Download TNF- and myocardial depression in endotoxemic rats

TNF-a and myocardial depression in endotoxemic rats:
temporal discordance of an obligatory relationship
XIANZHONG MENG, LIHUA AO, DANIEL R. MELDRUM, BRIAN S. CAIN,
BRIAN D. SHAMES, CRAIG H. SELZMAN, ANIRBAN BANERJEE, AND ALDEN H. HARKEN
Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado 80262
endotoxin; cardiac contractility; cycloheximide; dexamethasone; tumor necrosis factor binding protein; tumor necrosis
factor-a
ENDOTOXIN (lipopolysaccharide, LPS) depresses myocar-
dial contractility in laboratory animals (8, 11, 18, 20,
27, 28) and humans (29, 34) and is responsible for
cardiac dysfunction associated with sepsis (29). However, the mechanism by which LPS causes cardiac
dysfunction remains obscure. Previous studies have
suggested that sepsis-induced myocardial depression
was mediated by secondary factors (14, 15, 30). Indeed,
LPS stimulates monocytes and macrophages and
thereby elicits a cascade of proinflammatory cytokines.
Perhaps the proximal effectors of the cytokine cascade
include tumor necrosis factor-a (TNF-a) and interleukin-1. Dysregulated TNF-a production is critical for the
development of septic shock (36, 37). TNF-a has reThe 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. Section 1734
solely to indicate this fact.
R502
cently been demonstrated to be responsible for the in
vitro depression of cardiac myocyte contractility by
human septic shock serum (12). Furthermore, TNF-a
has been shown to depress the contractility of isolated
cardiac myocytes (10, 39) and in vivo instrumented
hearts (22, 23, 26). However, exogenous TNF-a appears
to induce immediate depression in vitro (5, 25) and
delayed depression in vivo (22). The temporal relationship between endogenous TNF-a and myocardial depression in endotoxemia remains to be determined.
Furthermore, the role of endogenous TNF-a in endotoxemic myocardial depression appears to be controversial. Pretreatment with a TNF-a-neutralizing antibody
has been reported to fail to prevent myocardial contractile dysfunction in a rabbit endotoxemic shock model
(24). Using low-dose LPS, we created a rat model of
endotoxemia not complicated by shock (18). We sought
to examine the influence of suppression of TNF-a
production and neutralization of TNF-a in this model to
determine the role of TNF-a in endotoxemic myocardial
depression.
Myocardial tissue produces TNF-a after a systemic
exposure to LPS (9, 19). Inhibition of protein synthesis
could be sufficient to abolish LPS-induced myocardial
TNF-a production. We have previously reported that
dexamethasone (Dex) suppresses LPS-induced increase in circulating and myocardial TNF-a levels (19),
and Brady and colleagues have demonstrated that
pretreatment with glucocorticoids prevents LPS-induced contractile dysfunction of guinea pig cardiac
myocytes (4). It is intriguing to examine whether
inhibition of protein synthesis with cycloheximide (CHX)
or immunosuppression with Dex prevents the contractile dysfunction of intact heart in this rat model of
endotoxemia without shock.
Soluble TNF receptors (also termed TNF binding
proteins, TNFBP) and antibodies to TNF-a can neutralize TNF-a and eliminate TNF-a bioactivities (10, 21,
38). In animal models of sepsis or endotoxemia, TNFBP
and antibodies to TNF-a can prevent shock (1, 37) or
mortality (1, 3, 16, 21, 38). TNFBP has also been shown
to abolish the in vitro negative inotropic properties of
TNF-a in cardiac myocytes (10). The influence of TNFBP
on in vivo endotoxemic myocardial depression remains
to be determined.
The purposes of this study were 1) to delineate the
temporal relationship of LPS-induced increase in circulating and myocardial TNF-a with myocardial depression, 2) to examine the influences of protein synthesis
inhibition and immunosuppression on LPS-induced
TNF-a production and myocardial depression, and 3) to
examine the influence of neutralization of TNF-a with
TNFBP on endotoxemic myocardial depression.
0363-6119/98 $5.00 Copyright r 1998 the American Physiological Society
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Meng, Xianzhong, Lihua Ao, Daniel R. Meldrum,
Brian S. Cain, Brian D. Shames, Craig H. Selzman,
Anirban Banerjee, and Alden H. Harken. TNF-a and
myocardial depression in endotoxemic rats: temporal discordance of an obligatory relationship. Am. J. Physiol. 275
(Regulatory Integrative Comp. Physiol. 44): R502–R508,
1998.—Exogenous tumor necrosis factor-a (TNF-a) induces
delayed myocardial depression in vivo but promotes rapid
myocardial depression in vitro. The temporal relationship
between endogenous TNF-a and endotoxemic myocardial
depression is unclear, and the role of TNF-a in this myocardial disorder remains controversial. Using a rat model of
endotoxemia not complicated by shock, we sought to determine 1) the temporal relationship of changes in circulating
and myocardial TNF-a with myocardial depression, 2) the
influences of protein synthesis inhibition or immunosuppression on TNF-a production and myocardial depression, and 3)
the influence of neutralization of TNF-a on myocardial depression. Rats were treated with lipopolysaccharide (LPS, 0.5
mg/kg ip). Circulating and myocardial TNF-a increased at 1
and 2 h, whereas myocardial contractility was depressed at 4
and 6 h. Pretreatment with cycloheximide or dexamethasone
abolished the increase in circulating and myocardial TNF-a
and preserved myocardial contractile function. Similarly,
treatment with TNF binding protein immediately after LPS
prevented myocardial depression. We conclude that endogenous TNF-a mediates delayed myocardial depression in
endotoxemic rats and that inhibition of TNF-a production or
neutralization of TNF-a preserves myocardial contractile
function in endotoxemia.
TNF-a AND ENDOTOXEMIC MYOCARDIAL DEPRESSION
MATERIALS AND METHODS
Fig. 1. Experimental protocols. LPS, lipopolysaccharide; TNF-a,
tumor necrosis factor-a; CHX, cycloheximide; Dex, dexamethasone;
TNFBP, TNF binding protein.
through the right atrium, and serum was prepared by centrifugation and stored at 270°C. Hearts were excised, and coronary blood vessels were flushed with 10 ml of phosphatebuffered saline (pH 7.4, 4°C) by retrograde perfusion through
the aortic root. After removal of the major vessels and atria,
ventricular (both left and right) tissue was frozen in liquid
nitrogen and stored at 270°C.
To examine the temporal relationship of cardiac contractile
dysfunction with changes in TNF-a levels, a group of rats was
treated with LPS (0.5 mg/kg ip) and another group with
bacteriostatic normal saline (0.4 ml ip). Rats were killed 2, 4,
or 6 h after the treatment. Hearts were isolated, and intrinsic
contractility was assessed by the Langendorff method.
Effects of protein synthesis inhibition on LPS-induced
changes in circulating and myocardial TNF-a levels and
cardiac contractile depression were evaluated by administration of CHX (0.5 mg/kg ip) 3 h before LPS. Serum and
ventricular myocardium were collected from a group of CHXpretreated rats at 1 h after LPS treatment for TNF-a assay.
Hearts were isolated from another group of CHX-pretreated
rats at 6 h after LPS treatment for the assessment of
contractile function. Four control rats were treated with CHX
alone (0.5 mg/kg ip). Serum and ventricular myocardium
were collected from two rats at 4 h (matching the time of CHX
treatment in rats treated with CHX and LPS) for TNF-a
assay. Hearts were isolated from two rats at 9 h (matching
time of CHX treatment in rats treated with CHX and LPS) for
the assessment of contractile function. The CHX dose used in
this study has been demonstrated to inhibit de novo protein
synthesis in rat tissues (31). Previous work demonstrated
that this dose of CHX was sufficient to abolish LPS-induced
myocardial resistance to ischemia/reperfusion (19).
Effects of immunosuppression with glucocorticoids on LPSinduced changes in circulating and myocardial TNF-a levels
and cardiac contractile depression were examined by administration of Dex (8.0 mg/kg iv) 30 min before LPS treatment.
Our previous work demonstrated that Dex at this dose was
effective in inhibiting LPS-induced TNF-a production (19). A
group of Dex-pretreated rats was killed at 1 h after LPS
treatment, and serum and ventricular myocardium were
prepared for TNF-a assay. Another group of Dex-pretreated
rats were killed at 6 h after LPS treatment, and hearts were
isolated for the assessment of contractile function. Four
control rats were treated with Dex alone (8.0 mg/kg iv).
Serum and ventricular myocardium were collected from two
rats at 1.5 h (matching time of Dex treatment in rats treated
with Dex and LPS) for TNF-a assay. Hearts were isolated
from the other two rats at 6.5 h (matching time of Dex
treatment in rats treated with Dex and LPS) for the assessment of contractile function.
Effects of TNFBP on LPS-induced cardiac contractile depression were examined by administration of TNFBP (40 or
80 µg/kg iv, ,7.5 or 15 nM in blood by calculation) immediately after LPS treatment. TNFBP used at a similar dose (11
nM) has been shown to completely abolish TNF-a cytotoxicity
to 1591-RE 3.5 cells and contractile depression induced by
TNF-a in isolated feline cardiac myocytes (10). Control rats
were treated with TNFBP alone (80 µg/kg iv). Rats were
killed at 6 h after treatment, and hearts were isolated for the
assessment of contractile function.
TNF-a assay. Immediately before TNF-a assay was performed, myocardium was homogenized with a tissue homogenizer (Tekmar, Cincinnati, OH) in four volumes of phosphatebuffered saline (pH 7.4, 4°C). After centrifugation at 2,500 g
at 4°C for 20 min, the supernatant was collected for TNF-a
assay. TNF-a levels in serum and myocardium were measured using an ELISA system containing a hamster anti-
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Animals. Male Sprague-Dawley rats, body weight 300–325
g (Sasco, Omaha, NE), were acclimated in a quarantine room
and maintained on a standard pellet diet for 2 wk before
initiation of the experiments. All animal experiments were
approved by the Animal Care and Research Committee of the
University of Colorado Health Sciences Center. All animals
received humane care in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory
Animals.
Chemicals and reagents. Dex was purchased from ElkinsSinn, (Cherry Hill, NJ). Human TNFBP was a generous gift
from Immunex. This TNFBP is a chimeric fusion protein
consisting of two molecules of human TNFBP2 linked by the
Fc portion of the human IgG1 (21). This chimeric dimer of
TNFBP2 has been shown to be more effective in neutralizing
TNF-a than monomeric TNFBP2 or TNFBP1 (10). The TNF-a
assay kit was obtained from Genzyme (Cambridge, MA). LPS
(from Salmonella typhimurium), CHX, and all other chemicals were obtained from Sigma (St. Louis, MO).
Experimental protocols. The rat model of endotoxemia used
in this study has been previously reported (18, 20). A single
sublethal dose of LPS (0.5 mg/kg ip) induces time-dependent
cardiac contractile depression. The cardiac contractility is
maximally depressed at 6 h after LPS exposure and completely normalized at 24 h. Mean arterial pressure is not
affected in this model, although this dose of LPS causes low
fever and body weight loss (18, 20).
The experimental protocols are depicted in Fig. 1. To
examine the effect of LPS on circulating and myocardial
TNF-a levels, a group of rats was treated with LPS (dissolved
in bacteriostatic normal saline, 0.5 mg/kg ip) and another
group with bacteriostatic normal saline (0.4 ml ip). Rats were
killed 1, 2, 4, or 6 h after the treatment. Blood was collected
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TNF-a AND ENDOTOXEMIC MYOCARDIAL DEPRESSION
Fig. 2. Temporal changes in circulating (s) and myocardial (k)
TNF-a levels after administration of a sublethal dose of LPS. Rats
were treated with LPS (0.5 mg/kg ip), and TNF-a in serum and
ventricular myocardium was determined by ELISA 1 (n 5 8 rats), 2
(n 5 6 rats), 4 (n 5 6 rats), and 6 h (n 5 5 rats) after LPS treatment.
Serum and myocardial TNF-a values of saline-treated rats were used
as baseline (time 0, n 5 8). Both circulating and myocardial TNF-a
increased at 1 and 2 h and returned to baseline level 4 h after LPS
treatment. Data are means 6 SE. * P , 0.01 vs. saline control.
RESULTS
Influence of protein synthesis inhibition on TNF-a
and myocardial depression. Because LPS-induced peak
TNF-a level was at 1 h and maximal myocardial
depression was at 6 h, the influences of interventions
on these parameters were determined at these two time
points after LPS treatment. CHX pretreatment attenuated the increase in serum TNF-a and abolished the
Temporal relationship of increase in TNF-a with
myocardial depression. The temporal changes in TNF-a
levels are shown in Fig. 2. Because saline injection and
time post saline injection do not appear to influence
circulating and myocardial TNF-a levels, data from
saline-treated rats were pooled and expressed as time
0. In saline-treated rats, TNF-a was barely detectable
in the serum, whereas a low level of TNF-a was
detected in the myocardium. After administration of
LPS, serum and myocardial TNF-a increased primarily
at 1 and 2 h. The peak serum TNF-a level (9.82 6 0.82
ng/ml, P , 0.01 vs. saline control) was observed at 1 h.
Myocardial TNF-a level increased fivefold at 1 h (P ,
0.01 vs. saline control). By 4 h after LPS treatment,
TNF-a in both serum and myocardium had returned to
baseline.
The temporal changes in cardiac contractility are
shown in Fig. 3. We observed previously that cardiac
contractility was not influenced by saline injection and
time after saline injection (18, 20). Thus contractility
data from saline-treated rats were pooled. LVDP was
101 6 3.4 mmHg in hearts isolated from saline-treated
rats. LVDP was not depressed at 2 h after administration of LPS. However, LVDP was significantly decreased at 4 and 6 h, with the maximal depression at
6 h (59.4 6 3.1 mmHg, P , 0.01 vs. saline control).
Fig. 3. Temporal changes in cardiac contractile function after administration of a sublethal dose of LPS. Rats were treated with LPS (0.5
mg/kg ip), and left ventricular developed pressure (LVDP) was
assessed by the Langendorff method 2, 4, and 6 h after administration of LPS. LVDP was attenuated at 4 and 6 h but not at 2 h after
LPS treatment. Data are means 6 SE. * P , 0.01 vs. saline control.
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mouse TNF-a antibody (cross-reaction with rat TNF-a). Recombinant murine TNF-a was used to construct a standard
curve. Absorbances of standards and samples were determined spectrophotometrically at 450 nm using a microplate
reader (Bio-Rad Laboratories, Hercules, CA). Results were
plotted against the linear portion of the standard curve.
Isolated heart perfusion and assessment of contractile
function. Intrinsic cardiac contractility was determined by a
modified isovolumetric Langendorff technique as described
elsewhere (18–20) and expressed as left ventricular developed pressure (LVDP). At the termination of the experiments,
beating hearts were rapidly excised into oxygenated KrebsHenseleit solution containing (in mmol/l) 5.5 glucose, 1.2
CaCl2, 4.7 KCl, 25 NaHCO3, 119 NaCl, 1.17 MgSO4, and 1.18
KH2PO4. Normothermic retrograde perfusion was performed
with the same solution in an isovolumetric and nonrecirculating mode. The perfusion buffer was saturated with a gas
mixture of 92.5% O2-7.5% CO2 to achieve a PO2 of 450 mmHg,
a PCO2 of 40 mmHg, and pH of 7.4. Perfusion pressure was
maintained at 70 mmHg. A latex balloon was inserted through
the left atrium into the left ventricle, and the balloon was
filled with water to achieve a left ventricular end-diastolic
pressure (LVEDP) of 5–10 mmHg (at peak and flat portion of
LVEDP-LVDP curve). Pacing wires were fixed to the right
atrium, and the heart was paced at 6.0 Hz. The myocardial
temperature was maintained by placing the heart in a
jacketed tissue chamber that was kept at 37°C by circulating
warm water. LVDP and LVEDP were continuously recorded
with a computerized pressure amplifier/digitizer (Maclab 8,
AD Instrument, Cupertino, CA).
Statistical analysis. Data were expressed as means 6 SE.
An ANOVA and a Bonferroni-Dunn post hoc test were performed. Differences were accepted as significant with P ,
0.05 verified by a Bonferroni-Dunn post hoc test.
TNF-a AND ENDOTOXEMIC MYOCARDIAL DEPRESSION
DISCUSSION
In a rat model of endotoxemia not complicated with
shock, the present study demonstrated that 1) myocardial depression was preceded by a transient increase in
circulating and myocardial TNF-a level, and myocardial depression occurred after TNF-a level had normalized; 2) inhibition of the increase in circulating and
myocardial TNF-a by protein synthesis blockade or
immunosuppression abolished LPS-induced cardiac contractile dysfunction; and 3) neutralization of TNF-a
with TNFBP also preserved cardiac contractile function. We conclude that TNF-a is a major but indirect
myocardial depressant factor in this model of endotoxemia and that suppression of TNF-a production or
neutralization of TNF-a preserves cardiac contractile
function in this model of endotoxemia.
LPS induces the synthesis and release of TNF-a by
monocytes and macrophages and thereby increases
circulating TNF-a level (40). LPS also induces TNF-a
Table 1. Effects of protein synthesis inhibition
and immunosuppression on TNF-a
and myocardial depression
Group
Serum TNF-a
Myocardial TNF-a
LVDP
Saline
LPS
CHX 1 LPS
Dex 1 LPS
CHX
Dex
0.03 6 0.02 (8)
9.82 6 0.82* (8)
0.91 6 0.72† (6)
1.06 6 0.39† (7)
0.01 6 0.01 (2)
0.02 6 0.01 (2)
0.28 6 0.04 (8)
1.43 6 0.25* (8)
0.23 6 0.06† (6)
0.07 6 0.02† (7)
0.13 6 0.05 (2)
0.39 6 0.15 (2)
101.2 6 3.4 (14)
59.4 6 3.1* (14)
94.0 6 3.5† (6)
94.3 6 2.8† (6)
98.0 6 2.9 (2)
99.6 6 3.9 (2)
Values are means 6 SE; n (no. of animals) in parentheses. LPS,
lipopolysaccharide; CHX, cycloheximide; Dex, dexamethasone; TNF-a,
tumor necrosis factor-a; LVDP, left ventricular developed pressure.
* P , 0.01 vs. saline; † P , 0.01 vs. LPS.
Fig. 4. Effects of TNFBP on LPS-induced myocardial depression.
Rats were treated with TNFBP (40 or 80 µg/kg iv) immediately after
administration of LPS (0.5 mg/kg ip). LVDP was assessed 6 h after
administration of LPS. TNFBP at either dose applied prevented
LPS-induced contractile dysfunction. Data are means 6 SE. * P ,
0.01 vs. saline control.
production by the myocardium (9, 19). In the present
study, administration of a sublethal dose of LPS induced a transient increase in circulating and myocardial TNF-a. TNF-a levels in both serum and myocardium increased primarily at 1 and 2 h and returned to
the baseline by 4 h. These temporal changes in circulating and myocardial TNF-a levels are consistent with
previous reports characterizing the time course of
circulating TNF-a of LPS-treated mice (40), rats (6, 7),
rabbits (24), and humans (17, 38). Myocardial contractility was not depressed 2 h after administration of LPS
when circulating and myocardial TNF-a levels remained elevated. Instead, myocardial contractility was
depressed at 4 h, and maximal depression appeared at
6 h. Although TNF-a has been shown to induce immediate contractile depression in cardiac muscle preparations (5) or cardiac myocytes (25) in vitro, the temporal
relationship of circulating and myocardial TNF-a levels
and contractile depression observed in the present
study indicates that endogenous TNF-a induces delayed cardiac dysfunction in endotoxemia.
LPS increases circulating and tissue TNF-a through
the activation of TNF-a gene transcription and ensuing
synthesis of TNF-a peptides in monocytes, macrophages, and other cell types (9, 40). LPS-induced
TNF-a gene transcription is regulated primarily by
transcription factor nuclear factor (NF)-kB (13, 35).
Two different approaches were applied in this study to
suppress TNF-a production. Glucocorticoids may inhibit TNF-a gene transcription by regulation of NF-kB
(2), and the protein synthesis blockade may inhibit the
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increase in myocardial TNF-a 1 h after administration
of LPS (Table 1). CHX alone administered 4 h before
sample collection did not influence either circulating or
myocardial TNF-a levels. Pretreatment of rats with
CHX also abolished cardiac contractile depression at
6 h after administration of LPS (Table 1), whereas
treatment of rats with CHX alone did not affect cardiac
contractility.
Influence of immunosuppression on TNF-a and myocardial depression. Dex pretreatment suppressed the
increase in serum TNF-a and abolished the increase in
myocardial TNF-a (Table 1). Pretreatment of rats with
Dex also prevented LPS-induced cardiac contractile
depression (Table 1). However, treatment with Dex
alone did not affect circulating and myocardial TNF-a
levels, nor did this treatment influence cardiac contractility.
Influence of TNFBP on myocardial depression. Treatment of rats with TNFBP at either dose abolished
LPS-induced cardiac contractile depression (LVDP was
94.0 6 4.2 mmHg with 40 µg/kg TNFBP and 92.0 6 4.0
mmHg with 80 µg/kg TNFBP, both P , 0.01 vs. LPS
alone and P . 0.05 vs. saline control; Fig. 4). Treatment
of rats with TNFBP alone at the higher dose did not
influence cardiac contractility (P . 0.05 vs. saline
control).
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TNF-a AND ENDOTOXEMIC MYOCARDIAL DEPRESSION
myocardial depressant factor in endotoxemia because
myocardial contractility was depressed when the elevated TNF-a level was no longer present. TNF-a may
depress cardiac contractility through the induction of a
secondary factor or secondary factors. Indeed, TNF-a
can induce the expression of inducible nitric oxide
synthase in the myocardium (32), and myocardial
depression associated with endotoxemic shock is accompanied by an enhanced nitric oxide synthase activity
and an increased nitric oxide level (4, 33). However,
nitric oxide may not be an important factor in this
model of endotoxemic myocardial depression because
nitric oxide synthase inhibitors failed to prevent or
reverse the contractile dysfunction (18). The secondary
factor or factors involved remain unknown. This unresolved issue will stimulate further research to determine the more distal factor or factors in endotoxemic
myocardial depression.
Perspectives
TNF-a has been proposed to be an important cardiodepressant factor in septic shock (12). Exogenous TNF-a
causes immediate myocardial depression in vitro (5)
and delayed myocardial depression in vivo (22). It has
long been recognized that endotoxemic myocardial dysfunction occurs late (8, 20, 28). The temporal relationship between endogenous TNF-a and myocardial depression is important to determine whether TNF-a is a
direct or indirect factor in endotoxemic myocardial
depression. Suppression of TNF-a production or neutralization of TNF-a may provide further information
about the role of this cytokine in endotoxemic myocardial dysfunction. Using a rat endotoxemia model, this
study demonstrated that increased circulating and
myocardial TNF-a levels precede myocardial contractile dysfunction and that myocardial contractile dysfunction occurs after TNF-a levels have already normalized. Myocardial contractile dysfunction is abolished by
inhibition of circulating and myocardial TNF-a. Neutralization of TNF-a with TNFBP also preserves myocardial contractile function. It appears that TNF-a is a
major but indirect myocardial depressant factor in this
model of endotoxemia. Further investigations using
different endotoxemia models with insight into downstream factors may help to determine the role of TNF-a
in myocardial depression related to trauma and sepsis
and the molecular mechanisms of the action of TNF-a.
The temporal discordance between TNF-a production
and myocardial contractile dysfunction suggests that a
therapeutic window may exist for the prevention of
cardiac dysfunction, particularly in surgical cases of
trauma or sepsis, through suppression of TNF-a production or neutralization of this cytokine.
The authors are grateful to Drs. Charles A. Dinarello and Verlyn
M. Peterson for constructive discussions.
This work was supported in part by National Heart, Lung, and
Blood Institute Grants HL-44186 and HL-43696 and National Institute of General Medical Sciences Grants GM-08315 and GM-49222.
Address for reprint requests: X. Meng, Dept. of Surgery, Box
C-320, Univ. of Colorado Health Sciences Center, 4200 East 9th Ave.,
Denver, CO 80262.
Received 14 January 1998; accepted in final form 22 April 1998.
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synthesis of TNF-a peptides. Indeed, pretreatment
with either Dex or CHX abolished the peak increase in
myocardial TNF-a and greatly blunted the peak increase in circulating TNF-a in endotoxemic rats. As a
result, pretreatment with either of these agents prevented the maximal myocardial depression at 6 h after
administration of LPS. Thus inhibition of TNF-a production by immunosuppression or protein synthesis blockade preserves myocardial contractile function in endotoxemic rats. It should be noted, however, that
circulating TNF-a remained slightly elevated after
immunosuppression or protein synthesis inhibition
whereas the increase in myocardial TNF-a was completely abolished. It is possible that the elevated level of
myocardial TNF-a is the primary contributor to contractile depression. Although the sources of circulating
TNF-a may be less sensitive to inhibition than those of
myocardial TNF-a, a low level of circulating TNF-a
alone is not sufficient to depress myocardial contractility.
TNFBP functions to bind free TNF-a and thereby
attenuates TNF-a cytotoxicity in vitro and in vivo (38).
The form of TNFBP used in this study has been shown
to abolish TNF-a cytotoxicity to 1591-RE 3.5 cells and
the negative inotropic properties of TNF-a in cultured
cardiac myocytes (10). To further examine the role of
TNF-a in this model of endotoxemic myocardial depression, TNFBP, at two doses comparable to that used
previously in vitro (10), was applied to rats immediately after administration of LPS. The maximal myocardial depression was abolished by TNFBP at either dose.
Taken together, the results of this study indicate that
endogenous TNF-a may be a major factor mediating
the delayed myocardial depression in this model of
endotoxemia. Our findings appear to differ from the
report by Nishikawa and colleagues (24), which demonstrated that pretreatment with antiserum against
TNF-a does not prevent cardiac dysfunction in a rabbit
endotoxemic shock model. The disparate findings may
be due to the use of different animal models and the
exact TNF-neutralizing agents used in these two separate studies. In the present study, TNFBP was applied
to a rat model of endotoxemia without shock whereas
the study by Nishikawa and colleagues (24) examined
the influence of antiserum against TNF-a on a rabbit
endotoxemic shock model. Perhaps TNFBP is more
effective than antiserum in preventing endotoxemic
cardiac contractile dysfunction. It is also possible that
some confounding factors are present in a shock model
but not in a nonshock model. Furthermore, the difference in the timing of TNF-neutralizing agent administration may account for the difference between our
findings and the previous study. In the present study,
TNFBP was administered immediately after injection
of LPS while antiserum against TNF-a was administered 1 h before injection of LPS in the study by
Nishikawa and colleagues (24).
TNF-a may exert its immediate negative inotropic
effect on myocardium by activation of the neutral
sphingomyelinase (25) or the constitutive nitric oxide
synthase (5). However, TNF-a is unlikely to be a direct
TNF-a AND ENDOTOXEMIC MYOCARDIAL DEPRESSION
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