect cardiomyocytes from
apoptosis are largely uncharacterized. Similarly,
calcineurin-transgenic hearts and AdCnA-infected
cardiomyocytes did not demonstrate a perturbation in the
Bcl-2–to–Bax ratio or the Bcl-xL–to–Bax
ratio. However, one mechanism whereby calcineurin signaling directly
protects cardiac myocytes is through activation of PKB/Akt. Akt is
known to downregulate caspase-9 activation, phosphorylate
BAD, and directly activate nuclear factor 
B to promote cell
survival.32 33 34 Although we have demonstrated that Akt
activation is associated with calcineurin-induced
hypertrophy, it is not clear how this activation occurs.
However, it is likely that calcineurin signaling first
activates the hypertrophic program, which then indirectly leads
to Akt activation, providing protection from cell death. NFAT3
adenoviral gene transfer also provided partial protection from
2-deoxyglucose–induced TUNEL labeling, suggesting an additional
mechanism whereby calcineurin provides protection. However, the direct
mechanism whereby NFAT3 provides protection from cell death is unknown,
but it can be speculated that protection is linked to the hypertrophic
response itself.
The observation that calcineurin protects cardiomyocytes
from apoptosis might also suggest that cyclosporine
treatment could promote myocardial cell apoptosis in vivo.
However, patients on chronic cyclosporine therapy have not
been reported to be at increased risk for heart failure, although an
increased propensity toward hypertrophy has been
described.40 We directly addressed this issue
experimentally by treating a mouse model that is prone to
apoptosis (Gq) with cyclosporine at 20 mg/kg per
day for 8 weeks.7 Characterization of TUNEL labeling did
not reveal an induction of cell death in wild-type or Gq-transgenic
mice with cyclosporine, yet aortic banding induced a
profound increase (Figure 10, J. Ross and G.W. Dorn,
unpublished data, 1999). In other cell types,
cyclosporine has been shown to inhibit apoptosis by
preventing mitochondrial pore transition, which is regulated by
cyclophilin-D (reviewed in Reference 4141 ). These data suggests that
cyclosporine has diverse intracellular effects, some of
which protect cells and others of which may be proapoptotic.
Indeed, calcineurin was recently shown to either protect cells from
apoptosis or to directly induce apoptosis depending on
cross talk between other intracellular signaling
pathways.42
Calcineurin has been shown to promote viability in multiple cell types by suppressing apoptosis,43 44 45 46 whereas other studies have shown that calcineurin can activate apoptosis in disparate cell types.47 48 49 50 These differing accounts suggest that the role of calcineurin in controlling cell viability is complex and integrated with other signaling pathways. In cardiomyocytes, it is likely that calcineurin functions in concert with other reactive signaling pathways to achieve a balanced activation of cellular hypertrophy, which is beneficial to cellular viability.
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Acknowledgments |
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This work was supported by NIH Grants HL-69562 and HL-62927, a Scholar Award from the Pew Foundation, a local American Heart Association affiliate grant-in-aid (to J.D.M.), and NIH Grants HL60665 and HL61550 (to R.N.K.). H.W.L. was supported by NIH Training Grant T32 ES07051. We thank the veterinary staff at Children’s Hospital Medical Center for excellent technical assistance.
Received July 6, 1999; accepted November 17, 1999.
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M. A. Sussman, A. McCulloch, and T. K. Borg Dance Band on the Titanic: Biomechanical Signaling in Cardiac Hypertrophy Circ. Res., November 15, 2002; 91(10): 888 - 898. [Abstract] [Full Text] [PDF]
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O. F. Bueno and J. D. Molkentin Involvement of Extracellular Signal-Regulated Kinases 1/2 in Cardiac Hypertrophy and Cell Death Circ. Res., November 1, 2002; 91(9): 776 - 781. [Abstract] [Full Text] [PDF]
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 P A J Krijnen, R Nijmeijer, C J L M Meijer, C A Visser, C E Hack, and H W M Niessen Apoptosis in myocardial ischaemia and infarction J. Clin. Pathol., November 1, 2002; 55(11): 801 - 811. [Abstract] [Full Text] [PDF]
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 B. Fiedler, S. M. Lohmann, A. Smolenski, S. Linnemuller, B. Pieske, F. Schroder, J. D. Molkentin, H. Drexler, and K. C. Wollert Inhibition of calcineurin-NFAT hypertrophy signaling by cGMP-dependent protein kinase type I in cardiac myocytes PNAS, August 20, 2002; 99(17): 11363 - 11368. [Abstract] [Full Text] [PDF]
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M. J. Donelan, G. Morfini, R. Julyan, S. Sommers, L. Hays, H. Kajio, I. Briaud, R. A. Easom, J. D. Molkentin, S. T. Brady, et al. Ca2+-dependent Dephosphorylation of Kinesin Heavy Chain on beta -Granules in Pancreatic beta -Cells. IMPLICATIONS FOR REGULATED beta -GRANULE TRANSPORT AND INSULIN EXOCYTOSIS J. Biol. Chem., June 28, 2002; 277(27): 24232 - 24242. [Abstract] [Full Text] [PDF]
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S. E. Hardt and J. Sadoshima Glycogen Synthase Kinase-3{beta}: A Novel Regulator of Cardiac Hypertrophy and Development Circ. Res., May 31, 2002; 90(10): 1055 - 1063. [Abstract] [Full Text] [PDF]
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 B. J Wilkins and J. D Molkentin Calcineurin and cardiac hypertrophy: Where have we been? Where are we going? J. Physiol., May 15, 2002; 541(1): 1 - 8. [Abstract] [Full Text] [PDF]
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O. F Bueno, E. van Rooij, J. D Molkentin, P. A Doevendans, and L. J De Windt Calcineurin and hypertrophic heart disease: novel insights and remaining questions Cardiovasc Res, March 1, 2002; 53(4): 806 - 821. [Abstract] [Full Text] [PDF]
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 H. FAUVEL, P. MARCHETTI, G. OBERT, O. JOULAIN, C. CHOPIN, P. FORMSTECHER, and R. NEVIERE Protective Effects of Cyclosporin A from Endotoxin-induced Myocardial Dysfunction and Apoptosis in Rats Am. J. Respir. Crit. Care Med., February 15, 2002; 165(4): 449 - 455. [Abstract] [Full Text] [PDF]
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 C. GILL, R. MESTRIL, and A. SAMALI Losing heart: the role of apoptosis in heart disease--a novel therapeutic target? FASEB J, February 1, 2002; 16(2): 135 - 146. [Abstract] [Full Text] [PDF]
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C. L. Antos, T. A. McKinsey, N. Frey, W. Kutschke, J. McAnally, J. M. Shelton, J. A. Richardson, J. A. Hill, and E. N. Olson Activated glycogen synthase-3beta suppresses cardiac hypertrophy in vivo PNAS, January 7, 2002; (2002) 231619298. [Abstract] [Full Text] [PDF]
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Y. Shizukuda and P. M. Buttrick Protein kinase C-zeta modulates thromboxane A2-mediated apoptosis in adult ventricular myocytes via Akt Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H320 - H327. [Abstract] [Full Text] [PDF]
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J. L. Gooch, Y. Tang, J. M. Ricono, and H. E. Abboud Insulin-like Growth Factor-I Induces Renal Cell Hypertrophy via a Calcineurin-dependent Mechanism J. Biol. Chem., November 2, 2001; 276(45): 42492 - 42500. [Abstract] [Full Text] [PDF]
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J. D. Molkentin Calcineurin, Mitochondrial Membrane Potential, and Cardiomyocyte Apoptosis Circ. Res., June 22, 2001; 88(12): 1220 - 1222. [Full Text] [PDF]
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 B. J. ARONOW, T. TOYOKAWA, A. CANNING, K. HAGHIGHI, U. DELLING, E. KRANIAS, J. D. MOLKENTIN, and G. W. DORN II Divergent transcriptional responses to independent genetic causes of cardiac hypertrophy Physiol Genomics, June 6, 2001; 6(1): 19 - 28. [Abstract] [Full Text] [PDF]
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L. J. De Windt, H. W. Lim, O. F. Bueno, Q. Liang, U. Delling, J. C. Braz, B. J. Glascock, T. F. Kimball, F. del Monte, R. J. Hajjar, et al. Targeted inhibition of calcineurin attenuates cardiac hypertrophy invivo PNAS, March 13, 2001; 98(6): 3322 - 3327. [Abstract] [Full Text] [PDF]
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O. F. Bueno, L. J. De Windt, H. W. Lim, K. M. Tymitz, S. A. Witt, T. R. Kimball, and J. D. Molkentin The Dual-Specificity Phosphatase MKP-1 Limits the Cardiac Hypertrophic Response In Vitro and In Vivo Circ. Res., January 19, 2001; 88(1): 88 - 96. [Abstract] [Full Text] [PDF]
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J. N. Muth, I. Bodi, W. Lewis, G. Varadi, and A. Schwartz A Ca2+-Dependent Transgenic Model of Cardiac Hypertrophy : A Role for Protein Kinase C{{alpha}} Circulation, January 2, 2001; 103(1): 140 - 147. [Abstract] [Full Text] [PDF]
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S. F. Steinberg The Cellular Actions of {beta}-Adrenergic Receptor Agonists : Looking Beyond cAMP Circ. Res., December 8, 2000; 87(12): 1079 - 1082. [Full Text] [PDF]
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J. D. Molkentin Calcineurin and Beyond : Cardiac Hypertrophic Signaling Circ. Res., October 27, 2000; 87(9): 731 - 738. [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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L. J. De Windt, H. W. Lim, S. Haq, T. Force, and J. D. Molkentin Calcineurin Promotes Protein Kinase C and c-Jun NH2-terminal Kinase Activation in the Heart. CROSS-TALK BETWEEN CARDIAC HYPERTROPHIC SIGNALING PATHWAYS J. Biol. Chem., April 28, 2000; 275(18): 13571 - 13579. [Abstract] [Full Text] [PDF]
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S. Saito, Y. Hiroi, Y. Zou, R. Aikawa, H. Toko, F. Shibasaki, Y. Yazaki, R. Nagai, and I. Komuro beta -Adrenergic Pathway Induces Apoptosis through Calcineurin Activation in Cardiac Myocytes J. Biol. Chem., October 27, 2000; 275(44): 34528 - 34533. [Abstract] [Full Text] [PDF]
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H. W. Lim, L. New, J. Han, and J. D. Molkentin Calcineurin Enhances MAPK Phosphatase-1 Expression and p38 MAPK Inactivation in Cardiac Myocytes J. Biol. Chem., May 4, 2001; 276(19): 15913 - 15919. [Abstract] [Full Text] [PDF]
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C. L. Antos, T. A. McKinsey, N. Frey, W. Kutschke, J. McAnally, J. M. Shelton, J. A. Richardson, J. A. Hill, and E. N. Olson Activated glycogen synthase-3beta suppresses cardiac hypertrophy in vivo PNAS, January 22, 2002; 99(2): 907 - 912. [Abstract] [Full Text] [PDF]
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T. Kakita, K. Hasegawa, E. Iwai-Kanai, S. Adachi, T. Morimoto, H. Wada, T. Kawamura, T. Yanazume, and S. Sasayama Calcineurin Pathway Is Required for Endothelin-1-Mediated Protection Against Oxidant Stress-Induced Apoptosis in Cardiac Myocytes Circ. Res., June 22, 2001; 88(12): 1239 - 1246. [Abstract] [Full Text] [PDF]
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