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1 Section of Critical Care Medicine, Department of Medicine, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612; and 2 Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G-2S2
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Previous studies have demonstrated the
existence of a circulating myocardial depressant substance during human
septic shock. We have recently identified this substance as a
synergistic combination of tumor necrosis factor-
(TNF-) and
interleukin-1
(IL-1). This study utilized an in vitro cardiac
myocyte assay to evaluate the potential mechanistic role of nitric
oxide (NO) and cGMP in depression of myocyte contractility induced by
TNF-, IL-1, TNF- + IL-1 (at low concentrations), and human
septic shock serum (HSS). TNF-, IL-1, TNF- + IL-1, and each
of 5 sera from patients with acute septic shock caused depression of
both maximum extent and peak velocity of cardiac myocyte shortening and
an increase in intracellular cGMP concentration during 30 min of
exposure (minimum P < 0.01). NO
synthetase (NOS) and guanylate cyclase inhibitors such as
N-methyl-L-arginine
(L-NMA) and methylene blue prevented these effects; an excess of
L-arginine with
L-NMA restored them (minimum
P < 0.01). In contrast,
D-arginine failed to reestablish cytokine-induced myocyte depression and cGMP accumulation prevented by
L-NMA. Exposure of myocytes to
TNF-, IL-1, or TNF- + IL-1 produced a
concentration-dependent increase in intracellular cGMP that paralleled
the depression of cardiac myocyte contractility (minimum
P < 0.001). In addition, TNF-,
IL-1, TNF- + IL-1, or HSS application to cardiac myocytes
resulted in increased NO gas generation, which was inhibited by
L-NMA (minimum
P < 0.01). Furthermore, unstimulated
cardiac myocytes were shown to harbor constitutive but not inducible
NOS activity. These data suggest that the sequential generation of NO
by a constitutive NOS and cGMP by guanylate cyclase represents an
important mechanism of cardiac myocyte depression by TNF-, IL-1,
TNF- + IL-1, and the myocardial depressant substance(s)
of septic shock.
myocardial depressant factor; cytokine; heart contractility; cyclic nucleotide; septicemia
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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SHOCK SECONDARY TO SEPSIS is a serious disorder with significant morbidity and mortality despite appropriate antibiotic and supportive therapy. The typical human cardiovascular response to septic shock is characterized by hypotension, decreased systemic vascular resistance, and elevated cardiac index. Recent studies using bedside radionuclide ventriculography and echocardiography have demonstrated that reversible myocardial depression manifested by reduction of both right and left ventricular ejection fractions and dilation of both ventricles is a common occurrence during human septic shock (44, 45).
Measurements of myocardial cell contraction in the presence of serum
from patients with septic shock demonstrate a depression of maximum
extent and peak velocity of myocyte shortening that correlates
quantitatively and temporally with the depression of ejection fraction
seen in the same patients (46, 48). This suggests the presence of a
circulating myocardial depressant substance or substances (MDS) during
the acute phase of septic shock that may be responsible for human
sepsis-induced myocardial dysfunction (46, 48). Previous studies
suggest that MDS may represent a protein or proteins in the 10- to
30-kDa-molecular weight range (34, 46, 48). The cytokines, tumor
necrosis factor- (TNF-) and interleukin-1 (IL-1), fit the
known physical characteristics of MDS. Serum levels of each are
elevated during sepsis and septic shock (12, 25). Using an in vitro
assay of cardiac myocyte function, we have recently demonstrated that
cardiac myocyte depression can be produced by extremely low
concentrations of TNF- and IL-1 in combination, concentrations
that are similar to those found circulating during human septic shock
(39). In addition, we have shown that the myocardial depressant
activity of human septic sera (HSS) obtained from patients with acute
septic shock can be eliminated by the immunoabsorption of TNF- and
IL-1, suggesting a central role for these two cytokines in human
septic myocardial depression (39).
Nitric oxide (NO) and cGMP are known to have significant physiological and pathophysiological roles in the vasculature. The role of NO and cGMP in the heart has not been as well defined. Recent data suggests that NO and cGMP may play a substantial part in physiological regulation of cardiac contractility (2, 3, 9, 17, 28, 30, 31, 36, 41, 42, 55, 61). Investigators have also shown that cytokine and endotoxin-mediated depression of contractility of in vitro myocardial tissue may be produced via an NO- and cGMP-dependent mechanism (4, 5, 8, 21, 22, 37, 40, 53). In contrast, other data argue against a role for NO in either the physiological regulation of cardiac contractility (67) or in endotoxin- or cytokine-driven pathophysiological myocardial depression (18, 68).
Given this conflicting data, this study was designed to evaluate the
possible mechanistic role of NO and cGMP in early cardiac myocyte
depression induced by TNF-, IL-1, low concentrations of TNF- + IL-1 together, and, in particular, HSS containing MDS activity.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Tissue culture and other methods utilized have been previously described (39, 46, 52). Spontaneously beating newborn rat myocardial cells were established using a modification of the technique described by Harary and Farley (29). Standard growth media consisted of 25% HEPES-buffered Medium 199 (GIBCO Laboratories, Grand Island, NY) and 10% heat-inactivated FCS (Sigma Chemical, St. Louis, MO) diluted in an L-arginine (L-Arg)-free balanced salt solution and supplemented with glutamine (Sigma Chemical), penicillin (Sigma Chemical), and streptomycin (Sigma Chemical). Medium 199 and FCS contained 0.33 and 0.21 mM L-Arg, respectively. The final concentration of L-Arg in standard growth media was 0.1 mM.
Latex microbeads were introduced into the culture of spontaneously beating myocytes and affixed themselves to cell membranes. Between 7 and 10 days after plating, the petri dish containing myocardial cells was fastened to the heated (37°C) stage of an inverted-optics, phase-contrast microscope attached to a side-arm video camera. A custom-built electronic tracking system was used to quantitate the movement of a latex bead selected from the many beads attached to the membranes of beating myocytes. The typical maximum initial extent of rhythmic displacement of the bead was between 5 and 8 µm, depending on the length of the myocyte to which it was adherent. The signal was relayed to an intervening electronic instrument (which derived peak contraction velocity from rate of change of bead displacement) and a two-channel strip chart recorder that printed an analog recording of the extent and velocity of bead displacement. To ensure a fixed contraction frequency, a custom-built alternating current electrical pulse generator was utilized to pace myocytes (12 V, maximum 40 mA, 0.7- to 7-ms pulse duration) at 1 Hz. The minimum current and pulse duration required to effectively pace cardiac myocytes were utilized for each experiment.
Each individual assay was performed as follows. After application of fresh growth media, plates were mounted on the microscope stage and myocytes were paced at 60 contractions/min. An appropriate bead was located, and the extent and velocity of myocyte shortening were measured for 5 min (baseline contractility). If the extent of cell contraction was stable (maximum 2.5% variation over 5 min), 1 ml of test solution was added. Test media used either 10% newborn calf serum (NCS) (GIBCO Laboratories) with the addition of designated concentrations of cytokines or 10% HSS in place of NCS. Control studies were performed with 10% NCS alone. After replacement of fresh growth media with test or control media, measurements of maximum extent and peak velocity of myocyte shortening were obtained every 5 min for 30 min.
All pipettes, plates, and other equipment used for preparation,
culture, or testing of cardiac myocytes was endotoxin-free and
disposable. All liquid media contained <1 pg/ml endotoxin. FCS used
for growth media contained no detectable endotoxin. NCS used in test
media contained 0.48 ng/ml endotoxin. All recombinant cytokines
contained <50 pg endotoxin/µg cytokine. Culture media, cytokine
solutions, and other test solutions were tested for endotoxin content
using a quantitative, chromogenic Limulus amebocyte lysate assay with a
detection limit of 5 pg/ml (Whitaker M. A. Bioproducts, Walkersville,
MD). TNF- and IL-1 concentrations in human sera were determined
using an enzyme-linked immunoabsorbent assay (TNF- by T Cell
Science, Boston, MA; IL-1 by Citron Biotechnologies, Pine Brook, NJ)
according to the instructions of the manufacturer.
Effect of
N-methyl-L-arginine
and methylene blue on contractility of cardiac myocytes exposed to
TNF-, IL-1, TNF- + IL-1, and HSS.
TNF- (50 ng/ml) (Sigma Chemical), IL-1 (500 ng/ml) (Sigma
Chemical), TNF- (0.05 ng/ml) + IL-1 (2.0 ng/ml), and HSS were tested. These cytokine concentrations match those used in a previous series of studies (39). The specified individual concentrations of
TNF- and IL-1 were used because they are within the range of
concentrations found circulating during human septic shock (13, 25,
64). Similarly, human septic shock is commonly associated with the very
low combined concentrations of TNF- and IL-1 used in the
experiment (13, 25, 64). HSS was obtained from 5 patients with acute
septic shock and marked reversible myocardial depression [mean
left ventricular ejection fraction 26 ± 7% (SD) during septic shock
and 55 ± 7% either before septic shock or after recovery; absolute
mean decrease 28 ± 10%]. Each HSS sample had previously been
confirmed to exhibit marked myocardial depressant activity in this in
vitro assay. The primary test solutions consisted of either standard
test media (10% NCS) plus the specified concentrations of TNF-
and/or IL-1 or standard media using 10% HSS in place of
10% NCS. Additional test solutions were composed of primary test
solution plus 1) 10 µM
N-methyl-L-arginine
(L-NMA), 2) 10 µM
L-NMA + 25 mM
L-Arg,
3) 10 µM
L-NMA + 25 mM
D-arginine (D-Arg) (not done with HSS), or
4) 2.5 µM methylene blue (MeB). In
the case of TNF- + IL-1,
L-NMA at 1 and 5 µM were also
run. Furthermore, a panel of control samples containing
1) 10 µM
L-NMA, 2) 25 mM
L-Arg,
3) 10 µM
L-NMA with 25 mM
L-Arg,
4) 25 mM
D-Arg, and
5) 2.5 µM MeB were also tested for
myocardial depressant activity.
[0, 0.125, 0.05, 0.2, 0.8, 3.2, 12.5, 25, 50, and 100 ng/ml (n = 4 each)], IL-1 [0, 2, 8, 32, 125, and 1,000 ng/ml
(n = 4 each)], and
TNF-:IL-1 [0:0, 0.003:0.125, 0.0125:0.5, 0.05:2, and 0.2:8 ng/ml (n = 4 each)] were placed
on cardiac myocytes for 30 min. Following a rapid wash with calcium-
and magnesium-free phosphate-buffered saline, myocytes were frozen by
direct exposure to liquid nitrogen and then stored at 
70°C.
In addition, randomly chosen plates from each test and control group of
the previously described contractility experiments (TNF-, IL-1,
TNF- + IL-1, or HSS and/or combinations of
L-NMA,
L-Arg,
D-Arg, and MeB) were similarly
frozen and stored (n = 4 each).
cGMP and cAMP concentrations were measured using the Biotrak cAMP
enzyme-immunoassay system (dual range) according to the protocol of the
manufacturer (Amersham International). Frozen tissue culture samples in
35-mm tissue culture petri dishes were rapidly thawed by the
application of a 1-ml volume of room-temperature assay buffer
(0.05 M sodium acetate, pH 5.8) containing 4 mM EDTA for
phosphodiesterase inhibition. Cells were scraped (as an intact tissue
sheet) and immediately immersed in boiling water for 10 min to denature
and precipitate protein (15). Following centrifugation at 3,000 g (4°C) for 10 min, 100 µl of
supernatant were utilized according to the nonacetylation protocol
instructions of the manufacturer.
Determination of NO synthetase presence and
activity. Routinely cultured neonatal rat cardiac
myocytes were grown to confluence. Between
day 7 and day
10, when myocytes appeared to exhibit
the density and robust spontaneous beating typical of cells used for assay of contractility, they underwent incubation for 30 min with 10%
NCS alone; 10% NCS with 50 ng/ml TNF-, 500 ng/ml IL-1, or 0.05 ng/ml TNF- + 2.0 ng/ml IL-1; and 10% HSS in place of 10% NCS
(n = 3 each). Subsequently, myocytes
were washed with phosphate-buffered saline, scraped, spun into a cell
pellet (~0.25 g each), frozen with liquid nitrogen, and stored at
75°C. A similar number of myocytes were also harvested
before the 30-min incubation with test or control media.
Calcium-dependent and -independent NO synthetase (NOS) activity was
determined by measuring the conversion of
L-[14C]Arg
to
L-[14C]citrulline
(detection limit <0.1 pmol citrulline · mg
protein
1 · min1)
as described by Schulz and colleagues (53, 54).
To determine whether detectable NO was being produced by cardiac
myocytes exposed to TNF-, IL-1, both cytokines together, and HSS,
cardiac myocytes were grown to confluence in 25-ml tissue culture
flasks in standard growth media. Cytokine test media solutions consisted of standard test media (10% NCS) with 50 ng/ml TNF-, 500 ng/ml IL-1, or 0.05 ng/ml TNF- + 2.0 ng/ml IL-1. Another three
test solutions contained 10% HSS from three patients in place of 10%
NCS. Additional test solutions contained the same cytokine or HSS
concentrations with 10 µM
L-NMA. The positive control was
standard media with 10% NCS ± 10 µM carbamylcholine (Sigma
Chemical). Headspace NO gas concentration was measured using a
modification of the techniques described by Archer et al. (1) and Brien
et al. (10). After washing with phosphate-buffered saline, the test
solution was introduced. The flask headspace was flushed for 5 min with
an NO-free gas mixture of 75% nitrogen, 20% oxygen, and 5% carbon
dioxide, and an airtight rubber cap was placed on top. Ten milliliters
of headspace gas were aspirated from the flask before and after 30-min
incubation with the test solutions (n = 4 each). NO in the sample was determined by chemiluminescence using a
NO gas analyzer (model 280A; Sievers Instruments, Boulder, CO) with a
detection limit of 2-5 parts per billion. NO gas production was
assessed by calculating the difference between headspace NO gas
concentration before and 30 min after introduction of the test solution.
Statistical analysis. By comparing the
maximum extent and peak velocity of shortening at each 5-min interval
to the baseline value, changes were referenced to initial
contractility. Data for the change in maximum extent and peak velocity
of cardiac myocyte shortening (percentage change from baseline) were
pooled and plotted as a function of time for each control and test
solution concentration. Linear regression analysis was utilized to fit a line for each resulting plot. The slopes of these lines were compared
with the slope of the line for the control solution by a two-tailed
Student's t-test to determine whether
these slopes were significantly different. In this manner, increased
depressant activity was indicated by a more negative value for slope of
the regression line. Where appropriate, a Bonferroni adjustment for multiple comparisons was made as indicated in the legends to
Figs. 1-7.
For studies of cGMP content derived from cardiac myocytes exposed to
increasing concentrations of TNF- and/or IL-1, individual cGMP values were plotted as a function of cytokine concentration. Logistic regression was utilized to fit a line to the data and the
slope of that line compared with a theoretical zero slope by Student's
t-test. A significant slope indicated
the existence of a concentration-dependent effect on intracellular cGMP content.
Multivariate analysis using data for all time points and concentrations
was used to determine whether a concentration-dependent reversal of
TNF-- + IL-1-mediated depression was caused by increasing concentrations of L-NMA.
All other analyses used Student's
t-test with a Bonferroni adjustment
for multiple comparisons where appropriate (as indicated in the legends
to Figs. 1-7).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Contractility, NO production, and cyclic nucleotide
content of TNF--stimulated cardiac
myocytes. Figure
1A shows
cardiac myocyte contractility (expressed as change in maximum extent of myocyte shortening) as a function of time for cells exposed to control
media, TNF-, TNF- + L-NMA,
TNF- + L-NMA + L-Arg, TNF- + L-NMA + D-Arg, or TNF- + MeB. The
same data are also shown in Fig. 1B,
where the slopes of the regression lines fit to the data for each test
group seen in Fig. 1A are plotted.
Control media (10% NCS) caused relatively little decrease of
contractility (decreased maximum extent of cardiac myocyte shortening).
TNF- resulted in marked depression compared with the control
(P < 0.005). The combination of
TNF- with L-NMA had
depression similar to control (P < 0.001 vs. TNF-). L-Arg in
combination with TNF- and
L-NMA reestablished depression
compared with either control (P < 0.001) or TNF- with L-NMA
(P < 0.001).
D-Arg with TNF- and
L-NMA did not reestablish
depression. Similar to L-NMA
with TNF-, MeB with TNF- reversed TNF-'s depressant
activity (P < 0.01 vs.
TNF-). Data for peak velocity of cardiac myocyte
shortening were entirely parallel and similarly significant (not
shown).
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Intracellular cGMP concentrations of cardiac myocytes from the
preceding experiment are shown in Fig.
1C. TNF- resulted in a
significant increase of intracellular cGMP concentration compared with
control media (P < 0.005). The
addition of L-NMA prevented this
increase (P < 0.005 vs. TNF-).
L-Arg (but not
D-Arg) combined with TNF- and
L-NMA reestablished the
increased cGMP concentration (P < 0.001 vs. control, P < 0.001 vs. TNF- + L-NMA). MeB
with TNF- also prevented the increase in cGMP seen with TNF-
(P < 0.005 vs. TNF-).
Intracellular cAMP levels were similarly assessed but did not show any
significant response to the interventions (not shown).
In addition, it having been previously demonstrated that TNF-
produces a concentration-dependent decrease in cardiac myocyte contractility (39), the effect of increasing concentrations of TNF-
on intracellular cGMP concentration of cardiac myocytes was assessed
(Fig. 1D). TNF- induced a highly
concentration-dependent increase in cGMP concentration
(r2 = 0.63, P < 0.001), which
paralleled the previously described decrease in cardiac myocyte
contractility (39).
NO production by myocytes stimulated with standard media alone,
carbamylcholine (positive control), and TNF- is displayed in Fig.
2. Significantly more NO was detected in
headspace gas above myocytes incubated with TNF- for 30 min than
myocytes not exposed to TNF- (P < 0.01) or myocytes exposed to TNF- + L-NMA (P < 0.01).
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Contractility, NO production, and cyclic nucleotide
content of IL-1-stimulated cardiac
myocytes. Figure
3A shows
cardiac myocyte contractility (expressed as change in maximum
extent of myocyte shortening) as a function of time for cells exposed
to control media, IL-1, IL-1 + L-NMA, IL-1 + L-NMA + L-Arg, IL-1 + L-NMA + D-Arg, or IL-1 + MeB. This
data is also shown in Fig. 3B, where
the slopes of the regression lines fit to the data for each group are
plotted. Control media (standard media) again caused relatively little
decrease of maximum extent of cardiac myocyte shortening. IL-1
resulted in marked depression of maximum extent of cardiac myocyte
shortening in comparison to the control
(P < 0.001). The combination of
IL-1 and L-NMA exerted
depression similar to control (P < 0.001 vs. IL-1). L-Arg (but
not D-Arg) in combination with
IL-1 + L-NMA reestablished
depression compared with either control
(P < 0.001) or IL-1 + L-NMA
(P < 0.001). Similar to
L-NMA, MeB reversed IL-1's
depressant activity (P < 0.001 vs.
IL-1). Data for peak velocity of cardiac myocyte shortening was
entirely parallel, with similar values for significance (not shown).
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Cardiac myocyte cGMP concentrations from the experiment are shown in
Fig. 3C. Similar to the TNF-
experiment, IL-1 resulted in a significant increase of intracellular
cGMP concentration compared with control media
(P < 0.001). The addition of
L-NMA prevented this increase
(P < 0.001 vs. IL-1).
L-Arg (but not D-Arg) in combination with
IL-1 + L-NMA reestablished
the increased cGMP concentration (P < 0.005 vs. control, P < 0.01 vs.
IL-1 + L-NMA). MeB with
IL-1 also prevented the increase in cGMP seen with IL-1
(P < 0.005 vs. IL-1).
Again, intracellular cAMP levels were similar in all groups (not shown).
The effect of increasing concentrations of IL-1 on intracellular
cGMP concentration of cardiac myocytes was assessed (Fig. 3D). In parallel to our previous
finding of concentration-dependent IL-1-mediated cardiac myocyte
depression, IL-1 also induced a highly concentration-dependent
increase in cGMP concentration (r2 = 0.83, P < 0.001).
Figure 2 shows NO production by myocytes stimulated with IL-1.
Significantly more NO was detected in headspace gas above myocytes
incubated with IL-1 for 30 min than myocytes exposed to standard
test media without IL-1 (P < 0.01) or myocytes exposed to IL-1 in the presence of
L-NMA
(P < 0.01).
Contractility, NO production, and cyclic nucleotide
content of cardiac myocytes exposed to TNF- + IL-1. Figure
4A shows the slopes of the regression lines representing change in myocyte shortening as a function of time for myocytes exposed to
TNF- (0.05 ng/ml) + IL-1 (2.0 ng/ml) in combinations as
specified with L-NMA (10, 5, and
1 µM), L-Arg (25 mM),
D-Arg (25 mM), and MeB
(2.5 µM). The combination of TNF- and IL-1 resulted in
substantial myocyte depression compared with myocytes exposed to
control media (P < 0.001). The
addition of increasing concentrations of
L-NMA serially reduced the
depressant effect of the cytokine combination, with 10 µM abrogating
the effect (P < 0.001 vs. TNF- + IL-1). A highly significant concentration-response relationship was
demonstrated to exist (P < 0.0001).
L-Arg (but not
D-Arg) in combination with TNF-/IL-1 + L-NMA
reestablished depression in comparison to both control
(P < 0.001) and TNF-/IL-1 + L-NMA
(P < 0.001). MeB also reversed the
depressant activity of the cytokine combination (P < 0.001 vs.
TNF-/IL-1). The findings for peak velocity of cardiac myocyte
shortening data were similar (not shown) and exhibited similar values
for significance.
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Cardiac myocyte cGMP concentrations from the experiment are shown in
Fig. 4B. Thirty minutes of cardiac
myocyte exposure to TNF-/IL-1 generated a significant increase of
intracellular cGMP concentration (P < 0.001 compared with control).
L-NMA at 10 µM
prevented this increase (P < 0.001 vs. TNF-/IL-1).
L-Arg (but not
D-Arg) with
TNF-/IL-1 + L-NMA restored the increased cGMP concentration (P < 0.005 vs. control, P < 0.001 vs.
TNF-/IL-1 + L-NMA). MeB
also prevented the increase in cGMP levels seen with the cytokine
combination (P < 0.005 vs.
TNF-/IL-1). Myocyte intracellular cAMP levels were not
significantly different (not shown).
The effect of increasing concentrations of TNF- and IL-1 on
intracellular cGMP concentration of cardiac myocytes is demonstrated in
Fig. 4C. A highly
concentration-dependent increase in cGMP concentration is shown to
exist in association with increasing concentrations of a combination of
TNF- and IL-1
(r2 = 0.62, P < 0.001). This finding
supports data in a previous manuscript (39) in which these same
increasing cytokine concentrations resulted in increasing amounts of
cardiac myocyte contractility depression.
As with TNF- and IL-1 individually, significantly more NO was
detected in headspace gas above myocytes incubated with the combination
of TNF- and IL-1 than control myocytes
(P < 0.01) or myocytes exposed to
the cytokine combination in the presence of
L-NMA
(P < 0.01) (Fig. 2).
Contractility, NO production, and cyclic nucleotide
content of HSS-stimulated cardiac myocytes. The effects
of 10% HSS alone and in combination with 10 µM
L-NMA, 10 µM
L-NMA + 25 mM
L-Arg, and 2.5 µM MeB on
cardiac myocyte contractility are shown in Fig. 5,
A-E.
In contrast to the TNF- and IL-1 experiments, only the regression-derived slopes derived from the plots of maximum extent and
peak velocity of cardiac myocyte shortening as a function of time are
shown. Similar results were found with each of the five HSS samples. In
each case, 10% HSS caused significant depression of both extent and
velocity of shortening compared with controls (minimum
P < 0.001).
L-NMA prevented this depressant
effect (minimum P < 0.01 vs. HSS,
but P = NS vs. control), whereas
L-Arg reestablished it (minimum
P < 0.001 vs. control,
P < 0.01 vs. TNF- + L-NMA). Similar to
L-NMA, MeB reversed the
depressant effects of HSS in each of the 5 sera tested (minimum
P < 0.01 vs. HSS).
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Three of the five patient sera (HSS) were assayed for cGMP (Fig. 6, A-C) and cAMP (not shown). Results were again similar in each case. HSS-exposed cardiac myocytes demonstrated a significant increase of intracellular cGMP content compared with myocytes exposed only to control media (minimum P < 0.01). Both L-NMA and MeB prevented this increase (for both, minimum P < 0.01 vs. HSS, but P = NS vs. control). L-Arg reestablished the increase in cGMP prevented by L-NMA (minimum P < 0.005 vs. HSS + L-NMA). In each case, intracellular concentration of cAMP was similar for each test group (not shown).
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NO production by HSS is demonstrated in Fig. 2. Overall, more NO was detected in headspace gas above HSS-stimulated cardiac myocytes than above myocytes exposed to control nonseptic serum (P < 0.01) or myocytes exposed to HSS with L-NMA (P < 0.01).
Effect of L-NMA, L-Arg, D-Arg, and MeB on contractility and cyclic nucleotide content of cardiac myocytes. The individual effects of L-NMA, L-Arg, L-NMA + L-Arg, D-Arg, and MeB on cardiac myocyte contractility are shown in Fig. 7A. None of these agents alone had a significant effect on either maximum extent or peak velocity (not shown) of cardiac myocyte shortening. No substance or combination of substances exerted a depressant or inotropic effect relative to control media. Similarly, Fig. 7B demonstrates that none significantly altered intracellular cGMP concentrations compared with control media. cAMP concentrations were similar in all groups also (not shown).
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1 · min1.
Calcium-dependent (cNOS) activity following 30-min incubation with
control media (10% NCS) or test media containing either 10% NCS with
specified concentrations of TNF- and/or IL-1 or 10% HSS
in place of 10% NCS was not significantly different from baseline. Concurrent mean calcium-independent [inducible NOS (iNOS)]
activity was 0.63 ± 0.09 pmol · mg
protein1 · min1
in the preincubation (baseline) sample. Values for postincubation test
and control samples were not significantly different.
Endotoxin and cytokine levels in test
media. The mean ± SE concentrations of TNF-,
IL-1, and endotoxin in HSS samples were 72 ± 6, 167 ± 18, and
440 ± 120 pg/ml, respectively. Media containing 10% NCS, including
those with cytokines, consistently demonstrated endotoxin
concentrations between 40 and 65 pg/ml (comparable to 10% HSS). Growth
media with 10% FCS demonstrated endotoxin concentrations below the
limit of detection.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The major finding of this study is that the in vitro myocyte depressant
activity of the circulating MDS of human septic shock [which we
have previously identified as a synergistic combination of TNF- and
IL-1 (39)] is mediated, at least in part, through the
sequential generation of NO by cNOS and cGMP by soluble guanylate cyclase. In addition, we have shown that the depressant activity of
TNF-, IL-1, and a combination of TNF- and IL-1 together (at
concentrations comparable to those found in serum during human septic
shock) is also mediated by the sequential generation of NO and cGMP.
Although others have previously demonstrated that early TNF--induced
cardiac myocyte depression may be NO dependent (22), early (as opposed
to late) IL-1-mediated depression has not been linked to NO and
cGMP. Perhaps most importantly, this study provides the first direct
evidence of a pathogenetic link between human septic serum-induced
depression of myocardial tissue and the generation of NO and cGMP.
Our conclusions are supported by a number of our observations. Human
septic serum, TNF-, IL-1, and low concentrations of TNF- + IL-1 together each result in a depression of maximum extent and peak
velocity of cardiac myocyte shortening. Along with contractility
depression, each also causes a parallel increase in measurable
intracellular cGMP. L-NMA, a
competitive inhibitor of both cNOS and iNOS, prevents both depression
of contractility and accumulation of intracellular cGMP.
L-Arg, the natural substrate of
NOS, reestablishes both. D-Arg
(the NOS-insensitive dextro-isomer) fails to reestablish cardiac
myocyte depression and cGMP accumulation in cardiac myocytes exposed to
either TNF- or IL-1 (alone or together) with
L-NMA. MeB, an irreversible
inhibitor of both NOS and guanylate cyclase, prevents depression of
myocyte contractility and intracellular accumulation of cGMP induced by
TNF-, IL-1, TNF- + IL-1, or HSS. These findings occur in
the absence of any significant changes in intracellular cAMP
concentrations. The potential role of cGMP in cytokine-induced cardiac
myocyte depression is reinforced by the demonstration that
concentration-dependent increases of intracellular cGMP induced by
TNF-, IL-1, or both parallel the concentration-dependent
decreases of cardiac myocyte contractility that we have demonstrated
previously (39). The role of NO is supported by the demonstration that
cultured cardiac myocytes exhibit substantial NOS activity. It is
further buttressed by observations that show that cardiac myocytes
stimulated with TNF-, IL-1, both together, or HSS produce
detectable NO gas in the culture flask headspace and that this NO
production is inhibited by the presence of
L-NMA.
We have previously demonstrated the finding of early (<10 min)
depression of HSS-exposed cardiac myocytes (46, 48). More recently, we
have shown that TNF-, IL-1, both TNF- and IL-1 together (at
extremely low concentrations consistent with human septic shock), and
supernatants of activated macrophages individually depress cardiac
myocyte contractility within the same period (38, 39). This rapid
timeframe for depressant response has been supported by other in vitro
studies of myocardial tissue, which also demonstrate the existence of
early depression following cytokine exposure (22, 37, 58, 68). This
would appear to be too short a period for de novo synthesis of iNOS.
This finding of early NO-sensitive myocyte depression along with early
NO production argues strongly that cNOS (as opposed to the inducible
form) is involved in NO generation in this model of septic myocardial
depression. This position is reinforced by our demonstration that
cultured cardiac myocytes exhibit significant calcium-dependent but not
calcium-independent NOS activity. Calcium-dependent NOS activity is
characteristic of cNOS isoforms, whereas calcium-independent activity
is typical of inducible NOS isoforms. The demonstration that measurable
cardiac myocyte cNOS and iNOS activity is unchanged following
incubation with TNF-, IL-1, TNF- and IL-1 together, and HSS
is also consistent with cNOS-, rather than iNOS-, mediated NO
generation. cNOS activity, which is measured under ideal conditions in
the L-citrulline assay, is
expected to be unchanged despite increased NO generation because cellular cNOS activity is substantially regulated by substrate and
cofactor conditions rather than fixed structural modifications or
increases in cellular enzyme content. In contrast, assayed iNOS
activity would be expected to increase with enhanced iNOS cellular
activity because cellular iNOS activity is primarily a function of the
amount of enzyme present (53, 54).
The probable clinical relevance of early septic serum-induced
cNOS-dependent cardiac myocyte depression is supported by the significant correlation Parrillo and colleagues (46, 48) have demonstrated between the in vivo depression of left ventricular ejection fraction among patients with acute septic shock and the in
vitro myocardial depressant activity of the same patients' sera.
Although it cannot be concluded that cNOS-dependent MDS activity (which
appears to be responsible for early depressant effects in vitro) is
solely responsible for relatively prolonged (up to 7-10 days)
myocardial depression seen during human septic shock, an association
between in vitro septic serum-induced cardiac myocyte depression and
clinical septic myocardial dysfunction is clear. Supporting the
potential clinical relevance of MDS-induced early cardiac myocyte
depression are in vivo canine studies that confirm the ability of
TNF- to induce early myocardial depression (within 1 h following
initiation of infusion) (20, 66). This myocardial depression occurs too
early to be caused by de novo iNOS generation. It is, however,
consistent with cytokine-stimulated cNOS activity.
Until recently, few data have been available regarding the
intracellular mechanisms underlying septic myocardial depression. Our
observations, like those of several others (4, 8, 9, 22, 54), generally
support the existence of direct NO-mediated depression of myocardial
tissue by a myocardial NOS stimulated by endotoxin or inflammatory
cytokines. Our work, however, also addresses the specific role of
myocardial cNOS in cytokine and HSS-induced depression of myocyte
contractility. Like ourselves, several investigators have also noted
early-onset (<30 min) depression of myocardial tissue exposed to
cytokines in vitro, consistent with cNOS involvement (22, 26, 68). In
particular, Finkel and colleagues (22) have shown early NO-dependent
depression of guinea pig papillary muscle exposed to cytokines,
including TNF-, IL-2, and IL-6. Goldhaber and colleagues (26) have
similarly shown that high concentrations of TNF- caused an early
NO-dependent depression of cardiac myocyte contractility. The rapidity
of the effect in each study suggests cNOS involvement. Yokoyama and
colleagues (68), however, have suggested that early TNF--induced
depression of adult cardiac myocytes is not dependent on NO. That same
group (43) has recently implicated sphingosine in the
immediate myocardial depressant effects of TNF- in adult feline
myocardial cells, a finding recently supported by Cain et al. (11)
using the combination of TNF- and IL-1 with human myocardial
tissue. The reason for the divergent results are not clear. No evidence
yet links the NO and sphingosine signaling pathways. Potential
differences that might account for the divergent results include the
model used (differing species and ages of animals from which cardiac
tissue was obtained) and differing NOS inhibitors and concentrations of
those inhibitors. It is also possible that myocyte pacing frequency may
play a role in cNOS activity in some studies. However, the pacing rate
of 1 Hz used in this study appears to be well below the 3-Hz threshold
that Kaye et al. (36) have suggested is required to demonstrate
pacing-induced cNOS activity.
In contrast to early, presumably cNOS-dependent myocardial depression,
a series of other studies have tended to support only later-onset
(hours to days) cytokine-driven depression of myocardial contractility
during inflammatory myocardial dysfunction (4, 14, 19, 21, 27, 33, 63).
Several investigators have shown that exposure of myocardial tissue in
vitro to TNF-, IL-1, and supernatants from activated macrophages
(which contain cytokines including TNF- and IL-1) results in
depression of contractility following a delay of several hours or days
(4, 14, 19, 21, 27, 33, 63). Disruption of -adrenergic signal
transduction due to an alteration in G protein interactions has been
implicated as a cause of this phenomenon by one group of investigators
(14, 27). The existence of increased inhibitory G protein subunits resulting in potential adrenoreceptor dysfunction has been confirmed in
catecholamine-resistant human septic shock (7, 49). The generation of
iNOS, NO, and cGMP in response to IL-1, TNF- with IL-1, and
supernatants of activated macrophages after a period of several hours
has also been implicated in later-onset depression of both baseline and
isoproterenol-stimulated contractility of in vitro myocardial
preparations (4, 5, 21, 33, 50, 53, 56, 57, 62, 63). Similarly, in vivo
endotoxemia results in the production of an iNOS and NO in the
myocardium, which is associated with a decrease in contractility of
excised myocardial tissue (8, 16, 53). Recent data suggest that iNOS-generated NO in these circumstances may act via cholinergic modulation of the inotropic response to -adrenergic stimulation (3,
28, 30, 31, 65). Both mechanisms of late depression (G protein
alteration and iNOS induction), although not mutually exclusive of each
other, appear to be distinct from those causing early depression of
cytokine-stimulated myocardial tissue in vitro.
A study by Kinugawa and colleagues (37) may help to reconcile these divergent findings of early versus late NOS-dependent myocardial depressant activity in models of sepsis. In their avian cardiac myocyte model, they were able to demonstrate both early (<30 min) and late (24 h) cardiac myocyte depression following IL-6 exposure. Myocyte depression appeared to be related to sequential cNOS activation followed by later iNOS generation. Such a possibility is also indirectly supported by evidence that suggests cNOS may potentially contribute to early septic or cytokine-mediated vascular dysfunction (23, 51, 59) even though iNOS is thought to be responsible for sustained septic vascular dysfunction (6, 32, 35, 47). The precise mechanism by which cytokines, supernatants of activated macrophages, and septic serum might stimulate myocardial cNOS remains undefined at this time. Potential mechanisms include those postulated for endothelial cNOS stimulation by shear stress, histamine, and vascular endothelial growth factor [heat shock protein 90 (24)] or by endotoxin (51) [endothelium-derived kinins (23) or platelet-activating factor (60)].
Clinical septic myocardial depression may represent a biphasic process
involving cytokine/MDS-stimulated cNOS production of NO in the early
phase, followed by cytokine/MDS-driven induction of iNOS in the later
phase. Both processes may culminate in myocardial depression through
the stimulation of guanylate cyclase and the production of cGMP, a
nucleotide with known myocardial depressant functions. The association
of MDS-produced early-onset cardiac myocyte depression in vitro
(decreased maximum extent and peak velocity of shortening) with
relatively prolonged clinical myocardial depression in vivo in septic
patients (as measured by ventricular ejection fraction) (46, 48) can be
explained by a dual action of the circulating myocardial depressant
substance of sepsis (i.e., TNF-/IL-1) in both activating
myocardial cNOS (early myocardial depressant effects) and stimulating
myocardial iNOS production (prolonged myocardial depressant effects).
| |
ACKNOWLEDGEMENTS |
|---|
The authors thank Anthony Suffredini, MD, and Richard Proctor, MD, for measurement of cytokine and endotoxin concentrations and Ronald Sorkness, PhD, for statistical assistance.
| |
FOOTNOTES |
|---|
A. Kumar was supported by a Research Fellowship Award of the Society of Critical Care Medicine (1992-1993) and a Research Fellowship from the Medical Research Council of Canada (1993-1994).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. Kumar, Section of Critical Care Medicine, Rush-Presbyterian-St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612.
Received 19 February 1998; accepted in final form 21 September 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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0.01. 
P < 0.005, 
P <.001 vs.
control.
