© 1999 American Heart Association, Inc.
Original Contribution |
Modulation of Cytokine-Induced Cardiac Myocyte Apoptosis by Nitric Oxide, Bak, and Bcl-x
From the Departments of Molecular and Cellular Pharmacology and Medicine (J.Z., K.A.W., N.H.B.), University of Miami School of Medicine, Miami, Fla; Falk Cardiovascular Research Center (D.J.I.), Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, Calif; and Department of Medicine (V.J.D.), Brigham and Women's Hospital, Boston, Mass.
Correspondence to Nanette H. Bishopric, MD, FACC, Associate Professor of Pharmacology and Medicine, Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine (R-189), PO Box 016189, Miami, FL 33101. E-mail nhb{at}chroma.med.miami.edu
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Abstract—Cytokine-induced NO production depresses myocardial contractility and has been shown to be cytotoxic to cardiac myocytes. However, the mechanisms of cytokine-induced cardiac myocyte cell death are unclear. To analyze these mechanisms in detail, we treated neonatal cardiac myocytes in serum-free culture with a combination of the macrophage-derived cytokines interleukin-1ß, tumor necrosis factor-
, and interferon-
. These cytokines
caused a time-dependent induction of cardiac myocyte apoptosis,
but not necrosis, beginning 72 hours after treatment, as determined by
nuclear morphology, DNA internucleosomal cleavage, and cleavage of
poly(ADP-ribose) polymerase, reflecting caspase activation.
Apoptosis was preceded by a >50-fold induction of inducible NO
synthase mRNA and the release of large amounts (5 to 8 nmol/µg
protein) of NO metabolites (NOx) into the medium. Cell death was
completely blocked by an NO synthase inhibitor and
attenuated by antioxidants (N-acetylcysteine and DTT)
and the caspase inhibitor ZVAD-fmk. Cytokines also
mediated an NO-dependent, sustained increase in myocyte expression of
the Bcl-2 homologs Bak and Bcl-x(L). The NO donor
S-nitrosoglutathione also induced apoptosis and
cell levels of Bak, but not of Bcl-x(L). All effects of
cytokines, including poly(ADP-ribose) polymerase cleavage,
could be attributed to interleukin-1ß; interferon- and tumor
necrosis factor- had no independent effects on apoptosis or
on NOx production. We conclude that cytokine toxicity
to neonatal cardiac myocytes results from the induction of NO and
subsequent activation of apoptosis, at least in part through
the generation of oxygen free radicals. The rate and extent of this
apoptosis is modulated by alterations in the cellular balance
of Bak and Bcl-x(L), which respond differentially to
cytokine-induced and exogenous NO and by the availability
of oxidant species.
Key Words: poly(ADP-ribose) polymerase • protein kinase G • nitric oxide • Bcl-x(L) • oxidative stress
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Introduction |
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Nitric oxide, a short-lived free radical–generating gas, is an important signal-transduction molecule in many cell types, regulating such diverse functions as vasomotor tone, neurotransmission, mediation of immune responses, and inflammatory cell adhesion to the vessel wall.1 2 3 In the cardiac myocyte, the major physiological role of NO appears to involve depression of contractility and electrophysiological stabilization through elevation of intracellular cGMP.2 4 5 6 A rise in myocardial NO levels may account for the contractile depression observed during sepsis7 8 and in isolated cardiac myocytes following exposure to macrophage-conditioned medium9 10 ; endogenous NO production has been shown to mediate the negative chronotropic effects of carbachol in cardiac myocytes.11
NO is generated by 2 different enzymes in myocardium, which
are macrophage-type, or inducible NO synthase (iNOS), and
endothelial-type NO synthase (eNOS).12 13
iNOS differs from eNOS in being constitutively active and regulated
primarily through transcriptional means, while eNOS is present but
inactive until exposed to elevated cytosolic calcium. Most cardiac
myocyte NO is thought to be produced by iNOS. iNOS is strongly induced
in cardiac myocytes exposed to macrophage-derived
cytokines, including tumor necrosis factor (TNF)-,
interferon (IFN)-, and interleukin
(IL)-1ß,10 12 14 15 16 17 18 and during allograft rejection, in
which cytokines are present in high levels.19
This strong induction may be attributed to the presence of more than 22
immune effector–responsive elements in the iNOS promoter, including
binding sites for nuclear factor 
B and IFN regulatory
factor-1.20
The high levels of NO produced by iNOS are cytotoxic and are an important part of the host defense against microbial invasion in many species. Cytokine-induced NO is lethal to cardiac myocytes in vitro21 and to several other cell types.22 23 24 25 26 27 In vivo models of cardiac allograft rejection show a correlation between induction of iNOS and increased apoptosis,21 28 while forced expression of human eNOS in rat myocardium induces features of apoptosis in transfected cardiac myocytes and neighboring cells.29 Elevation of iNOS in chronic congestive heart failure may contribute to myocardial dysfunction and disease progression.30 However, the effects of NO are highly tissue specific and may promote survival in specific cell types, including endothelial cells.31 32 33 34 35
Recently, we showed that atrial natriuretic peptide (ANP), via a cGMP-dependent pathway, induces cardiac myocyte apoptosis, accompanied by increased expression of the apoptosis modulator Mcl-1.36 We reasoned that NO should also activate programmed cell death, since cGMP mediates many of the effects of NO in other cell types.37 38 The experiments presented here were designed to determine the contribution of NO to cytokine-induced cardiac myocyte death and to determine whether NO effector systems alter expression of apoptosis-modulating genes in the heart.
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Materials and Methods |
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Materials
Expression vectors encoding Bcl-2, Bax, Bak, and Bcl-x(L/S), and a monoclonal antibody to Bak, were the kind gift of Dr Michael Kiefer (LXR Biotechnology, Richmond, Calif). The cDNA probe for murine macrophage iNOS was generously provided by Dr Charles Lowenstein (The Johns Hopkins University, Baltimore, Md). Antibodies to Bcl-x(L/S), Bax, and Bcl-2 were obtained from Oncogene Science (Uniondale, NY). The human GAPDH cDNA was purchased from Clontech Laboratories (Palo Alto, Calif).
Hoechst 33342 and propidium iodide dyes were purchased from Molecular
Probes. Human TNF- and rat IFN- were obtained from Genzyme
(Cambridge, Mass). Recombinant human IL-1ß and the protein kinase
G-specific inhibitor KT5823 were obtained from Calbiochem
(La Jolla, Calif). All other reagents were purchased from Sigma except
where indicated and were of the highest grade available.
Cell Culture
All procedures involving animals were performed in accordance
with institutional guidelines for the care and use of animals. Methods
for primary culture of neonatal rat cardiac myocytes have been
previously described.39 In brief, enriched cultures of
myocyte and nonmyocyte cells were obtained from 1- to 2-day-old
neonatal rats by stepwise trypsin dissociation and plated at a density
of 4x106/60-mm dish, or in 6-well plates at a
comparable density (2x105
cells/cm2), in MEM supplemented with 5% FCS,
penicillin, and streptomycin (MEM+5% FCS). On the following day, cells
were rinsed 3 times in MEM and transferred to a defined serum-free MEM
supplemented with transferrin, vitamin B12,
insulin, and 2 mmol/L L-arginine (DSF medium). The
final myocyte cultures contained >90% quiescent cardiac myocytes at
partial confluence; 0.1 mmol/L bromodeoxyuridine was included in
the medium for the first 3 days after plating to inhibit fibroblast
growth. In some experiments, nonmyocytes from the same cultures
(
95% fibroblasts, with small percentages of smooth muscle and
endothelial cells) were used to confirm the cell type
specificity of cytokine-mediated apoptosis. These cells
were used at passages 1 to 3 and maintained in MEM+5% FCS as
previously described.39
Experiments were performed beginning on days 1 to 3 of culture. Cells
were first transferred to DSF medium, after which cytokines
(TNF-, 25 ng/mL; IL-1ß, 5 ng/mL; and IFN-, 100 U/mL), or their
vehicle (water), were added with and without other reagents as
indicated in the text.
Quantitative Analysis of Apoptotic Nuclei
Cells were examined for morphological evidence of
apoptosis or necrosis after staining with the
fluorescent DNA-binding dyes H33342 and propidium iodide, as
previously described.36 Treated and control cell
monolayers were rinsed with PBS and stained with 5 µg/mL H33342 and 5
µg/mL propidium iodide for 15 minutes and then harvested by
trypsinization. Suspended cells were pelleted at 2000g,
resuspended in MEM, and visualized at x400 on a Zeiss IM
fluorescence microscope. Cells were scored as apoptotic
if they exhibited unequivocal nuclear chromatin condensation and/or
fragmentation, while propidium iodide-stained cells with normal nuclear
morphology were scored as necrotic. To quantify apoptosis, an
average of 500 nuclei from random fields were analyzed, and
apoptotic cell counts were expressed as a percentage of the
total number of nuclei counted. Samples were numbered to conceal the
identity of different treatment groups during scoring, and at least 3
samples were scored in each group.
Immunofluorescent Staining
In some experiments, apoptotic nuclei were localized
within cardiac myocytes by costaining with Hoechst 33342 and a
monoclonal antibody against sarcomeric myosin heavy
chain.36 Cardiac myocytes were cultured on uncoated Nunc
2-well coverslip dishes and treated for 96 hours with
IL-1ß. Control cells received an equal volume of diluent.
Cells were subsequently fixed in ice-cold methanol, rinsed, and stained
with anti-myosin heavy chain (MF-20, obtained from Developmental
Studies Hybridoma Bank, University of Iowa, Iowa City) and
Hoechst 33342, followed by an FITC-tagged anti-mouse IgG secondary
antibody. Cells were imaged and photographed on a Zeiss IM inverted
phase fluorescence microscope using a mounted Contax 35-mm
camera and ASA 400 Kodak color transparency film.
Analysis of DNA Fragmentation
Nuclear DNA was isolated from and examined for nucleosomal
fragmentation at predetermined time points after treatment with
cytokines and other reagents. Cells were lysed overnight in a
buffer containing 100 mmol/L NaCl, 10 mmol/L Tris-Cl, pH 8,
25 mmol/L EDTA, 0.5% SDS, and 0.15 mg/mL proteinase K. Lysates
were extracted with phenol/chloroform and precipitated with ammonium
acetate in 100% ethanol. The resulting pellet was resuspended and
treated with DNase-free RNase before reextraction and precipitation as
above. Nuclear DNA was then solubilized in Tris-EDTA buffer, and DNA
content was quantified by spectrophotometry at 260 nm. Samples were
then subjected to electrophoresis in 2% agarose gels and imaged by
ethidium bromide staining and digital photography. In some experiments,
before electrophoresis, DNA (1 µg) from each sample was 3'-end
labeled using 25 U terminal transferase (Boehringer Mannheim)
in a reaction buffer consisting of 0.2 mol/L potassium cacodylate,
25 mmol/L Tris-HCl, 250 µg/mL BSA, 5 mmol/L
CoCl2, and 50 µCi
[-32P]ddATP at 37°C for 1 hour. Reactions
were terminated by addition of EDTA, and DNA was precipitated twice
more in ethanol. Samples were then subjected to electrophoresis, dried
on a slab gel dryer, and exposed to x-ray film for 2 hours at -70°C.
In all cases, the extent of DNA fragmentation was quantified by
densitometry of subchromosomal DNA fragments on digitized images using
Adobe Photoshop 4.0 for Macintosh.
NO Analysis
The Griess reagent (2.9 mmol/L sulfanilic acid and 0.2
mmol/L N-[1-naphthyl] ethylenediamine-HCl in 5%
phosphoric acid) was used to measure the accumulation of NO metabolites
(predominantly NO2–) as a proxy
for NO synthesis. Equal volumes of medium and Griess reagent were mixed
and incubated at room temperature for 30 minutes. The red diazotization
product was quantified by spectrophotometry at 570 nm using known
concentrations of sodium nitrite as a standard.
Northern Blot Analyses
RNA transcript levels were measured by Northern analysis
using modifications of a standard protocol. Total cellular RNA was
extracted using the Trizol RNA isolation kit (Gibco-BRL) or by
guanidinium isothiocyanate and cesium banding. Electrophoresis,
blotting, and hybridizations were all as described
previously.40 cDNA probes were labeled with
5'-[32P]ATP by random priming (Prime-It,
Stratagene) to 
108 cpm/µg DNA. Hybridization
signals of specific mRNAs were normalized to those of 28S rRNA to
correct for differences in loading and/or RNA transfer.
Autoradiographic band intensity was quantified by image
digitization and analysis using a UMAX PowerLook II scanner and
Adobe Photoshop 4.0 for Macintosh as above.
Western Blot Analysis
For detection of Bak and Bcl-x proteins, cells were harvested in
PBS-trypsin, pelleted, and resuspended in ice-cold NP-40 lysis buffer
(50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.5% NP-40,
and 50 mmol/L NaF) with freshly added 1 mmol/L
Na3VO4, 1 mmol/L DTT,
1 mmol/L phenylmethylsulfonyl fluoride, 25 µg/mL
leupeptin, and 25 µg/mL aprotinin. Cells were lysed in a Dounce
homogenizer for 20 strokes, and the resulting lysates
were centrifuged at 10 000g for 10 minutes to
remove cell debris. Protein content was determined using a standard
colorimetric assay (BCA, Pierce Chemical). One hundred
micrograms of each lysate supernatant was fractionated on 12%
SDS-polyacrylamide gels and electroblotted to nitrocellulose
(Hybond, Amersham). Gels were stained with Ponceau Red to control for
equal transfer of proteins. Membranes were blocked for 1 hour at room
temperature with 5% nonfat milk in Tris-buffered saline (25
mmol/L Tris and 150 mmol/L NaCl) containing 0.05% Tween 20 and
incubated with antibodies specific to Bcl-x(L) (0.5 µg/mL, Santa Cruz
Biotechnology) or Bak (1.0 µg/mL) for 2 hours in the same buffer.
After washing, the blots were incubated for 1 hour with a 1:7500
dilution of horseradish peroxidase (HRP)–conjugated anti-rabbit IgG
(Bcl-x(L)) or HRP-conjugated donkey anti-goat IgG (Bak) and visualized
using an enhanced chemiluminescence detection system (Amersham).
Poly(ADP-Ribose) Polymerase Cleavage Assay
The poly(ADP-ribose) polymerase pADPRp antibody was supplied by
Dr Guy Poirier (Health and Environment Unit, Hospital Research
Center of University of Laval [CHUL], Quebec, Canada) and was used
according to the recommended protocol. Briefly, after rinsing with PBS,
the cells were collected by centrifugation and
suspended and sonicated on ice in 50 mmol/L Tris HCl, pH 6.8, 6
mol/L urea, 6% ß-mercaptoethanol, 3% SDS, and 0.0003%
bromophenol blue, at a concentration of 2x106
cells/mL. After incubating for 15 minutes at 65°C, aliquots were
subjected to 8% SDS-PAGE, transferred to nitrocellulose, and probed
with mouse monoclonal C-2-10 antibody against pADPRp. The blot was
developed with HRP-conjugated secondary antibody and visualized by
enhanced chemiluminescence.
Statistical Analysis
Results are expressed as mean±SEM. Differences between means
were evaluated by 2-tailed Student's t test. ANOVA was
carried out using InStat 2.0 statistical software for Macintosh.
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Morphological Study of Cytokine-Induced Apoptosis
The time course of cell death in cytokine-treated neonatal rat cardiac myocytes was evaluated by staining the cells as described in Materials and Methods. Both apoptotic and necrotic cells were scored. Under control conditions in DSF medium, myocyte cultures contained a low percentage of apoptotic cells (Figure 1A
, 
). Treatment of
the cells with a combination of cytokines (5 ng/mL IL-1ß, 100
U/mL IFN-, and 25 ng/mL TNF-) produced a time-dependent increase
in the percentage of apoptotic nuclei over untreated cells
(Figure 1A). The time of onset of
cytokine-induced apoptosis varied between 48 and 72
hours (compare Figures 1A, 2A, and 3A). In dual-staining experiments using
an anti-desmin antibody together with Hoechst 33342, these
apoptotic cells were clearly identified as cardiac myocytes
(not shown). As seen in Figure 1A, the increase in the
percentage of apoptotic myocytes was statistically significant
at 72 hours (19.1±3.5% versus 4.9±1.5%, n=3, P<0.05)
and remained nearly 8-fold greater than control at 120 hours
(18.5±3.4% versus 2.5±0.8%, n=3, P<0.02). Over the same
interval, there was no increase in the small (<1%) percentage of
necrotic cells (not shown), and there was a gradual decline in the
number of control myocytes undergoing spontaneous apoptosis
(Figure 1A, ). The time of onset of
cytokine-induced apoptosis varied between 48 and 72
hours.
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High levels of NO have been shown to be cytotoxic to many cell types.
To determine whether production of NO accounted for the
proapoptotic effects of cytokines, we used a
competitive inhibitor of NO synthase,
N-monomethyl-L-arginine
(L-NMMA). An excess concentration of L-NMMA (10 mmol/L) had no
effect on basal levels of apoptosis; however, L-NMMA blocked
induction of apoptosis by cytokines (Figure 1A
).
Cytokine-Induced Myocyte Apoptosis Correlates With
NO Production
Macrophage-derived cytokines have been shown
to exert negative inotropic and toxic effects on cardiac myocytes
through the transcriptional activation of iNOS12 14 15 21
and subsequent production of NO.7 14 21 41 42 As
expected, cytokine treatment of cardiac myocytes was followed
by a large increase in NOx metabolites in the cell culture medium. NOx
species were first detectable at 48 hours and reached levels of 5 to 8
nmol/µg protein at 96 hours after cytokine treatment. NOx
metabolites were not detected in the presence of L-NMMA (Figure 1B). The appearance of NOx reaction products preceded the
induction of apoptosis by 12 to 24 hours (Figure 1, compare panels A and B). Consistent with earlier studies,
cytokine-stimulated NOx production was preceded by the
appearance of the 4.4-kb iNOS mRNA transcript, peaking at 48 hours and
remaining significantly elevated at 72 hours (Figure 1C). iNOS
mRNA induction was accompanied by a sustained increase in the 130-kDa
iNOS protein by Western analysis, confirming previous
reports10 12 14 15 16 17 18 (not shown). These time-course studies
implicated iNOS-derived NO as a mediator of cardiac myocyte
apoptosis.
Cytokines Induce Myocyte DNA Laddering via NO
DNA cleavage into nucleosome-sized fragments is a hallmark of
apoptosis and results from the caspase-induced activation of a
specific DNase.43 To confirm the above results, DNA
isolated from cytokine-treated and control cells was end
labeled and analyzed on agarose gels as described in Materials
and Methods. Control cells exhibited a low level of DNA fragmentation
that declined over the course of the experiment. In contrast, DNA from
cytokine-treated cells exhibited a clear nucleosomal ladder
that was detectable within 48 hours after exposure and was completely
eliminated by L-NMMA (Figure 2A). In a second series of
experiments, the NO donor S-nitrosoglutathione (GSNO, 1
mmol/L) induced death in 30% to 50% of the cells, which was
associated with apoptotic nucleosomal cleavage (Figure 2B and 2D); the total number of affected cells was similar to
that for cytokines, but the kinetics appeared to be more rapid,
as indicated by the extent of nucleosomal cleavage at 24 hours (Figure 2B, last lane) and by direct identification of apoptotic
cells (28.3±3.5%, 1 mmol/L GSNO, versus control percentage of
5.2±0.5% at 4 hours; n=6, P<0.0001).
We have previously shown that the cell-permeable cGMP agonist
(8-Br-cGMP) directly induces myocyte apoptosis.36
To further characterize the cellular effects of cytokines, we
used methylene blue, an NO scavenger and soluble guanyl cyclase
inhibitor,44 and the cGMP-dependent protein
kinase inhibitor KT5728 to attempt to block
cytokine-induced DNA laddering, using densitometric
quantification of DNA cleavage products as an index. KT5728 did not
inhibit, and in some cases potentiated, cytokine-mediated
apoptosis but had no independent toxicity (Figure 2C).
In contrast, methylene blue, although cytotoxic during prolonged
exposure, consistently blunted the apoptotic effects of
cytokines (Figure 2C).
Additional evidence for a non-cGMP-dependent mechanism was observed
with GSNO. GSNO caused DNA laddering at 4 hours after treatment, in a
dose-dependent manner (Figure 2D). The effects of GSNO on
apoptosis were maximal at 1 mmol/L, the highest dose
tested (Figure 2D, lane 6), and were significantly reduced by
cotreatment with 2 different antioxidants, N-acetylcysteine
(NAC; Figure 2D, lane 8) and DTT (lane 10). These data are
consistent with a direct role for NO in
cytokine-mediated apoptosis that may not require
activation of cGMP-dependent protein kinase (eg, formation of
peroxynitrite).
An important feature of apoptosis is the sequential activation
of specific IL-converting enzyme-like proteases, or caspases.
Activation of the terminal protease, caspase-3 (CPP32, apopain, MACH-1,
or Yama) causes breakdown of key cell substrates and activation
of a specific nucleosomal DNase,45 which produces
the characteristic DNA ladder. Inhibition of caspase activity by the
peptide pseudosubstrate ZVAD-fmk strongly attenuated GSNO-induced
apoptosis (Figure 2D, lane 7). These data support the
requirement for formation of oxidative NO products such as
peroxynitrite in the induction of apoptosis by
cytokines and GSNO, as well as the activation of caspases
during this process.
IL-1ß Alone Induces Apoptosis
The cytokines used in this study and elsewhere include 3
proteins with distinct characteristics. IFN- has been shown to act
independently to induce apoptosis in many cell types; TNF-
and related molecules induce apoptosis by a specific
receptor-mediated, Fas-dependent mechanism,46 and TNF-
has been shown to induce apoptosis in isolated adult cardiac
myocytes.47 The actions of IL-1ß are more complex and
include both hypertrophic and antiproliferative effects depending on
the cell type.48 49 To better define the signal mechanisms
involved in cytokine-induced apoptosis, we examined
the independent effects of IL-1ß, TNF-, and IFN- on the
appearance of apoptotic nuclei. IL-1ß alone was sufficient to
activate apoptosis (Figure 3A). A statistically
significant increase in apoptosis was achieved earlier with the
combination of IL-1ß and IFN- (48 hours) than with IL-1ß alone
(Figure 3A, compare 
and 
; P<0.05 at 48
hours). This difference was offset by a relatively higher rate of
apoptosis in the IL-1ß-treated cells at 96 and 120 hours
(P<0.05, IL-1ß versus IL-1ß+IFN-). These results
indicate that IFN- accelerates the onset of apoptosis
induced by IL-1ß. However, neither TNF- nor IFN- had
independent proapoptotic effects on cardiac myocytes (Figure 3A).
The ability of specific cytokines to induce apoptosis
was closely tied to their ability to induce NO synthesis. As shown in
Figure 3B, IL-1ß stimulated a rapid sustained increase in NOx.
As with apoptosis, IFN- increased the production of
NO by IL-1ß at early (48 hours) but not at late (96 hours) time
points (Figure 3B, versus ; P<0.05 at 48
hours). However, IFN- had no effect on NO production or
apoptosis by itself. In contrast, TNF- had no effect on NO
production whether alone or in addition to the other
cytokines (Figure 3B, 
). Thus, the ability of each
cytokine to induce NO production correlated closely
with its ability to induce apoptosis (Figure 3, compare
panels A and B). Moreover, the onset of apoptosis after
cytokine treatment closely followed the appearance of
significant levels of NO metabolites in the culture medium.
IL-1ß Mimics Cytokine-Induced pADPRp Cleavage
A well-characterized substrate of caspase-3 is pADPRp, which
maintains the integrity of chromosomal DNA. To confirm the activation
of apoptotic pathways in cytokine-treated myocytes, we
examined cell lysates for pADPRp cleavage products (Figure 4). In control cells, bands corresponding
to both cleaved and uncleaved pADPRp were identified, with uncleaved
protein predominating. This ratio shifted markedly in favor of the
85-kDa cleavage product after treatment with either IL-1ß (Figure 4, top) or all 3 cytokines (Figure 4, bottom).
These data confirm that cytokines activate cysteinyl
proteases associated with apoptosis and that IL-1ß by itself
is sufficient for this activation.
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Both Proapoptotic and Antiapoptotic Genes Are
Upregulated by Cytokines
The relative abundance of cell survival-regulating factors
encoded by the Bcl-2 gene family can strongly affect, if not determine,
the decision to undergo apoptosis in response to an external
signal.50 Both proapoptotic and
antiapoptotic Bcl-2 homologs have been identified in
myocardium; evidence suggests that several of these
proteins and/or their mRNA transcripts are regulated during myocardial
development and aging as well as following ischemic
stress.51 52 53 54 However, it is not clear what molecular
effectors regulate the abundance of these proteins. We previously found
that myocyte Mcl-1 levels were down-regulated in response to ANP
treatment, coincident with the onset of
apoptosis.36 We therefore attempted to determine
whether cytokines and/or NO also influenced the balance of cell
survival factors.
In Northern analyses, Bak and Bcl-x(L) transcripts were
present in untreated cells and rose significantly within 24 hours
after treatment with both cytokines and IL-1ß alone (Figure 5). In contrast, mRNA transcripts for the
related proteins Bcl-2 and Bax were undetectable in both control and
cytokine-treated myocytes (not shown). Furthermore, although
both Bcl-2 and Bax proteins were identified on Western blots, their
levels were not modulated by cytokines (not shown).
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0.05, treated vs
control Bak mRNA levels; **P
The increase in Bak and Bcl-x(L) transcripts was accompanied by
induction of Bak and Bcl-x(L) proteins as determined by Western
analysis (Figure 6A through 6D).
A doublet band migrating at 30 kDa was visualized by the Bcl-x
antibody; no smaller protein bands were detected by this or by a second
Bcl-x(S)-specific antibody (not shown), suggesting absence of the
proapoptotic alternatively spliced product. The
cytokine-mediated increases in both Bak and Bcl-x(L) were NO
dependent, as they were blocked by L-NMMA (Figure 6B, lane 7,
and Figure 6D, 
). IL-1ß by itself was sufficient to induce
Bcl-x (Figure 6C, lanes 6 to 10, and Figure 6D, 
) and
Bak (Figure 6D, bottom panel), with kinetics similar to those of
cytokines.
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Surprisingly, however, the NO donor GSNO did not induce expression of
Bcl-x(L) either at 24 hours (Figure 6C, lanes 12 and 13) or at 4
hours (Figure 7), at times when DNA
laddering was easily detected (see Figure 2B and 2D). In
contrast, GSNO (1 mmol/L) induced Bak by 2.0-fold over control
(P<0.0001) by 4 hours after addition to the cultured cells
(Figure 6D). This increase was quantitatively similar to the
induction of Bak by IL-1ß at 48 hours and resulted in a significant
shift in the ratio of Bak to Bcl-x(L) in favor of
apoptosis.
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Discussion |
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Apoptosis has been strongly implicated in the toxicity of NO for cardiac myocytes.21 55 This study provides definitive evidence that cytokine-induced NO production transduces a proapoptotic signal in cardiac myocytes, as defined by morphological criteria, DNA laddering, and pADPRp cleavage. Our data further show that cytokines increase cardiac myocyte expression and abundance of Bak and Bcl-x(L), 2 proteins with opposing roles in the modulation of apoptosis. Interestingly, we were unable to induce apoptosis in cardiac fibroblasts obtained from the same cultures (not shown), suggesting that the induction of apoptosis was cell type specific, occurring predominantly or exclusively in cardiac myocytes. Significantly, the induction of apoptosis by ANP was also selective for cardiac myocytes in these mixed cultures.36 Since cardiac myocytes and fibroblasts both possess ANP receptors and soluble guanylate cyclase, it is likely that factors distal to these receptors render myocytes sensitive to ANP- and NO-induced apoptosis.
Several lines of evidence suggest that NO is, in fact, the effector of both cytokine-mediated apoptosis and Bak/Bcl-x induction. (1) The ability of individual cytokines to induce NO production in cardiac myocytes exactly paralleled their ability to activate programmed cell death. (2) The competitive iNOS antagonist, L-NMMA, prevented both NO production and apoptosis as determined by morphological criteria and DNA laddering and also blocked the expression of Bak and Bcl-x. (3) Methylene blue, an NO scavenger and inhibitor of soluble guanylate cyclase, attenuated cytokine and IL-1ß-induced DNA laddering. (4) An NO donor, GSNO, rapidly induced myocyte-specific apoptosis. (5) Cytokine- and IL-1ß-dependent induction of both Bak and Bcl-x were entirely blocked by L-NMMA, indicating that, like apoptosis, the regulation of these apoptosis-modulating factors was also dependent on NO.
NO exerts its effects on tissues through several mechanisms. It
activates guanylate cyclase, resulting in an
increase in intracellular cGMP; it modulates the L-type calcium channel
current; and it reacts with oxygen free radicals to generate
peroxynitrite, a potent oxidant.2 We have previously
demonstrated that other cGMP agonists, including the atrial and brain
natriuretic peptides zaprinast and 8-Br-cGMP, are
proapoptotic in cardiac myocytes.36 Oxidative
stress is also a potent inducer of apoptosis, and redox state
influences both cardiac myocyte signal transduction and gene
transcription.56 The present studies argue in favor of
an oxidative, rather than a cGMP-dependent, mechanism for
cytokine/NO-mediated apoptosis in these neonatal rat
cells. Most importantly, KT5823, a cGMP-dependent protein kinase-1
inhibitor,38 57 failed to prevent
cytokine/NO-mediated apoptosis, while having no
independent toxic effects. Although methylene blue inhibits soluble
guanylate cyclase,44 this inhibition is most
likely due to reactive destruction of NO through free radical
generation.58 59 Thus, the inhibition of
cytokine-mediated apoptosis by methylene blue can be
directly attributed to quenching of NO but not necessarily to
inhibition of guanylate cyclase. Since the degree to which
NO forms peroxynitrite is dependent on the availability of oxygen free
radicals,2 the redox state of individual myocytes may be
an important variable in determining the apoptotic response
to NO. This possibility is supported by the fact that the antioxidants
NAC and DTT were each able to inhibit GSNO-induced apoptosis
(see Figure 2D). Further studies will be required to determine
the relative importance of these mechanisms in the cardiac effects of
NO.
The process of apoptosis appears to require both an initial extrinsic signal and a complex intrinsic gene-regulated apoptotic program. Modulation of the abundance of apoptosis regulators of the Bcl-2 family plays a critical role in the determination of cell fate in response to apoptotic stress.60 61 62 These neonatal myocytes expressed predominantly Bcl-x(L) and Bak; low levels of Bcl-2 and Bax were not altered by cytokine treatment (not shown). Unlike Bcl-2, Bak is proapoptotic63 64 65 66 ; the mechanisms of Bak-associated cell death are incompletely understood, but they appear to involve both caspase-3–dependent and –independent pathways.63 Induction of Bak by NO would thus be predicted to potentiate cytokine-induced apoptosis.
In contrast, transcriptional induction of Bcl-x(L) is likely to be antiapoptotic. Bcl-x(L) protects against apoptosis in many cell types in response to a wide range of apoptosis effectors and following Fas-induced protease activation.67 68 69 70 In none of the experiments did we detect transcripts or protein corresponding to the proapoptotic splice variant Bcl-x(S).71 Bcl-x(L) acts upstream of the initiation of apoptosis and acts to delay its onset or reduce its probability, in part through altering mitochondrial membrane permeability.72 One transcriptional regulator of Bcl-x(L) is the tumor suppressor protein p53,73 and induction of Bcl-x in this setting could thus involve NO-dependent upregulation of p53.74 p53 upregulates Bcl-x(L) in the setting of DNA damage, possibly serving to delay the onset of apoptosis long enough to permit repair of salvageable cells.73 By analogy, NO-dependent, cytokine-mediated Bcl-x(L) induction might allow cardiac myocytes to survive transient or physiological rises in NO.
It is interesting in this regard that GSNO evoked apoptosis, DNA laddering, and Bak expression without induction of Bcl-x(L). The reason for the distinct effects of GSNO and cytokines on Bcl-x expression is not clear, but they may be due to differences in the kinetics or quantity of NO production by the 2 agents and differences in the regulation of the 2 genes. NO release from GSNO is rapid and affects all cells simultaneously, while NO production in response to cytokines requires a finite delay for transcription and protein synthesis. It is also possible that cytokine-activated signals in addition to NO generation are required for the induction of Bcl-x(L), but not Bak. The result is that the NO donor produces a relative increase in proapoptotic effector proteins compared with cytokines, and this may account in part for the difference in the speed and extent of apoptosis in response to the 2 agents. This difference also supports a role for Bcl-x(L) in promoting myocyte survival after cytokine exposure as proposed above.
The presence of reactive oxygen is a critical determinant of how NO behaves in biological systems. In a recent study, late myocardial preconditioning was mimicked by 2 different NO donors; protection was abolished by antioxidant treatment, suggesting that formation of peroxynitrite or OH was required for the beneficial effects of NO.75 In another study76 a third NO donor was shown to protect against stretch-induced superoxide production, contractile abnormalities, and programmed cell death. Both beneficial and potentially deleterious effects of NO and its oxidative products have thus been demonstrated in different systems. Differences in the rate and timing of NO release, as well as in the species and other experimental conditions, may account for the observed differences in the effects of NO. However, reactive oxygen concentration is clearly important. It is known that the presence of reactive oxygen determines the specific products of NO donor molecule breakdown (NO versus NO2– versus NO3–), as well as the reaction products and ultimately the protein targets of NO in the cell.77 It is likely that the seemingly inconsistent behavior of NO in these different studies is related to the strong dependence of NO signaling events on cell redox state.
In this study, induction of apoptosis was limited to
those cytokine combinations that generated NO. However, TNF-
has been reported to activate cardiac myocyte apoptosis
through cell surface receptors of the TNF receptor
family.47 TNF receptor ligands are able to
activate the cysteine protease cascade directly, using specific
adapter proteins (reviewed in References 78 and 7978 79 ). However, TNF-
was neither necessary nor sufficient to induce apoptosis (or
necrosis) in neonatal cardiac myocytes, in agreement with the earlier
study.47 This finding demonstrates the primacy of NO in
mediating neonatal rat cardiac myocyte apoptosis, as TNF- at
this dose was also unable to stimulate iNOS transcription or NO
production (Figure 2 and data not shown). It is possible
that neonatal myocytes lack 1 or more components required for TNF-
signal transduction. Consistent with this hypothesis,
transcripts for the type I TNF receptor have been reported to be
undetectable in cultured neonatal cardiomyocytes, in
contrast to adult myocytes.47 This would explain the lack
of NO production by TNF- in these cells.
Whether TNF- can induce cardiac myocyte apoptosis
through the induction of NO or other mechanisms in vivo remains to be
determined. Interestingly, most primary cell cultures are
resistant to TNF-mediated cytotoxicity, except under special
circumstances such as expression of viral oncogenes, oncogenic
transformation, or inhibition of protein synthesis.80
Indeed, TNF- was recently shown to induce hypertrophy
rather than apoptosis in adult cardiac myocytes in a manner
that depended on intact cell-substrate interaction.81
Although a line of transgenic mice overexpressing TNF- in
myocardium develops contractile abnormalities, this occurs
on the background of a chronic inflammatory state and in the absence of
significant apoptosis.82 The role of
apoptosis and of other TNF-–mediated effector systems in
the development and progression of congestive heart failure in vivo
remain to be established.
IL-1ß, in contrast, appears to be the key effector of
cytokine-mediated apoptosis in these cells,
consistent with its ability to induce NOx. IFN-, while
having no independent proapoptotic or antiapoptotic
effect, appeared to alter the kinetics of IL-1ß–induced
apoptosis without changing the total number of
apoptotic cells over the course of the experiment. The
proapoptotic properties of IL-1ß are distinct from its
previously reported trophic effect on cardiac myocytes, which is
mediated by a tyrosine kinase–dependent pathway.48 49
This observation is interesting in light of the hypothesis that
sustained hypertrophic stress leads to apoptosis83
and the possibly conflicting view that growth factors may present a
means for treating heart failure.84 85 86 87 Data
presented here and elsewhere suggest that, in fact, the
downstream signal pathways regulating cardiac myocyte
hypertrophy and apoptosis are dissociable. For
example, the antiapoptotic effects of ß-adrenergic
stimulation are calcium independent, while ß-adrenergic trophic
effects require calcium entry.36 40 Similarly, the
antiapoptotic and trophic effects of cardiotrophin-1 are
mediated by distinct intracellular effectors.88 The
precise relationship between cardiac myocyte growth and death signals,
and their effectors, remains to be elucidated.
|
Acknowledgments |
|---|
This work was supported by NIH grants HL49891 (to N.H.B.) and HL44578 (to K.A.W.), by grant 4RT-0134 from the California Tobacco-Related Diseases Research Program (to K.A.W. and N.H.B.), and by an American Heart Association Established Investigator Grant (to N.H.B.). Dr Ing is supported by a Research Fellowship from the Medical Research Council of Canada. We are grateful to Daryl Discher and Barbara Sato for expert technical assistance and to Dr Matt Pollman for help with the DNA laddering protocol. We also thank Drs Robert Furchgott and Larry Boise for helpful suggestions and discussions.
Received August 10, 1998; accepted October 20, 1998.
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1. Christopherson KS, Bredt DS. Nitric oxide in excitable tissues: physiological roles and disease. J Clin Invest. 1997;100:2424–2429.[Medline] [Order article via Infotrieve]
2.
Kelly RA, Balligand J-L, Smith TW. Nitric oxide and
cardiac function. Circ Res. 1997;79:363–380.
3. Robbins RA, Grisham MB. Nitric oxide. Int J Biochem Cell Biol. 1997;29:857–860.[Medline] [Order article via Infotrieve]
4. Hare JM, Keaney JF Jr, Balligand JL, Loscalzo J, Smith TW, Colucci WS. Role of nitric oxide in parasympathetic modulation of beta-adrenergic myocardial contractility in normal dogs. J Clin Invest. 1995;95:360–366.
5.
Mohan P, Brutsaert DL, Paulus WJ, Sys SU. Myocardial
contractile response to nitric oxide and cGMP. Circulation. 1996;93:1223–1229.
6.
Pabla R, Curtis MJ. Effects of NO modulation on
cardiac arrhythmias in the rat isolated heart. Circ
Res. 1995;77:984–992.
7.
Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG,
Simmons RL. Negative inotropic effects of cytokines on the
heart mediated by nitric oxide. Science. 1992;257:387–389.
8. Schulz R, Panas DL, Catena R, Moncada S, Olley PM, Lopaschuk GD. The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha. Br J Pharmacol. 1995;114:27–34.[Medline] [Order article via Infotrieve]
9. Balligand J-L, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T, Smith TW. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993;91:2314–2319.
10. Stein B, Frank P, Schmitz W, Scholz H, Thoenes M. Endotoxin and cytokines induce direct cardiodepressive effects in mammalian cardiomyocytes via induction of nitric oxide synthase. J Mol Cell Cardiol. 1996;28:1631–1639.[Medline] [Order article via Infotrieve]
11.
Balligand J-L, Kelly RA, Marsden PA, Smith TW, Michel
T. Control of cardiac muscle cell function by an endogenous
nitric oxide signaling system. Proc Natl Acad Sci U S A. 1993;90:347–351.
12. Schulz R, Nava E, Moncada S. Induction and potential biological relevance of a Ca2+-independent nitric oxide synthase in the myocardium. Br J Pharmacol. 1992;105:575–580.[Medline] [Order article via Infotrieve]
13. Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest. 1997;100:2146–2152.[Medline] [Order article via Infotrieve]
14.
Balligand J-L, Ungureanu-Longrois D, Simmons WW,
Pimental D, Malinski TA, Kapturczak M, Taha Z, Lowenstein CJ, Davidoff
AJ, Kelly RA, Smith TW, Michel T. Cytokine-inducible nitric
oxide synthase (iNOS) expression in cardiac myocytes. J Biol
Chem. 1994;269:27580–27588.
15.
Tsujino M, Hirata Y, Imai T, Kanno K, Eguchi S, Ito H,
Marumo F. Induction of nitric oxide synthase gene by
interleukin-1ß in cultured rat cardiocytes.
Circulation. 1994;90:375–383.
16. Shindo T, Ikeda U, Ohkawa F, Kawahara Y, Yokoyama M, Shimada K. Nitric oxide synthesis in cardiac myocytes and fibroblasts by inflammatory cytokines. Cardiovasc Res. 1995;29:813–819.[Medline] [Order article via Infotrieve]
17. Shindo T, Ikeda U, Ohkawa F, Takahashi M, Funayama H, Nishinaga M, Kawahara Y, Yokoyama M, Kasahara T, Shimada K. Nitric oxide synthesis in rat cardiac myocytes and fibroblasts. Life Sci. 1994;55:1101–1108.[Medline] [Order article via Infotrieve]
18.
Harding P, Carretero OA, LaPointe MC. Effects of
interleukin-1 beta and nitric oxide on cardiac myocytes.
Hypertension. 1995;25:421–430.
19. Wu CJ, Kurbegov D, Lattin B, Burchard E, Finkle C, Valantine H, Billingham ME, Starnes VA, Clayberger C. Cytokine gene expression in human cardiac allograft recipients. Transplant Immunol. 1994;2:199–207.[Medline] [Order article via Infotrieve]
20.
Xie Q-W, Whisnant R, Nathan C. Promoter of the mouse
gene encoding calcium-independent nitric oxide synthase confers
inducibility by interferon- and bacterial
lipopolysaccharide. J Exp Med. 1993;177:1779–1784.
21. Pinsky DJ, Cai B, Yang X, Rodriguez C, Sciacca RR, Cannon PJ. The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor ß. J Clin Invest. 1995;95:677–685.
22.
Pollman MJ, Yamada T, Horiuchi M, Gibbons GH.
Vasoactive substances regulate vascular smooth muscle cell
apoptosis: countervailing influences of nitric oxide and
angiotensin II. Circ Res. 1996;79:748–756.
23. Maciejewski JP, Selleri C, Sato T, Cho HJ, Keefer LK, Nathan CF, Young NS. Nitric oxide suppression of human hematopoiesis in vitro: contribution to inhibitory action of interferon-gamma and tumor necrosis factor-alpha. J Clin Invest. 1995;96:1085–1092.
24. Robaye B, Mosselmans R, Fiers W, Dumont JE, Galand P. Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro. Am J Pathol. 1991;138:447–453.[Abstract]
25. Damoulis PD, Hauschka PV. Nitric oxide acts in conjunction with proinflammatory cytokines to promote cell death in osteoblasts. J Bone Miner Res. 1997;12:412–422.[Medline] [Order article via Infotrieve]
26. Schulz JB, Matthews RT, Beal MF. Role of nitric oxide in neurodegenerative diseases. Curr Opin Neurol. 1995;8:480–486.[Medline] [Order article via Infotrieve]
27. Brune B, Messmer UK, Sandau K. The role of nitric oxide in cell injury. Toxicol Lett. 1995;82–83:233–237.
28. Yang X, Chowdhury N, Cai B, Brett J, Marboe C, Sciacca RR, Michler RE, Cannon PJ. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest. 1994;94:714–721.
29.
Kawaguchi H, Shin WS, Wang Y, Inukai M, Kato M,
Matsuo-Okai Y, Sakamoto A, Uehara Y, Kaneda Y, Toyo-oka T. In vivo gene
transfection of human endothelial cell nitric oxide
synthase in cardiomyocytes causes apoptosis-like
cell death: identification using Sendai virus-coated liposomes.
Circulation. 1997;95:2441–2447.
30.
Haywood GA, Tsao PS, von der Leyen HE, Mann MJ,
Keeling PJ, Trindade PT, Lewis NP, Byrne CD, Rickenbacher PR,
Bishopric NH, Cooke JP, McKenna WJ, Fowler MB. Expression of inducible
nitric oxide synthase in human heart failure. Circulation. 1996;93:1087–1094.
31. Chun SY, Eisenhauer KM, Kubo M, Hsueh AJ. Interleukin-1 beta suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology. 1995;136:3120–3127.[Abstract]
32. Beauvais F, Michel L, Dubertret L. The nitric oxide donors, azide and hydroxylamine, inhibit the programmed cell death of cytokine-deprived human eosinophils. FEBS Lett. 1995;361:229–232.[Medline] [Order article via Infotrieve]
33.
Kim YM, de Vera ME, Watkins SC, Billiar TR. Nitric
oxide protects cultured rat hepatocytes from tumor necrosis
factor-alpha-induced apoptosis by inducing heat shock protein
70 expression. J Biol Chem. 1997;272:1402–1411.
34.
Dimmeler S, Rippmann V, Weiland U, Haendeler J, Zeiher
AM. Angiotensin II induces apoptosis of human
endothelial cells: protective effect of nitric oxide.
Circ Res. 1997;81:970–976.
35. Tzeng E, Kim YM, Pitt BR, Lizonova A, Kovesdi I, Billiar TR. Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis. Surgery. 1997;122:255–263.[Medline] [Order article via Infotrieve]
36.
Wu C-F, Bishopric NH, Pratt RE. Atrial
natriuretic peptide in- duces apoptosis in rat
cardiac myocytes. J Biol Chem. 1997;272:14860–14866.
37. Loweth AC, Williams GT, Scarpello JH, Morgan NG. Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15. FEBS Lett. 1997;400:285–288.[Medline] [Order article via Infotrieve]
38.
Kim YM, Talanian RV, Billiar TR. Nitric oxide inhibits
apoptosis by preventing increases in caspase-3-like activity
via two distinct mechanisms. J Biol Chem. 1997;272:31138–31148.
39.
Bishopric NH, Kedes L. Adrenergic regulation of the
skeletal alpha-actin gene promoter during myocardial cell
hypertrophy. Proc Natl Acad Sci U S A. 1991;88:2132–2136.
40.
Bishopric NH, Sato B, Webster KA. Beta-adrenergic
regulation of a myocardial actin gene via a cyclic AMP-independent
pathway. J Biol Chem. 1992;267:20932–20936.
41. de Belder A, Moncada S. Cardiomyopathy: a role for nitric oxide? Int J Cardiol. 1995;50:263–268.[Medline] [Order article via Infotrieve]
42.
Ungureanu-Longrois D, Balligand J-L, Simmons WW, Okada
I, Kobzik L, Lowenstein CJ, Kunkel SL, Michel T, Kelly RA, Smith TW.
Induction of nitric oxide synthase activity by cytokines in
ventricular myocytes is necessary but not sufficient to
decrease contractile responsiveness to ß-adrenergic agonists.
Circ Res. 1995;77:494–502.
43. Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature. 1998;391:96–99.[Medline] [Order article via Infotrieve]
44. Hobbs AJ. Soluble guanylate cyclase: the forgotten sibling. Trends Pharmacol Sci. 1997;18:484–491.[Medline] [Order article via Infotrieve]
45. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S. A caspase-activated DNAse that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998;391:43–50.[Medline] [Order article via Infotrieve]
46. Nagata S. Apoptosis by death factor. Cell. 1997;88:355–365.[Medline] [Order article via Infotrieve]
47. Krown KA, Page MT, Nguyen C, Zechner D, Gutierrez V, Comstock KL, Glembotski CC, Quintana PJ, Sabbadini RA. Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes: involvement of the sphingolipid signaling cascade in cardiac cell death. J Clin Invest. 1996;98:2854–2865.[Medline] [Order article via Infotrieve]
48. Palmer JN, Hartogensis WE, Patten M, Fortuin FD, Long CS. Interleukin-1ß induces cardiac myocyte growth but inhibits cardiac fibroblast proliferation in culture. J Clin Invest. 1995;95:2555–2564.
49. Thaik CM, Calderone A, Takahashi N, Colucci WS. Interleukin-1ß modulates the growth and phenotype of neonatal rat cardiac myocytes. J Clin Invest. 1995;96:1093–1099.
50. Ellis RE, Yuan J, Horvitz R. Mechanisms and functions of cell death. Annu Rev Cell Biol. 1991;7:663–698.
51. Cheng W, Kajstura J, Nitahara JA, Li B, Reiss K, Liu Y, Clark WA, Krajewski S, Reed JC, Olivetti G, Anversa P. Programmed myocyte cell death affects the viable myocardium after infarction in rats. Exp Cell Res. 1996;226:316–327.[Medline] [Order article via Infotrieve]
52. Kajstura J, Mansukhani M, Cheng W, Reiss K, Krajewski S, Reed JC, Quaini F, Sonnenblick EH, Anversa P. Programmed cell death and expression of the protooncogene bcl-2 in myocytes during postnatal maturation of the heart. Exp Cell Res. 1995;219:110–121.[Medline] [Order article via Infotrieve]
53.
Olivetti G, Abbi R, Quaini F, Kajstura J, Cheng
W, Nitahara JA, Quaini E, Di Loreto C, Beltrami CA, Krajewski S, Reed
JC, Anversa P. Apoptosis in the failing human heart.
N Engl J Med. 1997;336:1131–1141.
54.
Misao J, Hayakawa Y, Ohno M, Kato S, Fujiwara T,
Fujiwara H. Expression of Bcl-2 protein, an inhibitor of
apoptosis, and Bax, an accelerator of apoptosis, in
ventricular myocytes of human hearts with myocardial
infarction. Circulation. 1996;94:1506–1512.
55.
Szabolcs M, Michler RE, Yang X, Aji W, Roy D, Athan E,
Sciacca RR, Minanov OP, Cannon PJ. Apoptosis of cardiac
myocytes during cardiac allograft rejection: relation to induction of
nitric oxide synthase. Circulation. 1996;94:1665–1673.
56. Wu X, Bishopric NH, Discher DJ, Murphy BJ, Webster KA. Physical and functional sensitivity of zinc finger transcription factors to redox change. Mol Cell Biol. 1996;16:1035–1046.[Abstract]
57. Kase H, Iwahashi K, Nakanishi S, Matsuda Y, Yamada K, Takahashi M, Murakata C, Sato A, Kaneko M. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun. 1987;142:436–440.[Medline] [Order article via Infotrieve]
58. Furchgott RF, Jothianandan, D. Endothelium-dependent and independent vasodilatation involving cyclic GMP: relaxation induced by NO, CO and light. Blood Vessels. 1991;28:52–61.[Medline] [Order article via Infotrieve]
59.
Kontos HA, Wei EP. Hydroxyl radical-dependent
inactivation of guanylate cyclase in cerebral arterioles by
methylene blue and by LY83583. Stroke. 1993;24:427–434.
60.
Yin XM, Oltvai ZN, Veis-Novack DJ, Linette GP,
Korsmeyer SJ. Bcl-2 gene family and the regulation of programmed cell
death. Cold Spring Harbor Symp Quant Biol. 1994;59:387–393.
61.
White E. Life, death, and the pursuit of
apoptosis. Genes Dev. 1996;10:1–15.
62. Boise LH, Gottschalk AR, Quintans J, Thompson CB. Bcl-2 and Bcl-2-related proteins in apoptosis regulation. Curr Top Microbiol Immunol. 1995;200:107–121.[Medline] [Order article via Infotrieve]
63.
McCarthy NJ, Whyte MK, Gilbert CS, Evan GI.
Inhibition of Ced-3/ICE-related proteases does not prevent cell death
induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak.
J Cell Biol. 1997;136:215–227.
64. Chittenden T, Harrington EA, O'Connor R, Flemington C, Lutz RJ, Evan GI, Guild BC. Induction of apoptosis by the Bcl-2 homologue Bak. Nature. 1995;374:733–736.[Medline] [Order article via Infotrieve]
65. Moss SF, Agarwal B, Arber N, Guan RJ, Krajewska M, Krajewski S, Reed JC, Holt PR. Increased intestinal Bak expression results in apoptosis. Biochem Biophys Res Commun. 1996;223:199–203.[Medline] [Order article via Infotrieve]
66. Ink B, Zornig M, Baum B, Hajibagheri N, James C, Chittenden T, Evan G. Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells. Mol Cell Biol. 1997;17:2468–2474.[Abstract]
67. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993;74:597–608.[Medline] [Order article via Infotrieve]
68. Shimizu S, Eguchi Y, Kosaka H, Kamiike W, Matsuda H, Tsujimoto Y. Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature. 1995;374:811–813.[Medline] [Order article via Infotrieve]
69.
Minn AJ, Boise LH, Thompson CB. Expression of Bcl-xL
and loss of p53 can cooperate to overcome a cell cycle checkpoint
induced by mitotic spindle damage. Genes Dev. 1996;10:2621–2631.
70.
Boise LH, Thompson CB. Bcl-x(L) can inhibit
apoptosis in cells that have undergone Fas-induced protease
activation. Proc Natl Acad Sci U S A. 1997;94:3759–3764.
71.
Minn AJ, Boise LH, Thompson CB. Bcl-x(S) antagonizes
the protective effects of Bcl-x(L). J Biol Chem. 1996;271:6306–6312.
72. Vander Heiden MG, Chandel NS, Williamson EK, Schumaker PT, Thompson CB. Bcl-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell. 1997;91:627–637.[Medline] [Order article via Infotrieve]
73. Zhan Q, Alamo I, Yu K, Boise LH, Cherney B, Tosato G, O'Connor PM, Fornace AJ Jr. The apoptosis-associated gamma-ray response of BCL-X(L) depends on normal p53 function. Oncogene. 1996;13:2287–2293.[Medline] [Order article via Infotrieve]
74. Messmer UK, Ankarcrona M, Nicotera P, Brune B. p53 expression in nitric oxide-induced apoptosis. FEBS Lett. 1994;355:23–26.[Medline] [Order article via Infotrieve]
75.
Takano H, Tang XL, Qiu Y, Guo Y, French BA, Bolli R.
Nitric oxide donors induce late preconditioning against myocardial
stunning and infarction in conscious rabbits via an
antioxidant-sensitive mechanism. Circ Res. 1998;83:73–84.
76. Kajstura J, Cheng W, Sarangarajan R, Li P, Li B, Nitahara JA, Chapnick S, Reiss K, Olivetti G, Anversa P. Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats. Am J Physiol. 1996;271(3 pt 2):H1215–H1228.
77. Feelisch M, Stamler JS. Donors of nitrogen oxides. In: Feelisch M, Stamler JS, eds. Methods in Nitric Oxide Research. New York, NY: John Wiley & Sons; 1996.
78. Wallach D. Placing death under control. Nature. 1997;388:123–126.[Medline] [Order article via Infotrieve]
79.
MacLellan WR, Schneider MD. Death by design: programmed
cell death in cardiovascular biology and disease.
Circ Res. 1997;81:137–144.
80.
Klefstrom J, Arighi E, Littlewood T,
Jäättelä M, Saksela E, Evan GI, Alitalo K. Induction
of TNF-sensitive cellular phenotype by c-Myc involves p53 and
impaired NF-B activation. EMBO J. 1997;16:7382–7392.[Medline]
[Order article via Infotrieve]
81.
Yokoyama T, Nakano M, Bednarczyk JL, McIntyre BW,
Entman M, Mann DL. Tumor necrosis factor-alpha provokes a hypertrophic
growth response in adult cardiac myocytes. Circulation. 1997;95:1247–1252.
82.
Kubota T, McTiernan CF, Frye CS, Slawson SE,
Lemster BH, Koretsky AP, Demetris AJ, Feldman AM. Dilated
cardiomyopathy in transgenic mice with
cardiac-specific overexpression of tumor necrosis factor-.
Circ Res. 1997;81:627–635.
83. Katz AM. The cardiomyopathy of overload: an unnatural growth response. Eur Heart J. 1995;16(suppl O):110–114.
84. Cittadini A, Grossman JD, Napoli R, Katz SE, Strömer H, Smith RJ, Clark R, Morgan JP, Douglas PS. Growth hormone attenuates early left ventricular remodeling and improves cardiac function in rats with large myocardial infarction. J Am Coll Cardiol. 1997;29:1109–1116.[Abstract]
85. Li Q, Li B, Wang X, Leri A, Jana KP, Liu Y, Kajstura J, Baserga R, Anversa P. Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy. J Clin Invest. 1997;100:1991–1999.[Medline] [Order article via Infotrieve]
86.
Duerr RL, McKirnan MD, Gim RD, Clark RG, Chien KR, Ross
J Jr. Cardiovascular effects of insulin-like growth
factor-1 and growth hormone in chronic left ventricular
failure in the rat. Circulation. 1996;93:2188–2196.
87.
Fazio S, Sabatini D, Capaldo B, Vigorito C, Giordano A,
Guida R, Pardo F, Biondi B, Sacca L. A preliminary study of growth
hormone in the treatment of dilated cardiomyopathy.
N Engl J Med. 1996;334:809–814.
88.
Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH,
Chien KR. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte
apoptosis via a mitogen-activated protein
kinase-dependent pathway: divergence from downstream CT-1 signals for
myocardial cell hypertrophy. J Biol Chem. 1997;272:5783–5791.
89. Boulakia CA, Chen G, Ng FW, Teodoro JG, Branton PE, Nicholson DW, Poirier GG, Shore GC. Bcl-2 and adenovirus E1B 19 kDA protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase. Oncogene. 1996;12:529–535.[Medline] [Order article via Infotrieve]
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|
 T. H. Tran, P. Andreka, C. O. Rodrigues, K. A. Webster, and N. H. Bishopric Jun Kinase Delays Caspase-9 Activation by Interaction with the Apoptosome J. Biol. Chem., July 13, 2007; 282(28): 20340 - 20350. [Abstract] [Full Text] [PDF]
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|
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D. Juric, P. Wojciechowski, D. K. Das, and T. Netticadan Prevention of concentric hypertrophy and diastolic impairment in aortic-banded rats treated with resveratrol Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2138 - H2143. [Abstract] [Full Text] [PDF]
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|
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|
 |
|
 E. Westphal, Li Chen, C. Pilowski, S. Koch, H. Ebelt, U. Muller-Werdan, K. Werdan, and H. Loppnow Endotoxin-activated cultured neonatal rat cardiomyocytes express functional surface-associated interleukin-1{alpha} Innate Immunity, February 1, 2007; 13(1): 25 - 34. [Abstract] [PDF]
|
|
|
|
 |
|
 A.-Y. Chong and G. Y.H. Lip Viewpoint: The prothrombotic state in heart failure: A maladaptive inflammatory response? Eur J Heart Fail, February 1, 2007; 9(2): 124 - 128. [Abstract] [Full Text] [PDF]
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|
|
|
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|
X. Zhu, H. Zhao, A. R. Graveline, E. S. Buys, U. Schmidt, K. D. Bloch, A. Rosenzweig, and W. Chao MyD88 and NOS2 are essential for Toll-like receptor 4-mediated survival effect in cardiomyocytes Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1900 - H1909. [Abstract] [Full Text] [PDF]
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 |
|
 X. Liao, J.-M. Liu, L. Du, A. Tang, Y. Shang, S. Q. Wang, L.-Y. Chen, and Q. Chen Nitric oxide signaling in stretch-induced apoptosis of neonatal rat cardiomyocytes FASEB J, September 1, 2006; 20(11): 1883 - 1885. [Abstract] [Full Text] [PDF]
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|
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|
|
|
Y. Z. Zhu, C. L. Chong, S. C. Chuah, S. H. Huang, H. S. Nai, H. T. Tong, M. Whiteman, and P. K. Moore Cardioprotective effects of nitroparacetamol and paracetamol in acute phase of myocardial infarction in experimental rats Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H517 - H524. [Abstract] [Full Text] [PDF]
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|
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A. Kumar, A. Kumar, P. Michael, D. Brabant, A. M. Parissenti, C. V. Ramana, X. Xu, and J. E. Parrillo Human Serum from Patients with Septic Shock Activates Transcription Factors STAT1, IRF1, and NF-{kappa}B and Induces Apoptosis in Human Cardiac Myocytes J. Biol. Chem., December 30, 2005; 280(52): 42619 - 42626. [Abstract] [Full Text] [PDF]
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|
|
 |
|
 K. Suzuki, B. Murtuza, J. R. Beauchamp, N. J. Brand, P. J. R. Barton, A. Varela-Carver, S. Fukushima, S. R. Coppen, T. A. Partridge, and M. H. Yacoub Role of Interleukin-1{beta} in Acute Inflammation and Graft Death After Cell Transplantation to the Heart Circulation, September 14, 2004; 110(11_suppl_1): II-219 - II-224. [Abstract] [Full Text] [PDF]
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S. FOGLI, P. NIERI, and M. C. BRESCHI The role of nitric oxide in anthracycline toxicity and prospects for pharmacologic prevention of cardiac damage FASEB J, April 1, 2004; 18(6): 664 - 675. [Abstract] [Full Text] [PDF]
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|
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|
T. C. Tung, G. Cui, K. Oshima, H. Laks, and L. Sen Balanced expression of mitochondrial apoptosis regulatory proteins correlates with long-term survival of cardiac allografts Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2832 - H2841. [Abstract] [Full Text] [PDF]
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|
|
|
A. Gonzalez, M. A Fortuno, R. Querejeta, S. Ravassa, B. Lopez, N. Lopez, and J. Diez Cardiomyocyte apoptosis in hypertensive cardiomyopathy Cardiovasc Res, September 1, 2003; 59(3): 549 - 562. [Abstract] [Full Text] [PDF]
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|
|
|
 |
|
 M. Singh and H. K. Saini Resident Cardiac Mast Cells and Ischemia-Reperfusion Injury Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2003; 8(2): 135 - 148. [Abstract] [PDF]
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|
|
|
|
|
M. A. Fortuno, A. Gonzalez, S. Ravassa, B. Lopez, and J. Diez Clinical implications of apoptosis in hypertensive heart disease Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1495 - H1506. [Full Text] [PDF]
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|
|
|
|
|
J. Heineke, T. Kempf, T. Kraft, A. Hilfiker, H. Morawietz, R. J. Scheubel, P. Caroni, S. M. Lohmann, H. Drexler, and K. C. Wollert Downregulation of Cytoskeletal Muscle LIM Protein by Nitric Oxide: Impact on Cardiac Myocyte Hypertrophy Circulation, March 18, 2003; 107(10): 1424 - 1432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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B. Tian, J. Liu, P. B. Bitterman, and R. J. Bache Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1958 - H1967. [Abstract] [Full Text] [PDF]
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|
|
|
|
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M. J. Szabolcs, J. Sun, N. Ma, A. Albala, R. R. Sciacca, G. B. Philips, J. Parkinson, N. Edwards, and P. J. Cannon Effects of Selective Inhibitors of Nitric Oxide Synthase-2 Dimerization on Acute Cardiac Allograft Rejection Circulation, October 29, 2002; 106(18): 2392 - 2396. [Abstract] [Full Text] [PDF]
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 |
|
 J. Bartunek, M. Vanderheyden, M. W. M. Knaapen, W. Tack, M. M. Kockx, and M. Goethals Deoxyribonucleic acid damage/repairproteins are elevated in the failing human myocardium due to idiopathic dilated cardiomyopathy J. Am. Coll. Cardiol., September 18, 2002; 40(6): 1097 - 1103. [Abstract] [Full Text] [PDF]
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|
|
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H. L. Li, J. Suzuki, E. Bayna, F.-M. Zhang, E. Dalle Molle, A. Clark, R. L. Engler, and W. Y. W. Lew Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT1 receptors Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H461 - H467. [Abstract] [Full Text] [PDF]
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V. Adams, B. Nehrhoff, U. Spate, A. Linke, P. C Schulze, A. Baur, S. Gielen, R. Hambrecht, and G. Schuler Induction of iNOS expression in skeletal muscle by IL-1{beta} and NF{kappa}B activation: an in vitro and in vivo study Cardiovasc Res, April 1, 2002; 54(1): 95 - 104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. C. Wollert, B. Fiedler, S. Gambaryan, A. Smolenski, J. Heineke, E. Butt, C. Trautwein, S. M. Lohmann, and H. Drexler Gene Transfer of cGMP-Dependent Protein Kinase I Enhances the Antihypertrophic Effects of Nitric Oxide in Cardiomyocytes Hypertension, January 1, 2002; 39(1): 87 - 92. [Abstract] [Full Text] [PDF]
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|
 C. R Holleyman and D. F Larson Apoptosis in the ischemic reperfused myocardium Perfusion, December 1, 2001; 16(6): 491 - 502. [Abstract] [PDF]
|
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|
|
|
|
E. J. Birks, N. Latif, V. Owen, C. Bowles, L. E. Felkin, A. J. Mullen, A. Khaghani, P. J.R. Barton, J. M. Polak, J. R. Pepper, et al. Quantitative Myocardial Cytokine Expression and Activation of the Apoptotic Pathway in Patients Who Require Left Ventricular Assist Devices Circulation, September 18, 2001; 104 (2009): I-233 - I-240. [Abstract] [Full Text] [PDF]
|
|
|
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|
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K. Suzuki, B. Murtuza, R. T. Smolenski, I. A. Sammut, N. Suzuki, Y. Kaneda, and M. H. Yacoub Overexpression of Interleukin-1 Receptor Antagonist Provides Cardioprotection Against Ischemia-Reperfusion Injury Associated With Reduction in Apoptosis Circulation, September 18, 2001; 104 (2009): I-308 - I-313. [Abstract] [Full Text] [PDF]
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|
|
 |
|
 X.-L. Ma, F. Gao, A. H. Nelson, B. L. Lopez, T. A. Christopher, T.-L. Yue, and F. C. Barone Oxidative Inactivation of Nitric Oxide and Endothelial Dysfunction in Stroke-Prone Spontaneous Hypertensive Rats J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 879 - 885. [Abstract] [Full Text]
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|
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|
|
F. Qin, N. K. Rounds, W. Mao, K. Kawai, and C.-s. Liang Antioxidant vitamins prevent cardiomyocyte apoptosis produced by norepinephrine infusion in ferrets Cardiovasc Res, September 1, 2001; 51(4): 736 - 748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. D. Hoit Two Faces of Nitric Oxide: Lessons Learned From the NOS2 Knockout Circ. Res., August 17, 2001; 89(4): 289 - 291. [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Wildhirt, M. Weis, C. Schulze, N. Conrad, S. Pehlivanli, G. Rieder, G. Enders, W. von Scheidt, and B. Reichart Coronary flow reserve and nitric oxide synthases after cardiac transplantation in humans Eur. J. Cardiothorac. Surg., June 1, 2001; 19(6): 840 - 847. [Abstract] [Full Text] [PDF]
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|
|
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M. J. Szabolcs, N. Ma, E. Athan, J. Zhong, M. Ming, R. R. Sciacca, J. Husemann, A. Albala, and P. J. Cannon Acute Cardiac Allograft Rejection in Nitric Oxide Synthase-2-/- and Nitric Oxide Synthase-2+/+ Mice : Effects of Cellular Chimeras on Myocardial Inflammation and Cardiomyocyte Damage and Apoptosis Circulation, May 22, 2001; 103(20): 2514 - 2520. [Abstract] [Full Text] [PDF]
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|
|
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K. Yamashita, J. Kajstura, D. J. Discher, B. J. Wasserlauf, N. H. Bishopric, P. Anversa, and K. A. Webster Reperfusion-Activated Akt Kinase Prevents Apoptosis in Transgenic Mouse Hearts Overexpressing Insulin-Like Growth Factor-1 Circ. Res., March 30, 2001; 88(6): 609 - 614. [Abstract] [Full Text] [PDF]
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|
|
|
P. Andreka, J. Zang, C. Dougherty, T. I. Slepak, K. A. Webster, and N. H. Bishopric Cytoprotection by Jun Kinase During Nitric Oxide-Induced Cardiac Myocyte Apoptosis Circ. Res., February 16, 2001; 88(3): 305 - 312. [Abstract] [Full Text] [PDF]
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|
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B. Tantini, F. Flamigni, C. Pignatti, C. Stefanelli, M. Fattori, A. Facchini, E. Giordano, C. Clo, and C. M. Caldarera Polyamines, NO and cGMP mediate stimulation of DNA synthesis by tumor necrosis factor and lipopolysaccharide in chick embryo cardiomyocytes Cardiovasc Res, February 1, 2001; 49(2): 408 - 416. [Abstract] [Full Text] [PDF]
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|
|
 |
|
 J.-F. Wang, X. Ren, J. DeAngelis, J. Min, Y. Zhang, T. G. Hampton, I. Amende, and J. P. Morgan Differential Patterns of Cocaine-Induced Organ Toxicity in Murine Heart versus Liver Experimental Biology and Medicine, January 1, 2001; 226(1): 52 - 60. [Abstract] [Full Text]
|
|
|
|
|
|
J. He, Y. Xiao, C. A. Casiano, and L. Zhang Role of Mitochondrial Cytochrome c in Cocaine-Induced Apoptosis in Coronary Artery Endothelial Cells J. Pharmacol. Exp. Ther., December 1, 2000; 295(3): 896 - 903. [Abstract] [Full Text]
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|
|
|
 |
|
 M. M. Santos, M. de Sousa, L. H. P. M. Rademakers, H. Clevers, J. J. M. Marx, and M. W. Schilham Iron Overload and Heart Fibrosis in Mice Deficient for Both {beta}2-Microglobulin and Rag1 Am. J. Pathol., December 1, 2000; 157(6): 1883 - 1892. [Abstract] [Full Text] [PDF]
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|
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S. Mustapha, A. Kirshner, D. De Moissac, and L. A. Kirshenbaum A direct requirement of nuclear factor-kappa B for suppression of apoptosis in ventricular myocytes Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H939 - H945. [Abstract] [Full Text] [PDF]
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D. B. Cowan, D. N. Poutias, P. J. Del Nido, and F. X. McGowan Jr CD14-independent activation of cardiomyocyte signal transduction by bacterial endotoxin Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H619 - H629. [Abstract] [Full Text] [PDF]
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D. A. Siwik, D. L.-F. Chang, and W. S. Colucci Interleukin-1{beta} and Tumor Necrosis Factor-{alpha} Decrease Collagen Synthesis and Increase Matrix Metalloproteinase Activity in Cardiac Fibroblasts In Vitro Circ. Res., June 23, 2000; 86(12): 1259 - 1265. [Abstract] [Full Text] [PDF]
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|
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A. Dorner, M. Pauschinger, P. L. Schwimmbeck, U. Kuhl, and H.-P. Schultheiss The shift in the myocardial adenine nucleotide translocator isoform expression pattern is associated with an enteroviral infection in the absence of an active T-cell dependent immune response in human inflammatory heart disease J. Am. Coll. Cardiol., June 1, 2000; 35(7): 1778 - 1784. [Abstract] [Full Text] [PDF]
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|
 |
|
 H. G. Seo, T. Nishinaka, and C. Yabe-Nishimura Nitric Oxide Up-Regulates Aldose Reductase Expression in Rat Vascular Smooth Muscle Cells: A Potential Role for Aldose Reductase in Vascular Remodeling Mol. Pharmacol., April 1, 2000; 57(4): 709 - 717. [Abstract] [Full Text]
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L. J. De Windt, H. W. Lim, T. Taigen, D. Wencker, G. Condorelli, G. W. Dorn II, R. N. Kitsis, and J. D. Molkentin Calcineurin-Mediated Hypertrophy Protects Cardiomyocytes From Apoptosis In Vitro and In Vivo : An Apoptosis-Independent Model of Dilated Heart Failure Circ. Res., February 18, 2000; 86(3): 255 - 263. [Abstract] [Full Text] [PDF]
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|
|
|
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X. Yang, N. Ma, M. J. Szabolcs, J. Zhong, E. Athan, R. R. Sciacca, R. E. Michler, G. D. Anderson, J. F. Wiese, K. M. Leahy, et al. Upregulation of COX-2 During Cardiac Allograft Rejection Circulation, February 1, 2000; 101(4): 430 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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C. Depre and H. Taegtmeyer Metabolic aspects of programmed cell survival and cell death in the heart Cardiovasc Res, February 1, 2000; 45(3): 538 - 548. [Abstract] [Full Text] [PDF]
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|
|
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G. Z. Feuerstein and P. R. Young Apoptosis in cardiac diseases: stress- and mitogen-activated signaling pathways Cardiovasc Res, February 1, 2000; 45(3): 560 - 569. [Abstract] [Full Text] [PDF]
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|
|
|
|
G. Taimor, B. Hofstaetter, and H.M. Piper Apoptosis induction by nitric oxide in adult cardiomyocytes via cGMP-signaling and its impairment after simulated ischemia Cardiovasc Res, February 1, 2000; 45(3): 588 - 594. [Abstract] [Full Text] [PDF]
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W. Song, X. Lu, and Q. Feng Tumor necrosis factor-{alpha} induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes Cardiovasc Res, February 1, 2000; 45(3): 595 - 602. [Abstract] [Full Text] [PDF]
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|
|
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M. Rezvani, J.D. Barrans, K.-S. Dai, and C.-C. Liew Apoptosis-related genes expressed in cardiovascular development and disease: an EST approach Cardiovasc Res, February 1, 2000; 45(3): 621 - 629. [Abstract] [Full Text] [PDF]
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G. Olivetti, E. Cigola, R. Maestri, C. Lagrasta, D. Corradi, and F. Quaini Recent advances in cardiac hypertrophy Cardiovasc Res, January 1, 2000; 45(1): 68 - 75. [Full Text] [PDF]
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A. M Shah Inducible nitric oxide synthase and cardiovascular disease Cardiovasc Res, January 1, 2000; 45(1): 148 - 155. [Full Text] [PDF]
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|
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S. A. Cook, P. H. Sugden, and A. Clerk Regulation of Bcl-2 Family Proteins During Development and in Response to Oxidative Stress in Cardiac Myocytes : Association With Changes in Mitochondrial Membrane Potential Circ. Res., November 12, 1999; 85(10): 940 - 949. [Abstract] [Full Text] [PDF]
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B. Yang, D. F. Larson, and R. Watson Age-related left ventricular function in the mouse: analysis based on in vivo pressure-volume relationships Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1906 - H1913. [Abstract] [Full Text] [PDF]
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M. A. Arstall, D. B. Sawyer, R. Fukazawa, and R. A. Kelly Cytokine-Mediated Apoptosis in Cardiac Myocytes : The Role of Inducible Nitric Oxide Synthase Induction and Peroxynitrite Generation Circ. Res., October 29, 1999; 85(9): 829 - 840. [Abstract] [Full Text] [PDF]
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D. J. Pinsky, W. Aji, M. Szabolcs, E. S. Athan, Y. Liu, Y. M. Yang, R. P. Kline, K. E. Olson, and P. J. Cannon Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture Am J Physiol Heart Circ Physiol, September 1, 1999; 277(3): H1189 - H1199. [Abstract] [Full Text] [PDF]
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H. Drexler Nitric Oxide Synthases in the Failing Human Heart : A Doubled-Edged Sword? Circulation, June 15, 1999; 99(23): 2972 - 2975. [Full Text] [PDF]
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C. Communal, W. S. Colucci, and K. Singh p38 Mitogen-activated Protein Kinase Pathway Protects Adult Rat Ventricular Myocytes against beta -Adrenergic Receptor-stimulated Apoptosis. EVIDENCE FOR Gi-DEPENDENT ACTIVATION J. Biol. Chem., June 16, 2000; 275(25): 19395 - 19400. [Abstract] [Full Text] [PDF]
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F. Sam, D. B. Sawyer, Z. Xie, D. L.F. Chang, S. Ngoy, D. A. Brenner, D. A. Siwik, K. Singh, C. S. Apstein, and W. S. Colucci Mice Lacking Inducible Nitric Oxide Synthase Have Improved Left Ventricular Contractile Function and Reduced Apoptotic Cell Death Late After Myocardial Infarction Circ. Res., August 17, 2001; 89(4): 351 - 356. [Abstract] [Full Text] [PDF]
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