From the University of Pittsburgh Medical Center, Division of Cardiology
and Division of Neuropathology (V.J.S.), Pittsburgh, Pa.
Correspondence to Arthur M. Feldman, MD, PhD, University of Pittsburgh Medical Center, Division of Cardiology, Scaife Hall S572, 200 Lothrop St, Pittsburgh, PA 15213. E-mail feldma{at}card2.cath.upmc.edu
Methods and Results—Left ventricular muscle
strips were obtained from seven patients with end-stage congestive
heart failure undergoing heart transplantation or insertion of a left
ventricular assist device. The muscle strips were incubated
at 37°C in 95% O2/5% CO2 and stimulated
with LPS (10 µg/mL). TNF-
Conclusions—Adenosine can significantly diminish
TNF levels in the failing human heart and may represent a new
pharmacological intervention in congestive heart failure.
Recently, studies have demonstrated that the phosphodiesterase
inhibitor vesnarinone,6 7 the
antiarrhythmic agent amiodarone,8 and the
cardiac glycoside ouabain9 inhibit the
production of TNF-
Measurement of TNF-
Immunohistochemistry
Statistical Analysis
Effect of Adenosine on TNF-
Interestingly, the response to LPS challenge was more pronounced for
muscle strips obtained from patients with ischemic
cardiomyopathy (5.09±0.72 pg ·
mL-1 · mg wt-1,
n=3) than for muscle strips from patients with dilated
cardiomyopathy (1.2±0.29 pg ·
mL-1 · mg wt-1,
n=4) (P<.001). There was no significant difference in age
(52 versus 49 years) or in medical regimen between the two groups. In
addition, adenosine had a significant effect
(P<.005) on TNF-
Because adenosine was given simultaneously with
LPS, it is undetermined whether adenosine can inhibit TNF-
Immunolocalization of TNF-
It was interesting to note that muscle strips from patients with
ischemic heart disease produced substantially more TNF-
In conclusion, these in vitro experiments suggest that
adenosine can significantly diminish TNF-
Received October 3, 1997;
revision received December 12, 1997;
accepted December 12, 1997.
© 1998 American Heart Association, Inc.
Brief Rapid Communications
Adenosine Inhibits Lipopolysaccharide-Induced Secretion of Tumor Necrosis Factor-
in the Failing Human Heart
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—The proinflammatory cytokine tumor
necrosis factor- (TNF-) has been implicated in the pathogenesis
of congestive heart failure. Recent studies have shown that
adenosine inhibits lipopolysaccharide (LPS)-induced
expression of TNF- in macrophages and rat
cardiomyocytes. The aim of this study was to determine
whether adenosine has a similar effect in the failing human
heart. release in the supernatant was measured
with ELISA, and muscle sections were stained for TNF-. Muscle strips
released TNF- in the absence of LPS (0.22±0.05 pg ·
mL-1 · mg wet wt-1). TNF- was
immunolocalized to the cardiac myocyte, suggesting that the myocyte is
a source for TNF- production. Adenosine (10
µmol/L) decreased TNF- by 40% (P<.05). The
selective adenosine A2 receptor agonist DPMA
(10 µmol/L) decreased TNF- release by 87%
(P<.001), whereas ITu (10 µmol/L), an
adenosine-regulating agent that increases
endogenous adenosine concentration, inhibited
TNF- release by 93% (P<.001).
Key Words: adenosine • tumor necrosis factor- • proteins • heart failure
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Clinical and basic
research studies support the hypothesis that the proinflammatory
cytokine TNF- plays an important role in the development of
dilated cardiomyopathy and congestive heart
failure: (1) patients with end-stage heart failure have elevated levels
of TNF-1 ; (2) TNF- has direct negative
inotropic effects on the myocardium2 ;
(3) myocytes from failing human hearts re-express
TNF-3 ; (4) elevations in left
ventricular filling pressures can stimulate TNF-
expression4 ; and (5) transgenic mice
overexpressing TNF- demonstrate interstitial fibrosis,
interstitial infiltrates, adrenergic desensitization, and
ventricular dilatation.5 However, the
biochemical mechanisms responsible for modulating myocardial TNF-
levels remain undefined. by human mononuclear cells after
challenge with LPS. A similar cytokine inhibitory
effect can be found with the naturally produced nucleoside
adenosine.10 Importantly,
adenosine has also been shown to inhibit LPS-stimulated
cytokine production by rodent myocardium,
an effect that appeared to be mediated through the adenosine
A2 receptor.11 However,
rodent hearts are often not representative of human
myocardium. Therefore, the present study was undertaken
to assess the ability of adenosine to regulate cytokine
production by failing human heart.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Human Trabecular Muscles
Human heart tissue was isolated from seven patients with
end-stage cardiomyopathy at the time of
transplantation or during insertion of an LVAD. The heart tissue was
transported at 4°C in St Thomas cardioplegic solution and
immediately cut into 
2x1x1-mm strips. The muscle strips were
incubated in DMEM/F12 medium containing 5% horse serum and
equilibrated for 1 hour at 37°C before experiments were begun. The
experiments were performed in 12-well plates at 37°C in 95%
O2/5% CO2. The wet weight
of muscle strips was determined at the end of the experiments.
LPS Escherichia coli 0127 (Sigma Chemical Co) (10
µg/mL) was used to induce production of TNF- in muscle
strips as previously described.11 At times 0 and
4 hours after addition of LPS, the supernatants were collected, frozen
in liquid nitrogen, and stored at -70°C until analysis.
TNF- in the supernatants was measured with a human TNF- ELISA kit
(R&D Systems). The accuracy of this ELISA kit was verified by repeat
measurements with a kit from a different company (Genzyme). Both kits
use a mouse monoclonal anti–TNF-, and they provided comparable
measurements. To lower the limit of detection to 1 pg/mL, all samples
were concentrated through centricon 10 concentrators (Amicon) as
previously described.11 A standard curve was
generated with each set of samples assayed. Linear regression
analysis of the standard curves yielded a correlation
coefficient of >.99.
Immunohistochemical staining of trabecular muscles
was performed as previously described.11 Tissue
sections were treated with chicken anti-human TNF- (Promega) and
rabbit anti-chicken secondary antibody (Jackson Laboratories).
Avidin-biotin complex (Vector Laboratories) was added, and
visualization of the reaction was achieved by treatment with
3-amino-9-ethyl cabazole and 0.03%
H2O2 in 0.1 mol/L acetate
buffer, pH 5.2. Sections were weakly counterstained with
hematoxylin.
Results are expressed as mean±SEM of determinations in muscle
strips of seven patients. All experiments were performed in triplicate.
Data were subjected to one-way ANOVA (Fisher test), and
P<.05 was considered statistically significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Clinical Characteristics
All seven patients were male, and their mean age was 50±7 years
(14 to 67 years). Three patients had ischemic
cardiomyopathy and four, dilated
cardiomyopathy. All patients were in NYHA
functional class IV and had a similar medical regimen consisting of
diuretics and intravenous inotropic agents. Six
patients were receiving dobutamine and two,
dobutamine and milrinone. One patient had an LVAD. Four
patients underwent orthotopic heart transplantation. Two patients
received a biventricular assist device and one, an
LVAD. Release
Trabecular muscles from failing human hearts released
TNF- in the absence of LPS (0.22±0.05 pg ·
mL-1 · mg wet
wt-1) (n=7). Furthermore, TNF- was
immunolocalized to the cardiac myocyte, suggesting that the myocyte was
a source for TNF- production (Fig 1
). As seen in Fig 2, adenosine 10 µmol/L
decreased TNF- by 40% (P<.05). Similarly, the selective
adenosine A2 receptor agonist DPMA
10 µmol/L decreased TNF- release by 87%
(P<.001), whereas ITu, an adenosine-regulating
agent that increases endogenous adenosine
concentration, inhibited TNF- by 93% (P<.001).
View larger version (142K):
[in a new window]
Figure 1. Muscle sections of cardiomyopathic
left ventricle were stained with anti–TNF- and counterstained with
hematoxylin (magnification x600). A, Control condition after 4-hour
incubation with diluent. Note faint positive staining for TNF-. B,
Muscle section treated with LPS (10 µg/mL) for 4 hours shows
increased staining for TNF-.
View larger version (12K):
[in a new window]
Figure 2. Muscle strips taken from seven patients with NYHA
functional class IV (three with ischemic and four with dilated
cardiomyopathy) were stimulated with 10 µg/mL LPS
at time 0. Where indicated, adenosine (ADO) 10 µmol/L,
A2 agonist DPMA 10 µmol/L, or adenosine
kinase inhibitor ITu 10 µmol/L was added at time 0.
After 4 hours, supernatants were collected and TNF- was measured
with ELISA. *P<.05, **P<.001 vs LPS
(n=7, ANOVA). release in the ischemic
cardiomyopathy group but did not alter TNF-
release in the dilated cardiomyopathy group. By
contrast, DPMA and ITu had a significant effect on TNF- in both the
ischemic and nonischemic groups.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
In the present study, thin slices of myocardium
from failing human heart expressed measurable levels of the
proinflammatory cytokine TNF-. This was not surprising,
because earlier studies by Torre-Amione et al3
demonstrated that failing but not nonfailing human heart re-expressed
abundant quantities of TNF-. However, the relatively modest level of
TNF- secretion by the tissue slices could be enhanced 10-fold by the
addition of LPS to the incubation medium. The LPS-stimulated TNF-
expression could be attenuated by adenosine and nearly
abrogated by the addition of the A2 receptor
agonist DPMA or the adenosine kinase inhibitor ITu.
The ability of adenosine to inhibit LPS-stimulated TNF-
secretion is consistent with earlier studies in the rat in
which adenosine inhibited myocardial TNF-
production, with maximal effect being observed with
adenosine A2-selective
agonists.11 However, in contrast to failing human
heart, normal rodent myocardium does not express TNF- in
the absence of LPS or other stimuli.
production once it is stimulated. In fact, we observed in
neonatal rat cardiomyocytes that adenosine is
effective only when given during the time period from 1 hour before to
1 hour after LPS challenge.11 Adenosine
was no longer effective when given 3 hours after LPS. This may limit
the clinical usefulness of adenosine. It is known that
milrinone inhibits TNF- secretion from human mononuclear
cells.12 In our study, there was no significant
effect of milrinone therapy on the secretion of TNF-. to the cardiac myocyte shows that the
myocyte was a source of TNF- production. These results in
the human heart are also consistent with studies by Kapadia and
colleagues4 demonstrating that stretch-induced
TNF- production is largely localized to the myocytes.
However, it should be noted that all seven patients had NYHA class IV
symptoms and were receiving inotropic support before cardiac
transplantation or LVAD insertion. Therefore, we cannot exclude the
possibility that myocardium isolated from patients with
mild to moderate heart failure might not express TNF- either at
baseline or in response to LPS. Nevertheless, our results suggest that
elevated serum levels of TNF- that have been observed in patients
with end-stage heart failure1 may be due in part
to myocardial secretion of TNF-. in
response to LPS than did strips from patients with dilated
cardiomyopathy. By contrast, baseline levels were
similar in both groups. The difference between the two groups is
difficult to explain. It is possible that in patients with
ischemic cardiomyopathy, some muscle
samples were obtained from ischemic myocardium.
This may have contributed to an enhanced secretion of TNF-, because
TNF- mRNA is increased in ischemic
myocardium.13 Greater fibroblast
proliferation in the ischemic heart may also have contributed
to the observed difference between the two groups. Adenosine
had a significant effect on TNF- release in the ischemic
cardiomyopathy group but did not alter TNF-
release in the dilated cardiomyopathy group. By
contrast, DPMA and ITu had a significant effect on TNF- in both
groups. It could be speculated that the diminished effect of
adenosine in dilated cardiomyopathy was due
to desensitization or downregulation of the adenosine
A2 receptor. However, because of the small sample
size, the significance of these findings is uncertain. levels in
myocardial tissue obtained from failing human heart. If confirmed in
vivo, adenosine and/or adenosine-regulating agents may
provide novel pharmacological strategies for the treatment of patients
with end-stage congestive failure.
Selected Abbreviations and Acronyms
DPMA
=
PD-125944
ITu
=
iodotubercidin
LPS
=
lipopolysaccharide
LVAD
=
left ventricular assist device
TNF
=
tumor necrosis factor
Acknowledgments
Part of this work was done during the tenure of a fellowship
from the American Heart Association, Nation's Capital Affiliate Inc
(Dr Wagner). We are thankful to the members of the Division of
Cardiothoracic Surgery of the University of Pittsburgh for providing
the heart muscle samples.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
and tumor necrosis factor
receptors in the failing human heart. Circulation. 1996;93:704–711.
gene and protein expression in adult feline
myocardium. Circ Res. 1997;81:187–195.
production. J
Clin Invest. 1994;94:1212–1217.
by human mononuclear cells: a possible mechanism for
its effect in heart failure. Circulation. 1997;96:1386–1389.
production and protect mice against endotoxin challenge.
J Immunol. 1993;151:389–396.[Abstract]
. Circ Res. 1998;82:47–56.
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