© 1999 American Heart Association, Inc.
Cellular Biology |
Cytokine-Mediated Apoptosis in Cardiac Myocytes
The Role of Inducible Nitric Oxide Synthase Induction and Peroxynitrite Generation
From the Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass.
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Abstract |
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Abstract—Increased production of nitric oxide (NO) after induction of the cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) in cardiac myocytes and other parenchymal cells within the heart may in addition to contributing to myocyte contractile dysfunction also contribute to the induction of programmed cell death (apoptosis). To investigate the mechanism(s) by which increased NO production leads to apoptosis, we examined the role of NO in primary cultures of neonatal rat ventricular myocytes (NRVMs) after induction by the cytokines interleukin-1ß (IL-1ß) and interferon
(IFN) or exposure to
the exogenous NO donor
S-nitroso-N-acetylcysteine (SNAC) or
peroxynitrite (ONOO-). Both SNAC (1 mmol/L) and
ONOO- (100 µmol/L), but not their respective
controls (ie, N-acetylcysteine and
pH-inactivated ONOO-), induced
apoptosis in confluent, serum-starved NRVMs at 48 hours.
Similarly, incubation of NRVMs with IL-1ß and IFN for 48 hours
resulted in an increase in iNOS expression, nitrite production,
and programmed cell death. Both the cytokine-induced
nitrite accumulation and myocyte apoptosis could be completely
prevented by the nonselective NOS inhibitor
L-nitroarginine (3 mmol/L) or the specific iNOS
inhibitor
2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT,
100 µmol/L). NO-mediated myocyte apoptosis was not
attenuated by the inhibition of soluble guanylyl cyclase with ODQ, nor
could apoptosis be induced by the incubation of NRVMs with
1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analogue. However,
NO-mediated apoptosis was significantly attenuated by the
superoxide dismutase mimetic and ONOO- scavenger
Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP, 100 µmol/L).
NO/ONOO--mediated apoptosis was associated with
increased expression of Bax with no change in Bcl-2 mRNA abundance.
Furthermore, apoptotic cell death was also confirmed in adult
rat ventricular myocytes (ARVMs) when grown in
heteroculture with IL-1ß– and IFN-treated rat cardiac
microvascular endothelial cells. Therefore,
cytokine-induced apoptosis in NRVMs and ARVMs is
mediated by iNOS induction, ONOO-, and associated with an
increase in Bax levels.
Key Words: nitric oxide • peroxynitrite • cytotoxicity • apoptosis • programmed cell death
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Introduction |
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Increased production of nitric oxide (NO) after induction of inducible nitric oxide synthase (iNOS or NOS2) has been implicated in the pathophysiology of myocardial dysfunction of a number of disease syndromes, including the systemic inflammatory response syndrome,1 inflammatory myocarditis,2 cardiac allograft rejection,3 4 and some forms of heart failure.5 6 7 Cardiac myocytes, as well as a number of other parenchymal cells within the myocardium, including the endothelium of the coronary microvasculature and the endocardium, and infiltrating inflammatory cells are all able to express iNOS in response to soluble inflammatory mediators, including specific cytokines and bacterial cell-wall components.8 9 In the presence of adequate levels of cofactors (heme, tetrahydrobiopterin) and substrates (O2 and L-arginine), very high levels of NO production can be achieved after iNOS induction,10 11 12 levels that may limit growth or prove lethal to invading pathogens, underscoring the importance of iNOS induction to the innate immune response.13 14 15 These levels of NO production may be detrimental to myocardial function, however, as iNOS induction has been demonstrated to diminish both basal and catecholamine-enhanced chronotropic and inotropic function in isolated myocytes and in the intact heart.16 The mechanisms by which NO mediates these effects include increased activation of soluble guanylyl cyclase and an increase in cGMP,17 inhibition of electron transport by the mitochondrial respiratory chain,18 19 S-nitrosation of functionally important thiol residues on proteins,20 21 22 and the production of oxidants, such as peroxynitrite (ONOO-).23
iNOS induction within the heart, as in other tissues and organs, may subserve an additional role as part of the innate immune response—programmed cell death or apoptosis of infected cells. NO generated by iNOS has been implicated in the induction of apoptosis in a number of cell types,24 25 including cardiac myocytes.26 27 Ing et al27 determined that iNOS-induced cardiac myocyte apoptosis appeared to be independent of cGMP and was possibly mediated by the generation of oxygen-derived free radicals. It is not known to what extent NO itself, or ONOO-, contributes to iNOS-dependent cell death in cardiac myocytes.
In this study, we confirm the data of Ing et al27 that a
combination of interleukin-1ß (IL-1ß) and interferon (IFN)
induces programmed cell death associated with increased Bax relative to
Bcl-2 expression in isolated neonatal rat ventricular
myocytes (NRVMs). We show that the mechanism is at least partially
dependent on the formation of ONOO-.
Furthermore, we demonstrate that iNOS-mediated apoptosis of
cardiac myocytes occurs in both neonatal and adult cells.
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Materials and Methods |
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Isolation of Primary Cultures
Animals were obtained from Charles River Laboratories (Lexington, Mass), and all protocols conformed to institutional guidelines. NRVMs from 1-day-old Sprague-Dawley rats were isolated28 and cultured to confluence. Cardiac microendothelial cells (CMECs) from adult rat hearts were isolated29 and grown to subconfluence. Calcium-tolerant ventricular myocytes were then isolated from adult rat ventricular myocytes (ARVMs)30 and plated on top of the CMECs as a heteroculture in defined medium.30
Biochemical Assays
Nitrite in the medium was measured by the Griess
reaction.12 Intracellular cGMP concentration was measured
using an enzyme immune assay kit (Cayman Chemical Co, Ann Arbor,
Mich).
Cell Survival by Metabolism of MTT: After a 2-hour coincubation with anhydrous MTT (Sigma), NRVMs were solubilized with dimethyl sulfoxide and Sorenson's glycine buffer, and the OD570nm was measured.
Immunoblotting for iNOS Protein
Equal quantities of total protein from the supernatant of
detergent-lysed NRVMs were separated by 7.5% SDS-PAGE and transferred
to a polyvinylidene difluoride membrane, which was blotted with
a mouse monoclonal anti-iNOS antibody (Transduction Laboratories,
Lexington, Ky), followed by a goat anti-mouse secondary antibody
conjugated with peroxidase (Sigma) and autoradiographed using
chemiluminescence (NEN Dupont, Bedford, Mass).
Northern Blotting for mRNA of Bax and Bcl-2
Total RNA was isolated from NRVMs using Trizol (Gibco BRL),
electrophoresed in a 1% agarose/formaldehyde gel, transferred to a
transfer membrane (NEN Life Science Products, Boston, Mass), and
hybridized using QuikHyb (Stratagene, La Jolla, Calif) with
32P-dCTP–labeled (ICN, Costa Mesa, Calif) cDNA
probe (Random primers DNA labeling system, Gibco BRL) for
Bax,31 GAPDH, and then Bcl-232 respectively,
followed by autoradiography. The quantity of Bax and
GAPDH mRNA was estimated by measurement of the integrated density of
the respective digitally scanned autoradiograph band using Scion Image
software.
Determination of Apoptosis
Nuclear Size Determination and Quantitation by Flow
Cytometry
Ethanol-fixed NRVMs were suspended in PBS containing propidium
iodide (Sigma) and RNAse A (Sigma), after which 10 000 cells were
analyzed by flow cytometry using a laser with an excitation
wavelength of 488 nm and an emission wavelength of >600 nm and forward
light scatter. The hypodiploid cells within one log of the
G0/G1 peak
(sub–G1 fraction) were considered
apoptotic.
Hoescht Nuclear Staining
Paraformaldehyde-fixed NRVMs were stained with
Hoescht 33258 and photographed under UV light.
Terminal Deoxynucleotidyl Transferase-Mediated
Fluorescein-dUTP Nick End-Labeling (TUNEL)
Staining
Cells were fixed with paraformaldehyde, and
newly formed free ends of DNA were nick-end labeled with
fluorescein-dUTP with a commercial kit
(Boehringer-Mannheim) before photography using an
epifluorescent microscope with excitation/emission wavelengths
of 495/520 nm. In the heterocultures of ARVMs and CMECs, the cells were
costained with Texas Red-X phalloidin (Molecular Probes), with
excitation/emission wavelengths of 591/608 nm.
DNA Gel Electrophoresis
The supernatant of detergent-lysed NRVMs was incubated with
DNAse free RNAse A (Sigma), then proteinase K (Sigma). Its DNA was
precipitated and electrophoresed on a 2.5% agarose gel, followed by
ethidium bromide staining and photography under UV light with an orange
filter.
Statistical Analysis
All replicate data are expressed as mean±SEM. In experiments
with comparison of two treatments, a nonpaired t test was
used. In experiments with multiple treatments, ANOVA was used followed
by Dunnett's multiple comparison test. In experiments comparing
different treatments for two sets of conditions, two-way ANOVA was
performed. Statistical significance was achieved at a value of
P<0.05 (two-tailed test).
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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NRVM Cytotoxicity by Apoptosis due to Exogenous NO or ONOO-
To determine the mechanism(s) contributing to the cytotoxicity of exogenous NO on NRVM primary cultures, either the NO donor S-nitroso-N-acetylcysteine (SNAC) or the nonnitrosylated control reagent N-acetylcysteine (NAC) was added to NRVMs in serum-free medium incubated another 48 hours. In triplicate experiments (Figure 1A
),
1 mmol/L (but not 100 µmol/L) SNAC resulted in significant
cell death, with cell survival only 31±23% of that of serum-starved
control NRVMs at 48 hours. (ANOVA, P=0.0067; Dunnett's
multiple comparison test, serum starved versus SNAC 1 mmol/L,
P<0.01). This cell death appeared to be due to
apoptosis (Figure 1B). In quadruplicate experiments, the
percentage of apoptotic cells, as determined by flow cytometric
analysis, was only 5.7±3.4% in serum-starved NRVMs and
7.6±3.1% and 5.1±1.8% after 48 hours of incubation with either
1 mmol/L NAC or 100 µmol/L SNAC, respectively. However, 48
hours of incubation with 1 mmol/L SNAC resulted in 33.2±5.6%
apoptotic cell death (ANOVA, P=0.0002; Dunnett's
multiple comparison test, serum-starved controls versus 1 mmol/L
SNAC, P<0.01). Hoescht 33258 staining of nuclei in parallel
experiments showed morphological changes also suggestive of
apoptosis (Figure 2).
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To further examine the mechanism by which NO induced NRVM
apoptosis, the related oxidant molecule
ONOO- or its pH-inactivated negative
control (see Materials and Methods) was incubated at increasing
concentrations in serum-starved NRVM primary cultures for 48 hours. In
triplicate experiments (Figure 3A), there
was no significant difference in cell survival between serum-starved
controls and 100 µmol/L of the negative control reagent for
ONOO- or between controls and cells treated with
1 µmol/L ONOO-. In contrast, a 48-hour
incubation with either 100 µmol/L or 10 µmol/L
ONOO- was associated with cell survivals of
40±17% and 83±17% compared with serum-starved NRVMs (ANOVA,
P=0.0094; Dunnett's multiple comparison test, serum starved
versus ONOO- 100 µmol/L,
P<0.01). As with the NO donor SNAC, cell death with
ONOO- was due to apoptosis (Figure 3B). Serum-starved NRVMs and myocytes exposed to the negative
control for ONOO- at 48 hours had rates of
apoptotic cell death of 7.9±4.1% and 11.4±6.1%,
respectively (quadruplicate experiments). Incubation with
ONOO- resulted in significant dose-dependent
increases in apoptosis to 5.8±2.1%, 10.5±3.6%, and
48.5±7.5% for 1 µmol/L, 10 µmol/L, and 100
µmol/L respectively (ANOVA, P=0.0001; Dunnett's multiple
comparison test, serum starved versus ONOO-
100 µmol/L, P<0.01). As with SNAC, Hoescht 33258
staining of nuclei in parallel experiments showed morphological changes
characteristic of apoptosis (Figure 2
).
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IL-1ß- and IFN-Induced NRVM Cytotoxicity Is Mediated by
iNOS
iNOS, normally not present in untreated NRVM primary cultures,
could be detected at 4 hours after the commencement of incubation with
recombinant human IL-1ß (rhIL-1ß) 4 ng/mL and recombinant murine
IFN (rmIFN) 0.05 U/mL (cytokines) in DMEM+1% ITS,
supplemented with 3 mmol/L arginine and 40 µmol/L
sepiapterin, and increased over time (Figure 4A). Similarly, although there was no
measurable nitrite in untreated, serum-starved NRVMs, an increase in
nitrite content could be detected at 6 hours, which increased with time
to 28.5 µmol/L after a 24-hour incubation (Figure 4B).
Coincubation of cytokine-treated NRVMs with 100 µmol/L
2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a
specific iNOS inhibitor,29 resulted in no
detectable nitrite in the medium after 24 and 48 hours (Figure 4B).
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To determine whether iNOS induction contributed to
cytokine-mediated NRVM cytotoxicity, serum-starved NRVMs were
treated with cytokines in the presence or absence of AMT.
Incubation of serum-starved NRVMs with arginine and sepiapterin alone
resulted in cell survival of 99±1% compared with serum-starved NRVMs
(n=8; P=NS) and no significant difference in the rate of
apoptosis (8.4±1.4% in serum-starved NRVMs and 10.9±1.8% in
arginine- and sepiapterin-treated NRVMs [n=12; P=NS]). AMT
alone, 100 µmol/L, had no effect on cell survival in
serum-starved NRVMs after 48 hours of incubation. Survival in
cytokine-treated NRVMs decreased over time to 81±2% (n=2) at
24 hours and 44±12% at 48 hours compared with serum-starved untreated
NRVMs (Figure 4C). However, in the presence of 100 µmol/L
AMT, cytokine treatment resulted in no significant decrease in
cell survival compared with serum-starved untreated NRVMs (94±0% at
24 hours and 114±16% at 24 and 48 hours, respectively).
To verify these findings, the effects on cytokine-induced
NRVM cytotoxicity and apoptosis of a nonselective NOS
inhibitor, L-nitroarginine (LNA), were compared
with those of the selective iNOS antagonist AMT.
Serum-starved NRVMs were incubated with cytokines and either
3 mmol/L LNA or 100 µmol/L AMT for 48 hours. Neither LNA
nor AMT alone was associated with any significant cell death (Figure 5A). Cytokine treatment resulted
in cell survival of 62±5% (n=8) compared with that of serum-starved
untreated NRVMs. NOS inhibition in cytokine-treated NRVMs
completely prevented cell death at 48 hours, with cell survival,
compared with untreated NRVMs of 100±3% (n=4) and 103±4% (n=4) in
LNA- (3 mmol/L) and AMT- (100 µmol/L) treated NRVMs,
respectively (two-way ANOVA: cytokine treatment,
P=0.0242; NOS inhibition, P<0.0001; interaction,
P<0.0001).
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The percentage of apoptotic cells in serum-starved untreated
NRVMs and those treated with either LNA or AMT alone was 5.2±1.2%
(n=20), 3.5±0.7% (n=5), and 2.5±0.8% (n=3) (Figure 5B).
Incubation with cytokines alone for 48 hours resulted in
29.2±4.8% apoptotic cell death (n=20 replicates), whereas
parallel incubations of cytokine-treated NRVMs in the presence
of LNA (3 mmol/L) or AMT (100 µmol/L) were 7.7±2.0%
(n=17) and 1.9±0.3% (n=4), respectively (two-way ANOVA:
cytokine treatment, P=0.0357; NOS inhibition,
P=0.0025; interaction, P=0.0113).
Further evidence of iNOS-mediated programmed cell death in NRVMs was
seen in parallel experiments by nuclear staining with Hoescht 33258
(Figure 2), TUNEL staining (Figure 6), and DNA electrophoresis (Figure 7). Cytokine-treated NRVMs
demonstrated morphological and biochemical evidence of
apoptosis after iNOS induction.
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Parallel experiments were performed comparing confluent NRVMs incubated in medium containing 10% FCS with myocytes incubated in serum-free medium. In the absence of cytokines, there were no significant differences in the extent of apoptosis in untreated cells by measurement of the sub–G1 fraction, TUNEL staining, or DNA electrophoresis (data not shown). Furthermore, cytokine treatment of serum-treated confluent NRVMs was associated with a similar rate of iNOS-mediated apoptotic cell death, as we observed in serum-free cells (data not shown).
iNOS-Induced Myocyte Apoptosis in ARVMs
ARVMs, in contrast to NRVMs and CMECs, produce low concentrations
of NO from cytokine-induced iNOS.16 As a
consequence, ARVM treatment with IL-1ß and IFN at the
concentrations used was not cytotoxic (data not shown). To determine
whether a high concentration of NO from an endogenous
paracrine cellular source would induce cytotoxicity in ARVMs, primary
heterocultures of ARVMs and CMECs were treated with cytokines
in the presence or absence of 100 µmol/L AMT for 72 hours. The
cells were then fixed for TUNEL staining. As shown in Figure 8, we qualitatively demonstrated that
iNOS-mediated apoptosis occurred in ARVMs within 72 hours in
triplicate experiments.
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iNOS-Induced Myocyte Apoptosis Is Not Mediated by
cGMP
To determine whether IL-1ß- and IFN-induced apoptosis
in NRVMs was mediated by an NO-dependent increase in cGMP,
serum-starved NRVMs were incubated with cytokines, arginine,
and sepiapterin in the presence or absence of the soluble guanylyl
cyclase inhibitor ODQ33 34 for 48 hours. The
intracellular cGMP concentration was 1.9±0.5 pmol/mg protein in
serum-starved NRVMs and 3.5±0.2 pmol/mg protein in NRVMs treated with
cytokines. ODQ (1 µmol/L) decreased myocyte cGMP content
to 2.0±0.8 pmol/mg protein (P=NS compared with control NRVM
cultures). ODQ incubation alone caused no significant decrease in cell
survival but did not influence cytokine-induced cell death
(Figure 9A). Cell survival in
cytokine-treated NRVMs was 39±10% (n=6) of untreated,
serum-starved NRVMs and 43±17% and 27±11% when coincubated with
either 1 µmol/L (n=4) or 10 µmol/L (n=4) ODQ,
respectively (two-way ANOVA: cytokine treatment,
P<0.0001; ODQ treatment compared with cytokines,
P=NS; interaction, P=NS). Similarly, the extent
of apoptosis in cytokine-treated NRVMs was not
decreased by coincubation with ODQ (Figure 9B). The percentage
of apoptotic cells increased from 6.3±2.6% (n=9) in
serum-starved, untreated NRVMs at 48 hours to 22.8±3.6% (n=6),
24.9±3.6% (n=6), and 29.9±6.9% (n=6) in cytokine-treated
serum-starved NRVMs and cytokine-treated myocytes coincubated
with 1 µmol/L and 10 µmol/L ODQ, respectively (two-way
ANOVA: cytokine treatment, P<0.0001; ODQ treatment,
P=NS; interaction, P=NS). The lack of an effect
of ODQ on cytokine-induced apoptosis was confirmed
by the absence of any attenuation in the extent of DNA laddering in
parallel experiments (Figure 7). These data were supported by
experiments with the cell-permeant cGMP analogue 8-bromo-cGMP (Figure 9). Incubation of NRVMs with 1 mmol/L 8-bromo-cGMP for 48
hours resulted in no significant change in cell survival of 88±4%
(n=4) compared with untreated, serum-starved cells (P=NS).
Similarly, there was no significant increase in the percentage of
apoptotic NRVMs when incubated with 1 mmol/L 8-bromo-cGMP
(5.4±1.8%, n=4) compared with untreated NRVMs (P=NS).
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iNOS-Induced Myocyte Apoptosis Is Mediated by
ONOO-
As we have shown that exogenous ONOO- was
capable of inducing apoptosis in NRVMs and that
cytokine-induced apoptosis was not mediated by
cGMP, we examined the possibility that ONOO- was
at least in part responsible for apoptotic cell death from
endogenous sources of NO and superoxide. We used the
cell-permeable porphyrin analogue Mn(III)tetrakis (4-benzoic acid)
porphyrin (MnTBAP), which has been shown in previous reports to be a
superoxide dismutase (SOD) mimetic and ONOO-
(but not an NO) scavenger35 36 37 with no direct effect on
iNOS activity.36
Serum-starved NRVMs were incubated for 24 to 48 hours, with or without
cytokines, arginine, and sepiapterin, in the presence or
absence of 100 µmol/L MnTBAP (Figure 10A). Cell survival in serum-starved
NRVMs incubated with MnTBAP in the absence of cytokines was
96±3%, whereas cytokines decreased NRVM cell survival to
47±8% (n=4 separate experiments). In contrast, coincubation of
cytokines with MnTBAP resulted in cell survival that was not
significantly different from serum-starved NRVMs alone, at 114±2%
(two-way ANOVA: cytokine treatment, P=0.01; MnTBAP
treatment, P<0.0001; interaction,
P<0.0001).
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There was a small but statistically significant increase in the rate of
apoptosis at 48 hours from 6.9±1.5% (n=11) in serum-starved
NRVMs to 12.5±2.3% (n=8) in MnTBAP-treated NRVMs
(P=0.007). However, as shown in Figure 10B, the
apoptotic rate decreased from 28.0±6.6% (n=11) in
cytokine-treated NRVMs to 16.4±3.8% (n=12) in NRVMs
coincubated with MnTBAP (two-way ANOVA: cytokine treatment,
P<0.0062; MnTBAP treatment, P=NS; interaction,
P=0.0413).
In parallel experiments (Figure 7), the extent of DNA laddering
in NRVMs was decreased by coincubation of cytokines with
100 µmol/L MnTBAP compared with cytokines alone. These
data indicate that an agent that scavenges both superoxide and
ONOO- could prevent cytokine-induced
toxicity by attenuating apoptotic cell death.
Increased iNOS-Mediated Bax Expression in Cytokine-Treated
NRVMs
In triplicate experiments, the expression of Bax and Bcl2 mRNA in
NRVMs was quantified 24 hours after cytokine treatment with or
without coculture of 100 µmol/L AMT. Bcl-2 expression remained
low at all conditions, making accurate quantification unreliable
(Figure 11A). In contrast, Bax
expression (Figure 11B) increased 9.5±3.4-fold 24 hours after
cytokine treatment and only increased 2.2±0.4-fold in NRVMs
treated with cytokines and AMT compared with untreated,
serum-starved NRVMs (ANOVA, P=0.027; Dunnett's multiple
comparison test: serum-starved controls versus cytokine-treated
NRVMs, P<0.05).
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Conclusions
We have demonstrated that both an exogenous NO donor (1
mmol/L SNAC) and exogenous ONOO- (100
µmol/L OONO-) induced predominantly programmed
cell death (apoptosis) within 48 hours in serum-starved primary
cultures of NRVMs. Furthermore, endogenous NO released by
these cells after induction of iNOS was responsible for programmed cell
death induced by the cytokine combination IL-1ß and IFN.
This iNOS-mediated cytotoxicity was not only confined to neonatal
myocytes but was also induced in the adult myocyte phenotype
cocultured with cardiac microvascular endothelial cells
after exposure to cytokines. The mechanisms by which NO
mediates apoptosis do not involve activation of guanylyl
cyclase but appeared to be due predominantly to the formation of
ONOO- and an associated increase in the ratio of
Bax to Bcl-2 expression. This confirms the recent report of Ing et
al27 and goes further to elucidate the mechanisms involved
in the activation of apoptotic pathways.
Despite the evidence presented in the present study suggesting a predominantly apoptotic mechanism for NO-mediated cardiac myocyte cytotoxicity, many of the assays for determination of apoptosis are not specific, making it impossible to exclude an element of necrosis in combination with apoptosis. The ratio of apoptosis to necrosis remains unclear.
There was a clear concentration threshold required to induce
apoptosis after exposure to exogenous NO in these serum-starved
NRVM primary cultures. Although the concentration of SNAC required to
induce apoptosis might imply the need for
supraphysiological concentrations of NO,
Matthews et al38 reported that free NO concentration
in solution 3 minutes after the addition of 1 mmol/L
S-nitroso-N-acetylpenicillamine (SNAP) released
70 nmol/L free NO. SNAC has a shorter half-life than SNAP,
suggestive of a faster release and higher concentration of free NO in
solution, although the concentration is unlikely to be more than
doubled. In addition, Messmer and Brune39
demonstrated that the integral of the NO concentration over time
accounted for the extent of apoptosis induced by NO donors in
macrophages. Therefore, a single, short exposure to a high
concentration of NO or ONOO- or a combination of
moderately high concentration of the same over a longer period of time,
such as occurs after iNOS induction (see Figure 4), could result
in programmed cell death.
Although NRVMs, like other cardiac myocyte phenotypes, do express a constitutive, calcium-dependent NOS (ie, endothelial NOS [eNOS]), it is clear that the relatively small amounts of NO generated by this enzyme would not give an adequate cumulative dose over the same period of time as iNOS and be capable of inducing apoptosis. This was suggested by the low rates of apoptosis in confluent, serum-starved NRVMs and the lack of effect of the eNOS inhibitor LNA on this baseline apoptosis. Furthermore, the choice of neonatal rather than adult rat ventricular myocyte phenotypes for the majority of the experiments reported in the present study was because of their low basal apoptotic rate, the high and sustained concentration of NO generated by iNOS after cytokine treatment, and the difficulty in performing many of the assays on ARVMs when grown in heteroculture with CMECs.
ONOO- formation occurs after the reaction of NO
with superoxide, the kinetics of which are faster than those of
superoxide with SOD.23 Exogenous NO donors have been
shown, even at low concentrations, to inhibit multiple enzymes involved
in mitochondrial electron transfer, resulting in increased superoxide
production and therefore increased substrate for the formation
of ONOO-,40 which itself may act as
an inhibitor of mitochondrial
respiration.19 41 Similarly, endogenous
sources of NO production in J774 cells treated with
lipopolysaccharide (LPS) and IFN have been shown to decrease
mitochondrial respiration, and the combination of
ONOO- and NO appeared to have additive
effects.35 Therefore, it is likely that excessive release
of NO after iNOS induction in NRVMs would result in increased
superoxide and, therefore, ONOO-
production in the absence of other inducers of oxidative
stress.
A number of studies have documented the roles of NO and/or
ONOO- in programmed cell death, in a time- and
concentration-dependent fashion. For example, exogenous NO donors have
been shown to induce apoptosis in HL-60,42 NA
cells,43 murine L929, and NIH3T3 cells,25 and
exogenous ONOO- has been shown to induce
apoptosis in HL-60 cells,44 PC12
cells,45 and rat thymocytes.46 A role for NO
in IL-1ß- and IFN-induced apoptosis has been demonstrated
in mixed neuronal and glial cell cultures47 and in cardiac
myocytes.26 27 Similarly, the combination of LPS- and
IFN-induced apoptosis in macrophages has been shown
to be mediated by NO,48 49 although some cells are
resistant to NO- or ONOO--mediated
programmed cell death.44 50
NO and ONOO- may initiate and/or regulate programmed cell death pathways at several levels. Although NO exposure to purified DNA does not cause single strand breaks even at concentrations as high as 1 mol/L,51 NO inactivates a DNA ligase, probably by nitrosation of a functionally critical lysine group,52 thereby decreasing normal DNA repair capability. In contrast, ONOO- itself can initiate DNA cleavage at every nucleotide,53 which is an obligatory stimulus for the activation of the nuclear enzyme poly(ADP-ribosyl)synthetase (PARS).27 54 55
Induction of iNOS in RAW 264.7 macrophages was shown to increase the activity of the tumor suppressor factor p53, which preceded apoptosis.48 56 Furthermore, Messmer and Brune57 demonstrated that there was a positive correlation between the concentration of an exogenous NO donor, p53 upregulation, and the extent of DNA fragmentation over time. These authors also demonstrated the facilitatory, but not obligatory, role of p53 with NO-induced apoptosis of a p53-negative cell line, U937.
Unlike p53, increased expression of Bcl-2 has been correlated with an increase in cellular resistance to exogenous NO-induced apoptosis,25 and transfection of Bcl-2 has led to resistance to both exogenous and endogenous NO-mediated apoptosis.25 58 We demonstrated no change in Bcl-2 expression, but increased iNOS-mediated Bax expression, which placed the cell in a "proapoptotic" state.31 59
The redox state of the intracellular environment appears to influence
NO-induced apoptosis, although the interactions are
consistent with the complexity of NO and
ONOO- chemistry.23 Significant
depletion of intracellular glutathione, an important intracellular
antioxidant, before incubation of J774 macrophages with LPS and
IFN, has been shown to exacerbate NO-mediated
cytotoxicity.60 Similarly, inhibition of Cu/Zn SOD
activity in PC12 cells after transformation with an antisense
oligonucleotide resulted in NO-mediated
apoptosis.61
The pathways by which ONOO- activates cardiac myocyte apoptosis via increased Bax expression require further elucidation and comparison with other cell types. Other non-cGMP effects of NO, such as S-nitrosation of thiols, which results in modulation of critical protein functions,62 have not been excluded in the present study. MnTBAP showed attenuation without complete inhibition of iNOS-induced apoptosis. This could be due to either partial superoxide and ONOO- scavenging of the compound at the concentration used or parallel NO, but non–peroxynitrite-mediated activation of apoptotic and/or necrotic pathways. However, these other possible NO-mediated effects appear to play a lesser role in NO-induced NRVM apoptosis than does the formation of ONOO-.
The relevance of NO and ONOO- generated by iNOS, in either myocytes or other cells such as microvascular endothelium and fibroblasts, to cardiac myocyte apoptosis will only be fully appreciated by in situ experimental animal models and in humans. Although there have been several reports indicating that iNOS expression and the rate of apoptosis are increased in patients with heart failure,1 5 6 7 63 64 there have been no data to date that clearly implicate NO released by iNOS in the pathogenesis of the heart failure syndrome in humans. It appears possible that if substantial expression of iNOS occurs in cardiac failure, the actions of the NO and ONOO- generated on mechanical function, energetics, and on pathways leading to programmed cell death in cardiac myocytes may all be contributing factors to its pathophysiology and progression.
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health (HL52320). Margaret Arstall was the recipient of the National Heart Foundation of Australia Ralph Reader Overseas Research Fellowship 1995-1998 and the Royal Australian College of Physicians JJ Billings Overseas Research Fellowship 1995.
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Footnotes |
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Reprint requests to Ralph A. Kelly, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.
Received July 6, 1999; accepted August 26, 1999.
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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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H. H. Hovels-Gurich, J. F. Vazquez-Jimenez, A. Silvestri, K. Schumacher, R. Minkenberg, J. Duchateau, B. J. Messmer, G. von Bernuth, and M.-C. Seghaye Production of proinflammatory cytokines and myocardial dysfunction after arterial switch operation in neonates with transposition of the great arteries J. Thorac. Cardiovasc. Surg., October 1, 2002; 124(4): 811 - 820. [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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 J. M. Pearl, D. P. Nelson, S. M. Schwartz, C. J. Wagner, S. M. Bauer, E. A. Setser, and J. Y. Duffy Glucocorticoids reduce ischemia-reperfusion-induced myocardial apoptosis in immature hearts Ann. Thorac. Surg., September 1, 2002; 74(3): 830 - 837. [Abstract] [Full Text] [PDF]
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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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 C.-F. Lin, H.-Y. Lei, A.-L. Shiau, H.-S. Liu, T.-M. Yeh, S.-H. Chen, C.-C. Liu, S.-C. Chiu, and Y.-S. Lin Endothelial Cell Apoptosis Induced by Antibodies Against Dengue Virus Nonstructural Protein 1 Via Production of Nitric Oxide J. Immunol., July 15, 2002; 169(2): 657 - 664. [Abstract] [Full Text] [PDF]
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G. W. De Keulenaer, Y. Wang, Y. Feng, S. Muangman, K. Yamamoto, J. F. Thompson, T. G. Turi, K. Landschutz, and R. T. Lee Identification of IEX-1 as a Biomechanically Controlled Nuclear Factor-{kappa}B Target Gene That Inhibits Cardiomyocyte Hypertrophy Circ. Res., April 5, 2002; 90(6): 690 - 696. [Abstract] [Full Text] [PDF]
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A. Ceriello, L. Quagliaro, M. D'Amico, C. Di Filippo, R. Marfella, F. Nappo, L. Berrino, F. Rossi, and D. Giugliano Acute Hyperglycemia Induces Nitrotyrosine Formation and Apoptosis in Perfused Heart From Rat Diabetes, April 1, 2002; 51(4): 1076 - 1082. [Abstract] [Full Text] [PDF]
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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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W. Wang, G. Sawicki, and R. Schulz Peroxynitrite-induced myocardial injury is mediated through matrix metalloproteinase-2 Cardiovasc Res, January 1, 2002; 53(1): 165 - 174. [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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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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H. Chen, D. Li, T. Saldeen, and J. L. Mehta TGF-{beta}1 modulates NOS expression and phosphorylation of Akt/PKB in rat myocytes exposed to hypoxia-reoxygenation Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1035 - H1039. [Abstract] [Full Text] [PDF]
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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]
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M. J. Shaw, H. Shennib, N. Bousette, E. H. Ohlstein, and A. Giaid Effect of endothelin receptor antagonist on lung allograft apoptosis and NOSII expression Ann. Thorac. Surg., August 1, 2001; 72(2): 386 - 390. [Abstract] [Full Text] [PDF]
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A. Iwata, S. Sai, Y. Nitta, M. Chen, R. de Fries-Hallstrand, J. Dalesandro, R. Thomas, and M. D. Allen Liposome-Mediated Gene Transfection of Endothelial Nitric Oxide Synthase Reduces Endothelial Activation and Leukocyte Infiltration in Transplanted Hearts Circulation, June 5, 2001; 103(22): 2753 - 2759. [Abstract] [Full Text] [PDF]
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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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M. M. Givertz, D. B. Sawyer, and W. S. Colucci Antioxidants and Myocardial Contractility : Illuminating the "Dark Side" of {{beta}}-Adrenergic Receptor Activation? Circulation, February 13, 2001; 103(6): 782 - 783. [Full Text] [PDF]
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P. Ferdinandy and R. Schulz Peroxynitrite: Toxic or Protective in the Heart? Circ. Res., February 2, 2001; 88 (2): e12 - e13. [Full Text] [PDF]
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M. Sata, M. Kakoki, D. Nagata, H. Nishimatsu, E. Suzuki, T. Aoyagi, S. Sugiura, H. Kojima, T. Nagano, K. Kangawa, et al. Adrenomedullin and Nitric Oxide Inhibit Human Endothelial Cell Apoptosis via a Cyclic GMP-Independent Mechanism Hypertension, July 1, 2000; 36(1): 83 - 88. [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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J. S. Beckman Parsing the Effects of Nitric Oxide, S-Nitrosothiols, and Peroxynitrite on Inducible Nitric Oxide Synthase-Dependent Cardiac Myocyte Apoptosis Circ. Res., October 29, 1999; 85(9): 870 - 871. [Full Text] [PDF]
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M. R Kibbe, J. Li, S. Nie, B. M. Choi, I. Kovesdi, A. Lizonova, T. R Billiar, and E. Tzeng Potentiation of nitric oxide-induced apoptosis in p53-/- vascular smooth muscle cells Am J Physiol Cell Physiol, March 1, 2002; 282(3): C625 - C634. [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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D. R. Pimentel, J. K. Amin, L. Xiao, T. Miller, J. Viereck, J. Oliver-Krasinski, R. Baliga, J. Wang, D. A. Siwik, K. Singh, et al. Reactive Oxygen Species Mediate Amplitude-Dependent Hypertrophic and Apoptotic Responses to Mechanical Stretch in Cardiac Myocytes Circ. Res., August 31, 2001; 89(5): 453 - 460. [Abstract] [Full Text] [PDF]
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G. W. De Keulenaer, Y. Wang, Y. Feng, S. Muangman, K. Yamamoto, J. F. Thompson, T. G. Turi, K. Landschutz, and R. T. Lee Identification of IEX-1 as a Biomechanically Controlled Nuclear Factor-{kappa}B Target Gene That Inhibits Cardiomyocyte Hypertrophy Circ. Res., April 5, 2002; 90(6): 690 - 696. [Abstract] [Full Text] [PDF]
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