 |
INTRODUCTION |
Tumor necrosis factor-
(TNF-
),1 a
pro-inflammatory cytokine with multiple biological actions (1, 2),
impairs contractile function in intact animals, isolated hearts, and
cardiomyocytes (3-5). TNF
-induced myocardial dysfunction has two
distinct phases. The early phase, which occurs within minutes after
TNF-
exposure, is related to sphingosine production leading to
disturbances of intercellular Ca2+ homeostasis (6). The
late phase, which occurs hours after TNF-
exposure, is mediated by
iNOS expression, apoptosis, and inhibition of both pyruvate
dehydrogenase activity and mitochondrial function (7, 8). In contrast
to the detrimental paradigm, treatment of TNF-
improves survival of
TNF-
/
mice infected with encephalomyocarditis virus
in a dose-dependent manner by increasing viral clearance
(9). Deficiency in TNF-
receptor function increased infarct size
after myocardial ischemia (10). These studies suggest that TNF-
has
a beneficial role in viral infections and myocardial ischemia.
Lipopolysaccharide (LPS) of Gram-negative bacteria has been recognized
as a causative agent in myocardial depression during sepsis.
Cardiomyocytes produce TNF-
in response to LPS exposure (4, 11).
However, the signaling pathways of LPS-induced TNF-
expression in
cardiomyocytes have not been fully defined. Previous studies have been
focused on Toll-like receptors (TLR) (12-14) and nuclear factor-
B
(NF-
B) in cardiomyocytes (14, 15). Little is known about the
signaling downstream of TLR and upstream of NF-
B. A crucial role of
p38 MAPK has been demonstrated in TNF-
expression induced by LPS in
neutrophils (16). However, whether p38 MAPK is involved in
LPS-stimulated TNF-
expression in cardiomyocytes remains to be determined.
Nitric-oxide synthase has three isoforms that serve different
physiological functions as a result of their pattern of expression and
the amount of nitric oxide (NO) produced upon activation (17, 18).
Neuronal nitric-oxide synthase is mainly expressed in neuronal cells
and regulates neurotransmission (19). Inducible nitric-oxide synthase
(iNOS) is expressed during inflammation or cytokine stimulation and
produces high levels of NO that may be toxic to undesired microbes or
tumor cells but may also harm healthy tissues (8, 17, 20). Endothelial
nitric-oxide synthase (eNOS) is constitutively expressed in a variety
of cells including endothelial cells and cardiomyocytes. Activation of
eNOS produces low levels of NO, which has been implicated as a
signaling molecule of many cellular processes, including
endothelium-dependent relaxation of blood vessels, angiogenesis, inhibition of platelet aggregation, and modulation of myocardial function (17, 21, 22). These actions of NO
have been ascribed to either the production of cGMP via soluble
guanylate cyclase (GC) and activation of cGMP-dependent protein kinase or the production of cAMP via adenylate cyclase (AC) and
activation of cAMP-dependent protein kinase (PKA) in cardiomyocytes (23, 24). Exogenous NO donors have been demonstrated to
induce TNF-
production in differentiated U937 cells and in the
myocardium (24, 25). However, it remains unknown if endogenously produced NO from eNOS modulates TNF-
expression during LPS
stimulation in cardiomyocytes.
The purpose of this study was to investigate the role of eNOS in
regulation of TNF-
expression upon LPS stimulation and to determine
the contribution of p38 MAPK, cGMP, as well as cAMP to LPS-induced
TNF-
expression in neonatal mouse cardiomyocytes.
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EXPERIMENTAL PROCEDURES |
Isolation and Culture of Neonatal Mouse
Cardiomyocytes--
C57BL/6 wild-type, iNOS
/
(stock
number 2609), and eNOS
/
(stock number 2684) mice were
purchased from The Jackson Laboratory. A breeding program was carried
out to produce neonates. All animals were used in accordance with the
guidelines of the Animal Care Committee at the University of Western
Ontario, Canada. The neonatal cardiomyocytes were prepared according to
methods we described previously (8). Briefly, ventricular myocardial
tissues from wild-type, iNOS
/
, or eNOS
/
mice born within 24 h were minced in a nominally Ca2+-
and Mg2+-free Hanks' balanced solution. Cardiomyocytes
were dispersed by 0.625 mg/ml collagenase (type II) at 37 °C for 40 min. The isolated cells were pre-plated for 90 min to remove
non-cardiomyocytes. The cardiomyocytes were plated in M199 medium
containing 10% fetal calf serum in 35-mm Petri dishes pre-coated with
1% gelatin. Cells were incubated at 37 °C in a humidified
atmosphere containing 5% CO2. After 48 h of cell
culture, cardiomyocytes were treated with different drugs either alone
or in combination.
Adenoviral Infection of Neonatal Mouse
Cardiomyocytes--
Cardiomyocytes from eNOS
/
mice were infected with adenoviral vectors containing bovine eNOS gene
(Adv-eNOS, a gift from Dr. Z. Katusic) or containing green fluorescence
protein (Adv-GFP, a gift from Dr. J. Lipp) as a negative control at a
multiplicity of infection of 10 plaque-forming units/cell.
Adenovirus-mediated gene transfer was implemented by adding a minimal
volume of the medium 199 with 2% fetal calf serum containing
gene-carrying adenoviruses. After culture for 2 h, the full volume
of culture medium containing 10% fetal calf serum was supplied. All
experiments were performed after 24 h of adenoviral infection.
Drugs--
Salmonella typhosa LPS,
N
-nitro-L-arginine methyl ester
(L-NAME),
1H-(1,2,4)oxadiazolo[4,3-
]quinoxalin-1-one (ODQ),
2,2'-(hydroxynitrosohydrazono)bis-ethanamine (DETA-NO),
S-nitroso-N-acetylpenicillamine (SNAP),
cycloheximide, and 8-Br-cGMP were purchased from Sigma. SB203580,
SB202190, 8-Br-cAMP, SQ22536, and H89 were purchased from
Calbiochem.
Measurement of TNF-
Protein and Intracellular cAMP and
cGMP--
TNF-
levels in the culture media were determined using a
mouse TNF-
ELISA Kit (ALPCO Diagnostics) according to the
manufacturer's instructions. The values were standardized with the
number of cardiomyocytes. Intracellular cAMP or cGMP levels were
measured by a cAMP or cGMP enzyme immunoassay (EIA) system (Amersham
Biosciences) following the manufacturer's instructions. The values
were presented as the levels of cAMP or cGMP to total proteins.
Analysis of TNF-
mRNA by Semi-quantitative Reverse
Transcriptase (RT)-PCR--
Total RNA was extracted from the
cardiomyocytes using the Trizol Reagent (Invitrogen) following the
manufacturer's instructions. Semi-quantitative RT-PCR was performed as
we described previously (8, 26) with modifications. The DNA
oligonucleotide primers for TNF-
were selected from the published
sequence of TNF-
gene (GenBankTM accession number
M13049). The sense and antisense primers for TNF-
gene were 5'-CCG
ATG GGT TGT ACC TTG TC-3' and 5'-GGG CTG GGT AGA GAA TGG AT-3',
respectively. Negative controls using H2O and samples
without reverse transcription were included in every run. Each sample
was amplified three times consequently in a thermal cycler (Hybaid). To
ensure a fixed amount of initial mRNA, parallel
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification was
performed using the following primers based on GAPDH gene sequence
(GenBankTM accession number M17701): sense 5' AAA
GGG CAT CCT GGG CTA CA 3', and antisense 5' CAG TGT TGG GGG CTG AGT TG
3'. Semi-quantitative RT-PCR detection was based on the optimal
conditions for each set of primers derived from calibration curves
(data not shown). cDNAs were exponentially amplified for 28 cycles
and 27 cycles for the TNF-
and GAPDH genes, respectively. PCR
products were separated on 1.2% agarose. The photographed DNA bands
were scanned and analyzed by a densitometer. TNF-
to GAPDH ratio for
each sample was calculated.
Analysis of p38 MAPK Phosphorylation--
Assessment of the
phosphorylation status of p38 MAPK in cardiomyocytes was accomplished
by Western blotting (26, 27). Aliquots containing 30 µg of protein
were subjected to SDS-PAGE using 12% gels, followed by electrotransfer
to nitrocellulose membranes. Blots were probed with antibodies against
p38 MAPK/phospho-p38 MAPK (New England Biolabs, 1:1000), followed by
incubation with horseradish peroxidase-conjugated secondary antibody
(Bio-Rad). Detection was performed using the enhanced chemiluminescence
detection method. Signals were determined by a densitometer. Levels of
phosphorylated p38 MAPK to total p38 MAPK were presented.
Nitrite Measurement--
The formation of nitrite in culture
medium was used as an indicator of NO release by neonatal
cardiomyocytes. The nitrite concentration in the supernatant was
measured by Griess reaction as described previously (8). The
concentration was expressed as nmol/mg cell protein.
Detection of iNOS Protein by Western Blot Analysis--
Total 30 µg of protein in each sample was subjected to SDS-PAGE using 10%
gels, followed by electrotransfer to ECL membranes. Expression of iNOS
protein was determined by probing the blots using specific antibodies
against iNOS (Transduction Laboratory, 1:2000), followed by an ECL
detection method (27). Inducible NOS was detected as a 130-kDa band.
Statistical Analysis--
All data were given as mean ± S.D. Differences between the two groups were compared by an unpaired
Student's t test. For multigroup comparisons, analysis of
variance followed by Student-Newman-Keuls test was performed. A value
of p < 0.05 was considered statistically significant.
 |
RESULTS |
Decreased basal TNF-
Expression in eNOS
/
Cardiomyocytes--
To determine basal levels of TNF-
expression in
cardiomyocytes from wild-type and eNOS
/
mice, levels of
TNF-
mRNA and protein were examined. TNF-
mRNA was
detectable in wild-type cardiomyocytes (Fig.
1, A and B). However, TNF-
mRNA expression was significantly decreased by 65.6% (p < 0.05) in eNOS
/
cardiomyocytes, compared with wild-type cardiomyocytes (Fig. 1B). Similarly, very low levels of TNF-
protein were
detected in the culture medium of wild-type cardiomyocytes, whereas no detectable TNF-
protein was found in the culture medium of
eNOS
/
cardiomyocytes (Fig. 1C). The results
demonstrate that deficiency in eNOS decreases basal TNF-
expression
at the transcriptional level in neonatal mouse cardiomyocytes.

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Fig. 1.
Basal TNF-
expression in neonatal cardiomyocytes. Neonatal
cardiomyocytes were prepared from wild-type and eNOS /
mice as indicated under "Experimental Procedures." After 48 h
of cell culture, TNF- protein was measured in culture medium by
ELISA, and TNF- mRNA was analyzed in cardiomyocytes by RT-PCR.
A, representative gel of TNF- and GAPDH amplification.
B, TNF- /GAPDH optical density ratio. TNF- mRNA
expression was significantly decreased in eNOS / mouse
cardiomyocytes, compared with the wild-type cardiomyocytes.
C, very low levels of TNF- protein were measured in the
culture medium of wild-type cardiomyocytes but were not detectable in
eNOS / mouse cardiomyocytes. Measurements were made in
triplicate. Data are means ± S.D. from 3 to 6 independent
experiments. *, p < 0.05 versus
wild-type.
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|
Attenuation of LPS-stimulated TNF-
Expression in
eNOS
/
Cardiomyocytes--
Previous studies have shown
that LPS administration induces production of TNF-
in cultured
cardiomyocytes (11) and in the myocardium (4). To investigate whether
LPS-stimulated TNF-
expression is modulated by eNOS, TNF-
mRNA and protein expression were measured in wild-type and
eNOS
/
cardiomyocytes after LPS treatment. Fig.
2A illustrates the time course
of TNF-
protein production induced by LPS (10 µg/ml) in cardiomyocytes. Up-regulation of TNF-
protein expression occurred in
less than 30 min and peaked around 4-6 h after LPS challenge. Based on
this time course response to LPS, 4 h were chosen for all
subsequent experiments. LPS dose-dependently induced
TNF-
protein release in wild-type cardiomyocytes (Fig.
2B). The levels of TNF-
mRNA (Fig. 2C) and
protein (Fig. 2D) in response to LPS were significantly
decreased by 49.2 and 33.9% (p < 0.05) in
eNOS
/
cardiomyocytes, compared with wild-type
cardiomyocytes. Consistent with basal TNF-
expression,
LPS-stimulated TNF-
expression is also decreased in
eNOS
/
cardiomyocytes.

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Fig. 2.
LPS induction of TNF-
expression in wild-type and
eNOS /
neonatal cardiomyocytes. After 48 h of culture,
cardiomyocytes were treated by LPS. A, time course of
LPS-induced TNF- protein production. TNF- protein was increased
within 30 min and reached the maximal levels after 4 h of LPS
treatment (10 µg/ml). B, LPS (0.1-10 µg/ml)
dose-dependently induced TNF- protein release in
wild-type cardiomyocytes. After LPS treatment (10 µg/ml), TNF-
mRNA (C) and protein release (D) were
significantly decreased in eNOS / neonatal
cardiomyocytes, compared with wild-type cardiomyocytes. LPS were
treated for 4 h in B-D. Measurements were made in
triplicate. Data are mean ± S.D. from 3 to 6 independent
experiments. *, p < 0.05 versus
wild-type.
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|
The Role of NO in LPS-stimulated TNF-
Expression--
To assess if enhancement of LPS-stimulated TNF-
expression in the wild-type cardiomyocytes was due to NO production
from eNOS, three lines of experiments were conducted. First, the NOS inhibitor L-NAME was employed in wild-type cardiomyocytes
to eliminate endogenously generated NO. Wild-type cardiomyocytes were
treated with LPS (10 µg/ml) and L-NAME (500 µM) for 4 h. L-NAME treatment decreased
LPS-induced TNF-
production to the levels similar to eNOS
/
cardiomyocytes in response to LPS
(p < 0.05, Fig.
3A). Second, basal nitrite
level in eNOS
/
cardiomyocytes was lower than that of
wild-type cardiomyocytes (8.3 ± 0.5 versus 14.4 ± 1.3 nmol/mg cell protein, n = 5 per group, p < 0.05). Addition of low concentration of DETA-NO (2 µM) raised nitrite levels (16.8 ± 2.5 nmol/mg cell
protein) in eNOS
/
cardiomyocytes to those of the
wild-type cardiomyocytes. DETA-NO increased basal TNF-
protein from
undetectable levels in eNOS
/
cardiomyocytes to 3.8 ± 2.7 pg/5 × 105 cells and increased LPS-induced
TNF-
protein production by 32.6% (p < 0.05, Fig.
3A). LPS-induced TNF-
protein production in both eNOS
/
and wild-type cardiomyocytes was completely
inhibited by protein synthesis inhibitor cycloheximide (Fig.
3A). Third, the effect of eNOS on LPS-induced TNF-
expression was further confirmed by using adenoviral mediated eNOS gene
transfer. Expression of re-introduced eNOS gene in
eNOS
/
cardiomyocytes was detected by RT-PCR using
bovine eNOS-specific primers (Fig. 3B). Adv-eNOS infection
increased TNF-
production by about 26% (p < 0.05, Fig. 3C) in response to LPS in eNOS
/
cardiomyocytes. Thus, the presence of NO was able to restore TNF-
production of eNOS
/
cardiomyocytes in response to
LPS.

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Fig. 3.
Effects of NO on LPS-induced
TNF- production in cardiomyocytes.
Neonatal cardiomyocytes were isolated from wild-type and
eNOS / mice and cultured for 48 h. A,
wild-type cardiomyocytes were treated with LPS (10 µg/ml) alone or in
the presence of nitric-oxide synthase inhibitor L-NAME (500 µM). L-NAME decreased LPS-induced TNF-
production in wild-type cardiomyocytes by 35% (*, p < 0.05 versus wild-type LPS). eNOS /
cardiomyocytes were treated with LPS (10 µg/ml) alone or in the
presence of NO donor DETA-NO (2 µM) for 4 h. DETA-NO
increased LPS-induced TNF- in eNOS / cardiomyocytes
by 30% (*, p < 0.05 versus
eNOS / LPS). Treatment of protein synthesis inhibitor
cycloheximide (20 µM) abolished LPS-induced TNF
production in both wild-type and eNOS / cardiomyocytes.
B, confirmation of eNOS mRNA expression by RT-PCR after
bovine eNOS gene was re-introduced in eNOS /
cardiomyocytes. DNA from Adv-eNOS was used as a positive control for
amplification of bovine eNOS expression and total RNA from
Adv-eNOS-infected eNOS / cardiomyocytes without RT as a
negative control. C, eNOS /
cardiomyocytes were infected with Adv-eNOS or Adv-GFP for 24 h before treatment with LPS. Adv-eNOS infection increased LPS-induced
TNF- protein production in eNOS / cardiomyocytes by
26% (*, p < 0.05 versus
LPS+Adv-GFP). Measurements were made in triplicate. Data are
means ± S.D. from 3 to 7 independent experiments.
|
|
Because LPS has been demonstrated to induce iNOS expression in
cardiomyocytes (7), we then investigated whether iNOS-derived NO was
involved in the regulation of LPS-stimulated TNF-
expression in our
experiments. Fig. 4A showed
that iNOS protein expression was only detectable at 6 h but not at
4 h after LPS treatment in cardiomyocytes. To exclude further the
contribution of iNOS in LPS-induced TNF-
production in our study,
iNOS
/
cardiomyocytes were employed. After 4 h of
LPS treatment, TNF-
production was not different between
iNOS
/
and wild-type cardiomyocytes (Fig.
4B), suggesting that iNOS was not involved in TNF-
production in the present study. Our results strongly support the
notion that NO production from eNOS enhances LPS-stimulated TNF-
expression.

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Fig. 4.
Involvement of iNOS in LPS-induced
TNF- protein production in
cardiomyocytes. A, the time course of iNOS protein
expression in cultured neonatal cardiomyocytes in response to LPS.
After addition of LPS (10 µg/ml) in culture medium, the cells were
harvested at different times for detection of iNOS protein by Western
blotting. Expression of iNOS protein (130 kDa) was detectable only
6 h after LPS treatment. The figure is representative of 3 independent Western blot experiments. B, wild-type and
iNOS / cardiomyocytes were treated with LPS for 4 h. TNF- production in the culture medium from both cardiomyocytes
was measured. Compared with wild-type cardiomyocytes, deficiency in
iNOS had no effect on LPS-induced TNF- protein production
(p = not significant). Measurements were made in
triplicate. Data are means ± S.D. from 4 independent
experiments.
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|
Role of cGMP in LPS-stimulated TNF-
Expression--
It has been
widely recognized that NO exerts cellular effects via both
cGMP-dependent and -independent mechanisms (23, 24). In the
present study, we tested the cGMP-dependent pathway by using a selective GC inhibitor ODQ and a membrane-permeable cGMP analogue 8-Br-cGMP. ODQ completely blocked NO donor SNAP-induced cGMP
production (Fig. 5A). However,
it did not have any effect on LPS-induced TNF-
protein production in
wild-type cardiomyocytes (Fig. 5B). Furthermore, no effect
of 8-Br-cGMP was observed on LPS-induced TNF-
expression in
eNOS
/
cardiomyocytes (Fig. 5B). The results
suggest that eNOS-derived NO regulates LPS-stimulated TNF-
expression through a mechanism independent of cGMP.

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Fig. 5.
Effects of cGMP on LPS-stimulated
TNF- expression. Wild-type and
eNOS / cardiomyocytes were cultured for 48 h after
isolation. A, wild-type mouse cardiomyocytes were treated
with a selective guanylate cyclase inhibitor ODQ (100 µM)
alone or in combination with the NO donor SNAP (10 µM)
for 2 h. Intracellular cGMP level was measured by ELISA. SNAP
increased cGMP levels by 2.5-fold, compared with the basal level in
cardiomyocytes (*, p < 0.01 versus control,
n = 3). ODQ completely blocked SNAP-stimulated increase
in cGMP production ( , p < 0.01 versus
SNAP, n = 3). B, wild-type mouse
cardiomyocytes were pretreated with ODQ (100 µM) for 30 min followed by incubation of LPS (10 µg/ml) for 4 h.
Pretreatment with ODQ did not have any effect on TNF- production
induced by LPS in wild-type cardiomyocytes (p = not significant). Treatment of 8-Br-cGMP (10 µM)
for 4 h did not have any effect on TNF- production induced by
LPS in eNOS / cardiomyocytes (p = not significant). Measurements were made in triplicate. Data are
means ± S.D. from 3-5 independent experiments.
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|
Role of cAMP in LPS-stimulated TNF-
Expression--
Studies
have demonstrated that NO also activates cAMP-dependent
pathway (23). We therefore hypothesized that deficiency in eNOS
resulted in decreased cAMP levels, leading to attenuated TNF-
expression in response to LPS in cardiomyocytes. Consistent with this
hypothesis, our data showed that intracellular cAMP levels were
decreased in eNOS
/
cardiomyocytes compared with
wild-type cardiomyocytes (Fig. 6). DETA-NO (2 µM) treatment elevated cAMP levels in
eNOS
/
cardiomyocytes (Fig. 6). Furthermore, LPS-induced
TNF-
protein was decreased by inhibition of cAMP production using a
selective AC inhibitor SQ22536 in a dose-dependent manner
and by a specific PKA inhibitor H89 in wild-type cardiomyocytes (Fig.
7A). A membrane-permeable cAMP
analogue 8-Br-cAMP (10-100 pM) enhanced LPS-induced
TNF-
expression in eNOS
/
cardiomyocytes (Fig.
7B). These results demonstrated that NO enhances LPS-induced
TNF-
expression via cAMP-dependent pathway in
cardiomyocytes.

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Fig. 6.
Effects of DETA-NO on intracellular cAMP
production in cardiomyocytes. Intracellular cAMP levels were
decreased in eNOS / compared with wild-type
cardiomyocytes (*, p < 0.05 versus
wild-type control). Addition of NO donor DETA-NO (2 µM)
to eNOS / cardiomyocytes for 30 min elevated the
intracellular cAMP levels similar to wild-type cardiomyocytes ( ,
p < 0.05 versus eNOS /
control). LPS (10 µg/ml) had no effect on intracellular cAMP
production in wild-type cardiomyocytes after treatment for 30 min.
Measurements were made in triplicate. Data are means ± S.D. from
4 independent experiments.
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Fig. 7.
Effects of cAMP and PKA on LPS-stimulated
TNF- expression. Wild-type and
eNOS / cardiomyocytes were cultured for 48 h after
isolation. A, wild-type mouse cardiomyocytes were pretreated
with a selective adenylate cyclase inhibitor SQ22536 (0.2-2
mM) or a specific PKA inhibitor H89 (20 µM)
for 30 min followed by incubation of LPS (10 µg/ml) for 4 h.
Pretreatment with SQ22536 or H89 attenuated TNF- production induced
by LPS (*, p < 0.05 versus LPS alone).
B, eNOS / cardiomyocytes were treated with
LPS alone or 8-Br-cAMP (10 to 100 pM) for 4 h.
Treatment of 8-Br-cAMP (10-100 pM) enhanced LPS-induced
TNF- production (*, p < 0.05 versus LPS
alone). Measurements were made in triplicate. Data are mean ± S.D. from 3 to 5 independent experiments.
|
|
Essential Role of p38 MAPK in LPS-stimulated TNF-
Expression--
It has been shown that p38 MAPK is required for
LPS-stimulated TNF-
expression in neutrophils (16). However, the
role of p38 MAPK in LPS-induced TNF-
expression in cardiomyocytes is not clear. In the present study, we demonstrated that LPS induced activation of p38 MAPK. A time course response to LPS showed that activation of p38 MAPK in cardiomyocytes occurred within 5 min, reaching the maximal response at 30 min, and returned to basal level
after 6 h of LPS challenge (Fig.
8A). The activation of p38
MAPK preceded the production of TNF-
protein in cardiomyocytes (Fig.
2B). LPS treatment dose-dependently increased
phosphorylation of p38 MAPK in cardiomyocytes (Fig. 8B).
Moreover, inhibition of p38 MAPK by using a selective inhibitor,
SB203580 or SB202190 (5 µM), abrogated TNF-
mRNA (Fig. 8C) and protein expression (Fig.
8D) induced by LPS in wild-type cardiomyocytes, indicating that LPS-induced TNF-
expression is mediated by activation of p38
MAPK in cardiomyocytes.

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Fig. 8.
Requirement of p38 MAPK activation for
LPS-induced TNF- expression in wild-type
neonatal cardiomyocytes. A, time course of LPS-induced
phosphorylation of p38 MAPK. Phosphorylation of p38 MAPK was increased
within 5 min, reaching the maximal levels within 15 min and returning
to basal levels after 6 h of LPS treatment (10 µg/ml).
B, LPS (0.1-10 µg/ml) dose-dependently
induced phosphorylation of p38 MAPK in cardiomyocytes after 4 h of
treatment. Incubation with a p38 MAPK-specific inhibitor SB203580 or
SB202190 (5 µM) abrogated TNF- mRNA (C)
and protein expression (D) in response to LPS (10 µg/ml).
Measurements were made in triplicate. Data are means ± S.D. from
3 to 5 independent experiments. *, p < 0.05 versus LPS alone.
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|
NO Enhances TNF-
Expression via cAMP-mediated p38 MAPK
Pathway--
To demonstrate further whether the effects of NO on
LPS-induced TNF-
expression were mediated by p38 MAPK activation via cAMP-dependent pathway, the following experiments were
performed. 1) Inhibition of NOS by L-NAME (500 µM, Fig. 9A),
inhibition of AC by SQ22536 (200 µM, Fig. 9B),
or inhibition of PKA by H89 (20 µM, Fig. 9B)
attenuated LPS-induced phosphorylation of p38 MAPK in wild-type
cardiomyocytes (p < 0.05). 2) NO donor DETA-NO (2 µM) enhanced the effects of LPS on phosphorylation of p38
MAPK in eNOS
/
cardiomyocytes (p < 0.05, Fig. 9C). 3) Neither ODQ nor 8-Br-cGMP had any effect
on LPS-induced activation of p38 MAPK (Fig. 9, A and
C). 4) A cAMP analogue 8-Br-cAMP (100 pM)
increased phosphorylation of p38 MAPK in the presence or absence of LPS
in eNOS
/
mouse cardiomyocytes (p < 0.05, Fig. 9, D and E), mimicking the effect of
NO (Fig. 9C). The results demonstrate that NO enhances phosphorylation of p38 MAPK via cAMP-dependent pathway in
response to LPS.

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Fig. 9.
NO enhances LPS-induced p38 MAPK activation
via cAMP/PKA-dependent pathway. A,
pretreatment with a NOS inhibitor L-NAME (500 µM) for 30 min but not a selective guanylate cyclase
inhibitor ODQ (100 µM) attenuated phosphorylation of p38
MAPK induced by LPS (10 µg/ml) in wild-type cardiomyocytes.
B, pretreatment with a selective adenylate cyclase inhibitor
SQ22536 (200 µM) or a specific PKA inhibitor H89 (20 µM) for 30 min attenuated phosphorylation of p38 MAPK
induced by LPS (10 µg/ml) in wild-type cardiomyocytes.
C, NO donor DETA-NO (2 µM) increased
phosphorylation of p38 MAPK and enhanced the effects of LPS (10 µg/ml) on p38 phosphorylation in eNOS /
cardiomyocytes. However, 8-Br-cGMP (10 µM) had no effect.
D and E, 8-Br-cAMP (100 pM) alone or
in the presence of LPS enhanced p38 MAPK phosphorylation in
eNOS / cardiomyocytes, mimicking the effect of NO.
Measurements were made in triplicate. Data are mean ± S.D. from 3 to 6 independent experiments. *, p < 0.05 versus LPS alone; , p < 0.05 versus control.
|
|
 |
DISCUSSION |
The major findings of the present study are that endogenous NO
production from eNOS enhanced basal and LPS-stimulated TNF-
expression in cultured neonatal mouse cardiomyocytes. TNF-
expression was preceded by increased phosphorylation of p38 MAPK, and
selective inhibition of p38 MAPK abrogated TNF-
expression
stimulated by LPS, suggesting an important role of p38 MAPK in
LPS-induced TNF-
expression. We further demonstrated that the
effects of NO were mediated through the cAMP/PKA-dependent
p38 MAPK pathway. Our study demonstrated a novel signaling mechanism by
which endogenously produced NO from eNOS modulates LPS-stimulated
TNF-
expression in cardiomyocytes.
NO from eNOS Enhances LPS-induced TNF-
Expression via
cAMP-dependent Pathway--
Cardiomyocytes constitutively
express eNOS, which plays an important role in the regulation of
myocardial function (21). In the present study we demonstrated that
eNOS-derived NO enhanced LPS-stimulated TNF-
expression in neonatal
mouse cardiomyocytes. This conclusion was based on the following three
lines of evidence. First, deficiency in eNOS decreased both basal and
LPS-stimulated TNF-
mRNA and protein expression in mouse
cardiomyocytes. Second, treatment of NOS inhibitor L-NAME
in wild-type cardiomyocytes decreased LPS-induced TNF-
expression to
the level similar to that of eNOS
/
cardiomyocytes.
Third, either NO donor DETA-NO or Adv-eNOS infection was able to
restore LPS-stimulated TNF-
production in eNOS
/
cardiomyocytes to a similar level of wild-type cardiomyocytes. In the
present study, TNF-
was measured at 4 h after LPS treatment when iNOS was not detectable. This is consistent with previous demonstrations that induction of iNOS in cardiomyocytes or heart occurs
at late stages of LPS administration (4, 15). In addition, deficiency
in iNOS did not influence the response to LPS in terms of TNF-
production in cardiomyocytes. Thus, the contribution of iNOS in
LPS-induced TNF-
expression is negligible in the present study.
Signal transductions of NO are mediated by both
GC/cGMP-dependent and -independent pathways (23). A recent
study (24) showed that exogenous NO donors induced TNF-
expression
in cardiomyocytes via the GC/cGMP-dependent pathway.
However, in the present study 8-Br-cGMP failed to mimic the effects of
eNOS-derived NO on TNF-
expression in eNOS
/
cardiomyocytes. ODQ abrogated NO donor-stimulated cGMP production but
did not affect TNF-
biosynthesis in wild-type cardiomyocytes in
response to LPS. The results indicate that the effect of eNOS-derived NO on LPS-stimulated TNF-
expression is independent of cGMP in cardiomyocytes. Indeed, cGMP-independent effects of NO on LPS-induced TNF-
expression have been demonstrated in non-cardiomyocytes such as
neutrophils (28). Studies have also shown that low levels of NO
activate AC and increase cAMP by a cGMP-independent mechanism in rat
adult cardiomyocytes (23). Consistent with this notion, our data
demonstrated that DETA-NO at low concentrations increased intracellular
cAMP levels in eNOS
/
cardiomyocytes. Moreover,
deficiency in eNOS decreased cAMP levels in cardiomyocytes. We further
demonstrated that the effects of eNOS-derived NO on LPS-induced TNF-
production were mediated through AC/cAMP-dependent pathways
in cardiomyocytes. Inhibition of cAMP production attenuated LPS-induced
TNF-
expression in wild-type cardiomyocytes. cAMP analogue 8-Br-cAMP
mimicked the effects of NO by increasing TNF-
production in
eNOS
/
cardiomyocytes. These results were somewhat
unexpected as previous studies (29) have shown that cAMP analogues at
10 µmol/liter concentrations inhibited LPS-induced TNF-
expression
in cardiomyocytes. It is important to note that the physiological level
of cAMP in cardiomyocytes is ~6 pmol/mg protein (23). To mimic the
endogenous level of cAMP, we used low concentrations of cAMP analogue
(10 and 100 pmol/liter) in the present study. Our results demonstrated that low levels of cAMP enhance basal as well as LPS-induced TNF-
production. Thus, cAMP may have completely different effects depending on its intracellular concentrations in cardiomyocytes. Whereas high
concentrations of cAMP analogue (at micromolar range) inhibit LPS-induced TNF-
production, low levels of cAMP enhance LPS-induced TNF-
expression in cardiomyocytes.
Activation of p38 MAPK Is Required for LPS-induced TNF-
Expression--
An important finding in the present study is that
LPS-stimulated TNF-
expression is mediated through p38 MAPK
activation in neonatal cardiomyocytes. LPS-induced phosphorylation of
p38 MAPK was less than 5 min, reaching a maximum within 30 min, which preceded the release of TNF-
production. More importantly,
inhibition of p38 MAPK abrogated TNF-
expression induced by LPS. The
mechanism by which LPS induces phosphorylation of p38 MAPK in
cardiomyocytes remains elusive. It is possible that LPS stimulates
p38 MAPK by activating protein tyrosine kinases through CD14 (30) and
TLRs (13), and by inducing oxidative stress through
Rac1-dependent pathway (31).
NO from eNOS Promotes LPS-stimulated p38 MAPK
Activation via cAMP/PKA-dependent
Pathway--
Recent studies (32) have shown that cAMP activates p38
MAPK via a PKA-dependent mechanism in adult mouse
cardiomyocytes. PKA inhibits Ser/Thr protein phosphatases and increases
p38 MAPK activity (33, 34). However, it is not known if
cAMP/PKA-dependent p38 MAPK activation is responsible for
the effects of NO on LPS-induced TNF-
expression. In the present
study, inhibition of either NOS or AC activities attenuated LPS-induced
phosphorylation of p38 MAPK and TNF-
release in cardiomyocytes.
Addition of a NO donor or a cAMP analogue at low concentrations
increased phosphorylation of p38 MAPK and TNF-
release induced by
LPS in eNOS
/
cardiomyocytes. Furthermore, inhibition of
PKA decreased phosphorylation of p38 MAPK and TNF-
expression
induced by LPS. Our results demonstrated that eNOS-derived NO enhances
LPS-induced p38 MAPK and TNF-
expression via a
cAMP/PKA-dependent pathway.
In summary, the present study demonstrated for the first time that
endogenously produced NO from eNOS enhances LPS-stimulated TNF-
expression in cultured neonatal mouse cardiomyocytes. Activation of p38
MAPK is essential in LPS-stimulated TNF-
expression. Moreover, the
effects of NO on LPS-stimulated TNF-
expression are mediated through
cAMP/PKA-dependent p38 MAPK mechanisms (Fig.
10). These findings provide novel
pathways leading to LPS induction of TNF-
in cardiomyocytes. The
significance of the finding that eNOS-derived NO enhances LPS-induced
TNF-
expression in cardiomyocytes in sepsis is presently not clear.
High levels of TNF-
have been demonstrated to have detrimental
effects on myocardial function. However, TNF-
is required for innate
resistance to invading pathogens (2). It is possible, albeit
speculative, that increased expression of TNF-
by eNOS leads to an
enhancement of innate immune response in the heart, which in turn
promotes the recruitment of neutrophils, monocytes, and natural killer
cells to the areas of the myocardium where the pathogens are located.
Increased TNF-
expression has also been shown to induce manganese
superoxide dismutase expression (35) which may reduce oxidative stress
and tissue injury during sepsis. Therefore, eNOS enhancement of TNF-
production may confer some beneficial effects. However, the
pathophysiological significance of the eNOS/cAMP/p38 MAPK pathway in
sepsis requires further investigation.

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Fig. 10.
Proposed model of signaling pathways leading
to TNF- expression during LPS stimulation in
neonatal mouse cardiomyocytes. Activation of p38 MAPK is required
for LPS-induced TNF- expression. NO produced from eNOS enhances
LPS-stimulated TNF- expression through activation of adenylate
cyclase and cAMP/PKA-dependent p38 MAPK pathway.
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