(Circulation. 2000;102:1440.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Congenital Deficiency of Nitric Oxide Synthase 2 Protects Against Endotoxin-Induced Myocardial Dysfunction in Mice
From the Department of Anesthesia and Critical Care (R.U., F.I., W.M.Z., Z.M.N.Q.) and the Cardiology Division (M.S.-C., K.D.B., M.H.P.), Cardiac Ultrasound Laboratory (M.S.-C., M.H.P.), and Cardiovascular Research Center (K.D.B., F.I., H.N., Z.M.N.Q.) of the Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston.
Correspondence to Warren M. Zapol, MD, Reginald Jenney Professor of Anaesthesia, Department of Anesthesia and Critical Care, Massachusetts General Hospital, 32 Fruit St, Boston, MA 02114. E-mail zapol{at}etherdome.mgh.harvard.edu
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Abstract |
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Background—Sepsis can be complicated by severe myocardial dysfunction and is associated with increased nitric oxide (NO) production by inducible NO synthase (NOS2). To investigate the role of NOS2 in endotoxin-induced myocardial dysfunction in vivo, we studied wild-type and NOS2-deficient mice.
Methods and Results—Serial echocardiographic
parameters of myocardial function were measured before and
at 4, 7, 16, and 24 hours after an endotoxin challenge. Seven hours
after challenge with either endotoxin or saline, systemic and left
ventricular pressures were measured, and the first
derivative of left ventricular developed pressure (dP/dt),
slope of the end-systolic pressure–dimension relationship
(SlopeLVESPD), and time constant of isovolumic relaxation
(
) were calculated. Endotoxin challenge in wild-type mice decreased
left ventricular fractional shortening, velocity of
circumferential shortening, dP/dtmax,
SlopeLVESPD, and dP/dtmin and increased time
constant . Endotoxin-induced myocardial dysfunction was associated
with increased ventricular NOS2 gene expression and cGMP
concentrations. Seven hours after endotoxin challenge, NOS2-deficient
mice had greater fractional shortening, dP/dtmax, and
SlopeLVESPD than did endotoxin-challenged wild-type mice.
Measures of diastolic function, dP/dtmin and
time constant , were preserved in endotoxin-challenged
NOS2-deficient mice. After endotoxin challenge in wild-type mice, early
(3-hour) inhibition of NOS2 with
L-N6-(1-iminoethyl)lysine
hydrochloride prevented, whereas later (7-hour) inhibition could not
reverse, endotoxin-induced myocardial dysfunction.
Conclusions—These results suggest that NOS2 is required for the development of systolic and diastolic dysfunction in murine sepsis.
Key Words: echocardiography • heart failure • inflammation • inhibitors • sepsis
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Introduction |
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During sepsis, patients can have decreased left ventricular (LV) ejection fraction, ventricular dilation, increased cardiac output, and decreased systemic vascular resistance.1 Although myocardial depression of sepsis has been well described, its pathogenesis is incompletely understood. During infection, bacterial mediators, such as endotoxin, can activate a cascade of endogenous cytokines. Subsequently, endotoxin and cytokines stimulate the production of large amounts of nitric oxide (NO), which appears to be a mediator of cytokine-induced myocardial depression.2 3 4 5
Incubating rat myocytes with inflammatory mediators increases NO production and decreases their contractile response to ß-adrenergic agents.2 Furthermore, inhibitors of NO synthesis reverse the negative inotropic effects of cytokines in rat and guinea pig myocytes in culture and in hamster papillary muscle.2 3 4 5 In dogs, intracoronary injection of interleukin-1ß bound to microspheres produced myocardial depression and increased cardiac NO production and formation of peroxynitrite, a potentially toxic product of the reaction between NO and superoxide.6 Others, however, have questioned the role of NO in cytokine-induced myocardial depression. Inhibition of NO synthesis failed to improve myocardial depression in tumor necrosis factor–challenged dogs and cats.7 8 Thus, the role of NO in sepsis-induced myocardial depression is incompletely defined.
Another area of controversy is the source of increased NO during cytokine-induced myocardial depression. Several studies have suggested that NO synthase 3 is the source of NO in cytokine-induced myocardial impairment,3 4 whereas others have proposed that increased expression of the inducible isoform of NO synthase (NOS2) contributes to cytokine-induced myocardial dysfunction.2 9 10 11
In the present study, we investigated the role of NOS2 on endotoxin-induced myocardial depression in vivo. Cardiac function was studied in mice with and without a congenital deficiency of NOS2 before and after endotoxin challenge. We also examined the ability of selective pharmacological inhibition of NOS2 to prevent or reverse endotoxin-induced myocardial dysfunction. After endotoxin challenge, wild-type mice developed profound systolic and diastolic myocardial impairment. In contrast, in NOS2-deficient mice, endotoxin-induced myocardial dysfunction was attenuated. Furthermore, specific pharmacological inhibition of NOS2 with L-N6-(1-iminoethyl)lysine hydrochloride (L-NIL) in wild-type mice, when provided early after endotoxin challenge (3 hours), could prevent endotoxin-induced myocardial dysfunction, whereas when given later (7 hours), L-NIL could not reverse it. Our results suggest that NOS2 is an important mediator of murine endotoxin-induced myocardial dysfunction.
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Methods |
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Experimental Subjects
After approval by the Massachusetts General Hospital Subcommittee on Research Animal Care, we studied SV129/B6F1 wild-type mice (F1-generation of the parental strains of NOS2-deficient mice, SV129 and C57 BL/6, Jackson Laboratories, Bar Harbor, Me) and NOS2-deficient mice with an SV129 and C57BL/6 background, generously provided by Dr Carl Nathan (Cornell University Medical School, New York, NY) and by Dr John Mudgett (Merck Research Laboratories, Whitehouse Station, Rahway, NJ). In supplemental studies, NOS2-deficient mice, backcrossed 10 generations onto a C57BL/6 background (C57BL/6-Nos2tmlLau, N10-backcross generation, Jackson Laboratories, Bar Harbor, ME) and wild-type C57BL/6 mice were studied. Mice were aged 2 to 5 months and weighed 18 to 30 g. Sex was matched among groups.
Experimental Protocol
Serial Echocardiographic Measurements
Echocardiographic measurements were obtained in
wild-type (n=20) and NOS2-deficient (n=21) mice before and 4 and 7
hours after endotoxin challenge. In a subset of wild-type (n=6 at 16
hours and n=4 at 24 hours) and NOS2-deficient (n=6) mice,
echocardiographic measurements were obtained at 16 and
24 hours after endotoxin challenge. At time 0, Escherichia
coli 0111:B4 endotoxin (50 mg/kg, DIFCO Laboratories) was injected
intraperitoneally.
Invasive Hemodynamics
Invasive measurements of systemic and LV pressures were obtained
7 hours after saline or endotoxin challenge in wild-type (n=11 and n=9,
respectively) and NOS2-deficient (n=12 and n=10, respectively) mice.
Additionally, C57BL/6 wild-type mice and
C57BL/6-Nos2tmlLau mice were studied 7 hours
after saline or endotoxin challenge (n=5 for each group).
L-NIL Treatment
Wild-type mice were injected
intraperitoneally with L-NIL (5 mg/kg, A.G.
Scientific),12 a selective inhibitor of NOS2,
either early (3 hours, n=6) or late (7 hours, n=6) after endotoxin
challenge. Serial echocardiograms were performed at 4 and 7 hours after
saline or endotoxin challenge. Invasive hemodynamics
were measured 7 hours (4 hours after L-NIL administration) after saline
(n=4) or endotoxin (n=5) challenge. In a subset of wild-type mice,
invasive hemodynamics were measured 7 hours after
saline (n=4) or endotoxin (n=8) challenge, and measurements were
repeated 0.5 hours after administration of L-NIL at 7 hours.
Serial Echocardiographic Measurements
Echocardiography was performed by using a
13-MHz ultrasound probe (Sequoia, Acuson) in sedated mice
(ketamine, 0.05 mg/g IP) as previously
described.13 LV end-diastolic diameter, LV
end-systolic diameter, heart rate, and ejection time were
measured on M-mode echocardiograms. Fractional shortening (FS),
ejection time, and velocity of circumferential shortening corrected for
heart rate (Vcfc) were calculated as previously
described.13
Invasive Hemodynamics
Mice were anesthetized with ketamine (0.1 mg/g),
xylazine (0.01 mg/g), and pancuronium (0.002 mg/g)
intraperitoneally. Tracheostomy,
arterial catheterization, and mechanical
ventilation were performed as previously described.12 A
1.4F high-fidelity pressure catheter (Millar Instruments) was advanced
into the LV via the carotid artery. The first derivative of the
developed LV pressure (dP/dt) was calculated by differentiation of the
digitized analog LV pressure tracing (Windaq, Dataq Instruments). The
time constant of LV isovolumic relaxation () was calculated by using
the method of Weiss et al.14 LV end-systolic
internal diameter and LV systolic pressure were recorded
simultaneously by using
echocardiography and
micromanometer measurements, as described by
Williams et al.15 Reductions in end-systolic
internal diameter and systolic pressure were produced by
transient mechanical occlusion of the inferior vena cava
through a small laparotomy. Five to 6 end-systolic
pressure–dimension points were generated in each animal, a regression
line was determined, and slope and intercept were
calculated.15
Ventricular NOS2 mRNA
Ventricular mRNA was extracted by the guanidine
isothiocyanate–cesium chloride method. RNA (10 µg) was fractionated
in formaldehyde agarose gels and transferred to nylon membranes.
Membranes were hybridized initially with a
32P-labeled 0.3-kb mouse NOS2 cDNA probe
(nucleotides 3101 to 3552)16 and subsequently
with a 15-fold excess of a 32P-labeled
oligonucleotide complementary to rat 18S RNA.
Ventricular cGMP Concentrations
Ventricles were homogenized with 10%
trichloroacetic acid and centrifuged. Supernatants were
extracted with water-saturated ether, and cGMP concentrations were
measured by use of a radioimmunoassay (Biomedical Technologies Inc).
Trichloroacetic acid–precipitable protein was quantified by a Bradford
assay. Tissue cGMP levels are expressed as picomoles of cGMP per
milligram trichloroacetic acid–precipitated protein.
Ventricular Nitrotyrosine Immunohistochemistry
Cardiac tissue, cut at the midventricular level, was
frozen in 2-methylbutane chilled with liquid nitrogen. Sections
(10 µmol/L) were reacted with a rabbit polyclonal
anti-nitrotyrosine antibody (Upstate Biotechnology). Bound antibody was
detected with the use of goat anti-rabbit IgG antibody linked to
horseradish peroxidase. As a negative control, sections were incubated
with 2% BSA in PBS instead of the primary antibody. As a positive
control, sections were reacted with 10 mmol/L peroxynitrite
(Upstate Biotechnology) before reaction with the primary antibody.
Statistical Analysis
All data are expressed as mean±SEM. Differences between groups
were determined by ANOVA for repeated measurements. When significant
differences were detected by ANOVA, a post hoc Fisher test was used. A
value of P<0.05 indicated a significant difference.
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Results |
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Baseline Studies and Clinical Effects of Endotoxin
Baseline studies of wild-type and NOS2-deficient mice showed similar mean values for all echocardiographic and invasive hemodynamic measurements (Figures 1
and Tables 1 and 2).
The changes in echocardiographic and invasive
hemodynamics produced by endotoxin were similar in
SV129B6F1 and C57BL/6 wild-type strains and in NOS2-deficient mice with
an SV129/B6F1-hybrid background and NOS2-deficient mice backcrossed for
10 generations on a C57BL/6 background (data not shown).
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), and endotoxin-challenged NOS2+/+
mice treated with L-NIL at 3 hours after challenge (
).
*P<0.01 vs baseline for each group;
P<0.05 vs endotoxin-treated wild-type mice.
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After endotoxin challenge, animals manifested weakness, lethargy, piloerection, and diarrhea. At 24 hours after endotoxin challenge, mortality was similar between wild-type (10 [62%] of 16) and NOS2-deficient (6 [40%] of 15) mice.
Serial Echocardiography in Sedated
Mice
To determine the time course of endotoxin-induced myocardial
dysfunction, echocardiography was performed
serially. After endotoxin challenge, FS and Vcfc
decreased in both wild-type and NOS2-deficient mice. Endotoxin-induced
cardiac dysfunction was evident at 4 hours, pronounced at 7 hours, and
gradually returned to near baseline values at 24 hours after challenge
(P<0.05 at 4, 7, and 16 hours; Table 1
and Figure 1). However, 4 and 7 hours after endotoxin challenge,
NOS2-deficient mice had a greater FS than did wild-type mice (52±2%
versus 44±3% at 4 hours and 39±3% versus 26±2% at 7 hours, both
P<0.05; Table 1 and Figure 1). In addition, 4
and 7 hours after endotoxin challenge, Vcfc was
greater in NOS2-deficient mice than in wild-type mice (3.4±0.2 versus
2.8±0.2 circumferences per second at 4 hours and 2.3±0.2 versus
1.6±0.2 circumferences per second at 7 hours, P<0.05;
Table 1).
After endotoxin challenge, wild-type and NOS2-deficient mice had
decreases in heart rate and increases in ejection time that were
evident at 7 hours, more pronounced at 16 hours, and returned toward
baseline by 24 hours (P<0.05, Table 1).
Invasive Hemodynamics and
Echocardiography in Anesthetized Mice
To further characterize endotoxin-induced changes in myocardial
function, we measured systemic and LV pressures with
simultaneous M-mode echocardiography to
generate heart rate–independent and load-independent
parameters of cardiac function. Seven hours after
challenge, FS, dP/dtmax, and
dP/dtmin were markedly decreased in
endotoxin-treated wild-type compared with saline-treated wild-type mice
(all P<0.05, Table 2 and Figure 2). Additionally, 7 hours after
challenge, endotoxin-treated wild-type mice had decreased mean systemic
arterial pressure (PSA), LV
end-systolic pressure (PLVES), and increased LV
end-diastolic pressure (PLVED) compared with
saline-treated wild-type mice (P<0.05, Table 2). In
contrast, dP/dtmax,
dP/dtmin, PSA,
PLVES, and PLVED did not
differ in NOS2-deficient mice challenged with endotoxin or saline.
Seven hours after challenge, endotoxin-challenged NOS2-deficient mice
had lower FS than did saline-challenged NOS2-deficient mice (42±3%
versus 49±1%, P<0.05). However, 7 hours after endotoxin,
NOS2-deficient mice compared with wild-type mice had a greater FS
(42±3% versus 32±4%) and a higher dP/dtmax
and dP/dtmin (all P<0.05, Table 2 and Figure 2).
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P<0.05).
The slope of the LV end-systolic pressure–diameter
relationship (SlopeLVESPD), a load-independent
measure of systolic function, was shifted downward in
endotoxin-challenged (79±22 mm Hg/mm) compared with
saline-challenged (144±9 mm Hg/mm) wild-type mice
(P<0.01, Figure 3A). In
contrast, the SlopeLVESPD did not differ in
NOS2-deficient mice challenged with endotoxin (113±11 mm Hg/mm)
or saline (136±7 mm Hg/mm, Figure 3B). After endotoxin
challenge, the SlopeLVESPD was higher in
NOS2-deficient mice than in wild-type mice (113±11 versus 79±22
mm Hg/mm, P<0.05; Figure 3A and 3B).
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The heart rate–independent and load-independent measurement of
diastolic function, time constant , was similar in
wild-type and NOS2-deficient mice after saline administration. However,
7 hours after endotoxin challenge, the value was markedly impaired
in wild-type mice but not in NOS2-deficient mice treated with endotoxin
(P<0.05, Table 2).
Inhibition of NOS2 with L-NIL
To determine whether pharmacological inhibition of NOS2 alters
cardiac function, wild-type mice were treated with L-NIL. In
saline-treated wild-type mice, L-NIL when administered early (at 3
hours) or late (at 7 hours) did not alter myocardial function or
hemodynamics, as assessed by
echocardiography or invasive
hemodynamic measurements (data not shown). However,
when L-NIL was given 3 hours after endotoxin challenge, FS and
Vcfc decreased less in L-NIL-treated wild-type
mice than in untreated mice (P<0.05 at 4 hours and 7 hours
after endotoxin challenge, Table 1 and Figure 1). In
wild-type mice treated with L-NIL at 3 hours and studied 7 hours after
endotoxin challenge, there were no changes in
PSA, PLVES,
PLVED, dP/dtmax,
dP/dtmin, SlopeLVESPD, and
time constant compared with those values in saline-treated
wild-type mice (Table 2 and Figure 3C). However, 7 hours
after endotoxin challenge, L-NIL–treated (at 3 hours) wild-type mice
had a higher dP/dtmax,
dP/dtmin, and SlopeLVESPD
and lower values than did endotoxin-challenged wild-type mice not
treated with L-NIL (all P<0.05, Table 2 and Figure 3A and 3C).
To determine whether inhibition of NOS2 activity reverses endotoxin-induced cardiac dysfunction once it is established, echocardiograms and invasive hemodynamics were obtained in wild-type mice 7 hours after endotoxin challenge and again 30 minutes after L-NIL administration. When administered late (7 hours), L-NIL did not alter hemodynamic and echocardiographic parameters (data not shown).
Ventricular NOS2 Gene Expression and cGMP
Concentrations
To examine whether endotoxin-induced myocardial dysfunction in
wild-type mice was associated with increases in ventricular
NOS2 expression, we measured cardiac NOS2 mRNA and cGMP concentrations.
After saline challenge (control, Figure 4A), ventricles of wild-type mice had
undetectable levels of NOS2 mRNA. In contrast, NOS2 gene expression was
markedly increased in mice challenged with endotoxin compared with
saline-challenged mice. Ventricular cGMP levels were
increased in a time-dependent manner in endotoxin-challenged wild-type
mice compared with saline-challenged mice (Figure 4B). Treatment
with L-NIL 3 hours after endotoxin prevented the endotoxin-induced
increase in ventricular cGMP levels.
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Effect of Endotoxin on Ventricular Nitrotyrosine
Immunoreactivity
To determine whether endotoxin-induced myocardial dysfunction in
wild-type mice was associated with increased formation of
peroxynitrite, nitrotyrosine immunoreactivity in
ventricular sections of wild-type mice 7 hours after
endotoxin or saline challenge was assessed. No increase in
ventricular nitrotyrosine immunoreactivity was detected in
endotoxin- or saline-challenged wild-type mice.
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Discussion |
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Despite evidence that NO produced in response to cytokines may mediate decreases in cardiac contractility during sepsis or endotoxemia, there are no data firmly establishing a role for NOS2 in the pathogenesis of endotoxin-induced myocardial impairment. Most studies implicating NO in endotoxin- or cytokine-induced myocardial depression have been performed in vitro and have used NO synthase inhibitors that are not isoform specific.2 3 4 5 11 17 18 Unfortunately, these agents can inhibit all 3 NO synthase isoforms and may affect coronary perfusion and heart rate and produce a series of side effects (neurotoxicity, hypertension, or hepatotoxicity).7 19 20 In the present study, we took advantage of mice congenitally deficient in the gene encoding NOS2, thereby avoiding the incomplete selectivity of pharmacological inhibitors of NOS. Using congenitally NOS2-deficient mice, we demonstrate a clear role for NOS2 in endotoxin-induced myocardial dysfunction.
After endotoxin challenge, wild-type mice developed decreases in FS and
Vcfc that were pronounced at 7 hours and improved
by 24 hours. However, endotoxin-induced decreases in FS and
Vcfc were less marked in NOS2-deficient mice than
in wild-type mice (Table 1 and Figure 1). Seven hours
after endotoxin challenge, wild-type mice exhibited profound
systolic dysfunction manifested by a decrease in
dP/dtmax (Table 2 and Figure 2) and
a downward shift of SlopeLVESPD (Figure 3A) as well as diastolic impairment, indicated by
decreases of dP/dtmin and increases in time
constant (Table 2). In contrast, NOS2-deficient mice were
protected from endotoxin-induced changes in systolic and
diastolic function (Table 2 and Figure 3A and 3B). Our observations differ from those of Nicholson et
al,21 who, using echocardiography and
lower doses of endotoxin in mice, observed that endotoxin induced an
increase in cardiac output in wild-type mice but not in NOS2-deficient
mice. Possibly, differences in the models and measurements obtained, as
well as the higher dose of endotoxin used in the present study
causing profound myocardial dysfunction, permitted us to identify
differences between wild-type and NOS2-deficient
mice.21
It is of interest that in NOS2-deficient mice 7 hours after endotoxin administration, echocardiographic measurements revealed a modestly decreased FS and Vcfc but that heart rate–independent and load-independent hemodynamic measurements showed no evidence of systolic cardiac dysfunction (dP/dtmax and SlopeLVESPD). It is unlikely that differences in the echocardiographic and invasive hemodynamic parameters are attributable to differences in the anesthetics used, inasmuch as echocardiographic measurements in sedated animals and in anesthetized animals were similar 7 hours after saline or endotoxin challenge. FS and Vcfc depend on changes in heart rate, preload and afterload, as well as on myocardial contractility. In contrast, dP/dtmax and SlopeLVESPD are relatively independent of heart rate and load conditions. Therefore, the differences in echocardiographic and invasive hemodynamic parameters of cardiac function measured in NOS2-deficient mice challenged with endotoxin might reflect the different dependence of these measurements on preload and afterload conditions.
The inhibition of NOS2 with L-NIL at 3 hours after endotoxin challenge
prevented endotoxin-induced decreases in FS,
dP/dtmax, and the downward shift of
SlopeLVESPD, as well as decreases of
dP/dtmin and increases in time constant
(Figures 1 through 3). Although early inhibition of NOS2
prevented subsequent endotoxin-induced ventricular
dysfunction, L-NIL did not reverse established myocardial dysfunction
when administered 7 hours after endotoxin challenge. These findings are
in agreement with reports that S-methylisothiourea (a
relatively selective inhibitor of NOS2) partially prevented
endotoxin-induced myocardial dysfunction in rats when administered at
the time of endotoxin challenge but not when given after myocardial
dysfunction had been established.22 Taken together,
these findings suggest that inhibition of NOS2 activity cannot reverse
endotoxin-induced ventricular dysfunction after it is
established.
Our findings strongly suggest that a product of NOS2 contributes to
endotoxin-induced myocardial dysfunction. NOS2 produces large
quantities of NO, which reacts with various targets in myocardial
cells. One such target, soluble guanylate cyclase, augments
intracellular levels of the second messenger cGMP.
Ventricular cGMP concentrations increased in wild-type mice
challenged with endotoxin (Figure 4B). Moreover, treatment with
L-NIL at 3 hours after endotoxin prevented the accumulation of cGMP
(Figure 4B) and the development of myocardial dysfunction.
Therefore, it is conceivable that expression of NOS2 produces
myocardial dysfunction via cGMP-dependent mechanisms, including reduced
myofilament sensitivity to calcium via activation of cGMP-dependent
protein kinase, decreased activity of L-type calcium channels, and
stimulation of cGMP-sensitive phosphodiesterases, thereby increasing
cAMP degradation.23 24 25
Superoxide, also a product of NOS2, could have contributed to endotoxin-induced cardiac dysfunction, inasmuch as it has been shown to have a role in interleukin-1ß–induced myocardial dysfunction.6 In addition, NO can also interact with superoxide and generate peroxynitrite. Peroxynitrite can react with a variety of proteins participating in contractile function, leading to the formation of nitrotyrosine.26 In the present study, nitrotyrosine was not detected in the hearts of wild-type mice 7 hours after endotoxin. These findings differ from those of Oyama et al,6 who reported that 2 days after intracoronary administration of interleukin-1ß in dogs, myocardial nitrotyrosine levels, assessed by high-performance liquid chromatography, correlated with the degree of ventricular dysfunction. Possible explanations for these differing findings are that high-performance liquid chromatography is more sensitive than immunohistochemistry or that prolonged exposure to endotoxin and/or cytokines is needed before detectable myocardial nitrotyrosine accumulates.
Interestingly, although genetic deficiency and pharmacological inhibition of NOS2 protected mice against endotoxin-induced myocardial dysfunction, they did not alter endotoxin-induced mortality. Our results are similar to earlier reports demonstrating that NOS2-deficient mice are not protected from endotoxin-induced death.27 These findings suggest that endotoxin challenge causes mortality in these mice by mechanisms independent of myocardial dysfunction and that other organ systems may be more vulnerable to endotoxin injury.
In summary, using mice congenitally lacking the gene for NOS2, we have demonstrated the role of NOS2 in endotoxin-induced myocardial dysfunction. Selective pharmacological inhibition of NOS2 activity with L-NIL prevented, but did not reverse, endotoxin-induced myocardial dysfunction. These findings suggest a potential strategy for the pharmacological prevention of sepsis-induced myocardial dysfunction.
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Acknowledgments |
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This study was supported by US Public Health Service grants HL-42397 and HL-55377 (Drs Zapol and Bloch). Dr Ullrich is supported by the Max Kade Foundation, and Dr Scherrer-Crosbie is supported by the Harold M. English Fellowship (Harvard Medical School). Dr Bloch is an Established Investigator of the American Heart Association. The authors thank John Hromi, Rebecca Nowak, and Sunbin Song for technical assistance and Dr Yu Chiao Chang for statistical advice.
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Footnotes |
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1 Drs Ullrich and Scherrer-Crosbie contributed equally to this investigation.
Received February 1, 2000; revision received April 13, 2000; accepted April 27, 2000.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Parker MM, Shelhamer JH, Bacharach SL, et al. Profound but reversible myocardial depression in patients with septic shock. Ann Intern Med. 1984;100:483–490.
2. Balligand JL, Ungureanu D, Kelly RA, et al. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993;91:2314–2319.
3.
Kumar A, Thota V, Dee L, et al. Tumor necrosis factor
alpha and interleukin 1 beta are responsible for in vitro myocardial
cell depression induced by human septic shock serum. J Exp
Med. 1996;183:949–958.
4.
Finkel MS, Oddis CV, Jacob TD, et al. Negative
inotropic effects of cytokines on the heart mediated by nitric
oxide. Science. 1992;257:387–389.
5.
Brady AJ, Poole-Wilson PA, Harding SE, et al. Nitric
oxide production within cardiac myocytes reduces their
contractility in endotoxemia. Am J
Physiol. 1992;263:H1963–H1966.
6. Oyama J, Shimokawa H, Momii H, et al. Role of nitric oxide and peroxynitrite in the cytokine-induced sustained myocardial dysfunction in dogs in vivo. J Clin Invest. 1998;101:2207–2214.[Medline] [Order article via Infotrieve]
7.
Quezado ZM, Karzai W, Danner RL, et al. Effects of
L-NMMA and fluid loading on TNF-induced cardiovascular
dysfunction in dogs. Am J Respir Crit Care Med. 1998;157:1397–1405.
8. Yokoyama T, Vaca L, Rossen RD, et al. Cellular basis for the negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian heart. J Clin Invest. 1993;92:2303–2312.
9. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994;78:915–918.[Medline] [Order article via Infotrieve]
10. Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest. 1997;100:2417–2423.[Medline] [Order article via Infotrieve]
11.
Balligand JL, Ungureanu-Longrois D, Simmons WW, et al.
Cytokine-inducible nitric oxide synthase (iNOS) expression in
cardiac myocytes: characterization and regulation of iNOS expression
and detection of iNOS activity in single cardiac myocytes in vitro.
J Biol Chem. 1994;269:27580–27588.
12. Ullrich R, Bloch KD, Ichinose F, et al. Hypoxic pulmonary blood flow redistribution and arterial oxygenation in endotoxin-challenged NOS2-deficient mice. J Clin Invest. 1999;104:1421–1429.[Medline] [Order article via Infotrieve]
13.
Scherrer-Crosbie M, Steudel W, Ullrich R, et al.
Echocardiographic determination of risk area size in a
murine model of myocardial ischemia. Am J
Physiol. 1999;277:H986–H992.
14. Weiss JL, Frederiksen JW, Weisfeldt ML. Hemodynamic determinants of the time-course of fall in canine left ventricular pressure. J Clin Invest. 1976;58:751–760.
15. Williams RV, Lorenz JN, Witt SA, et al. End-systolic stress-velocity and pressure-dimension relationships by transthoracic echocardiography in mice. Am J Physiol. 1998;274:H1828–H1835.
16.
Lyons CR, Orloff GJ, Cunningham JM. Molecular cloning
and functional expression of an inducible nitric oxide synthase from a
murine macrophage cell line. J Biol Chem. 1992;267:6370–6374.
17.
Flesch M, Kilter H, Cremers B, et al. Effects of
endotoxin on human myocardial contractility involvement
of nitric oxide and peroxynitrite. J Am Coll Cardiol. 1999;33:1062–1070.
18. Joe EK, Schussheim AE, Longrois D, et al. Regulation of cardiac myocyte contractile function by inducible nitric oxide synthase (iNOS): mechanisms of contractile depression by nitric oxide. J Mol Cell Cardiol. 1998;30:303–315.[Medline] [Order article via Infotrieve]
19. Meng X, Ao L, Brown JM, et al. Nitric oxide synthase is not involved in cardiac contractile dysfunction in a rat model of endotoxemia without shock. Shock. 1997;7:111–118.[Medline] [Order article via Infotrieve]
20.
Cobb JP, Natanson C, Hoffman WD, et al.
N-
-Amino-L-arginine, an inhibitor of
nitric oxide synthase, raises vascular resistance but increases
mortality rates in awake canines challenged with endotoxin. J Exp
Med. 1992;176:1175–1182.
21. Nicholson SC, Hahn RT, Grobmyer SR, et al. Echocardiographic and survival studies in mice undergoing endotoxic shock: effects of genetic ablation of inducible nitric oxide synthase and pharmacologic antagonism of platelet-activating factor. J Surg Res. 1999;86:198–205.[Medline] [Order article via Infotrieve]
22. McDonough KH, Smith T, Patel K, et al. Myocardial dysfunction in the septic rat heart: role of nitric oxide. Shock. 1998;10:371–376.[Medline] [Order article via Infotrieve]
23.
Vila-Petroff MG, Younes A, Egan J, et al. Activation of
distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in
cardiac myocytes. Circ Res. 1999;84:1020–1031.
24. Quignard JF, Frapier JM, Harricane MC, et al. Voltage-gated calcium channel currents in human coronary myocytes: regulation by cyclic GMP and nitric oxide. J Clin Invest. 1997;99:185–193.[Medline] [Order article via Infotrieve]
25.
Han X, Kobzik L, Balligand JL, et al. Nitric oxide
synthase (NOS3)-mediated cholinergic modulation of
Ca2+ current in adult rabbit
atrioventricular nodal cells. Circ Res. 1996;78:998–1008.
26.
Xia Y, Zweier JL. Superoxide and peroxynitrite
generation from inducible nitric oxide synthase in macrophages.
Proc Natl Acad Sci U S A. 1997;94:6954–6958.
27.
Laubach VE, Shesely EG, Smithies O, et al. Mice lacking
inducible nitric oxide synthase are not resistant to
lipopolysaccharide-induced death. Proc Natl Acad Sci
U S A. 1995;92:10688–10692.
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