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is
mediated by a sphingosine signaling pathway
1 Department of Medicine/Cardiology, University of Würzburg, 97080 Würzburg; and 2 Department of Cardiology/Pulmonology, University of Göttingen, 37075 Göttingen, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study investigated the
effect of tumor necrosis factor (TNF)-
on myocardial energy
metabolism as estimated by myocardial oxygen consumption
(M
O2).
MO2 of electrically stimulated isolated trabeculae of right ventricular Wistar rat myocardium was
analyzed using a Clark-type oxygen probe. After the initial data
collection in the absence of TNF-, measurements were repeated after
TNF- stimulation. In separate experiments, pretreatment with the
nitric oxide (NO) synthase inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME) or the ceramidase inhibitor n-oleoylethanolamine (NOE) was done to investigate
NO/sphingosine-related effects. TNF- impaired myocardial economy at
increasing stimulation frequencies without altering baseline
MO2. Incubation with TNF- in the
presence of L-NAME further impaired myocardial economy. NOE
preincubation abrogated the TNF- effect on myocardial economy. Moreover, the negative inotropic effect of TNF- was observed in
NOE-pretreated but not L-NAME-pretreated muscle fibers.
Exogenous sphingosine mimicked the TNF- effect on mechanics and
energetics. We conclude that TNF- impairs the economy of
chemomechanical energy transduction primarily through a
sphingosine-mediated pathway.
cytokines; nitric oxide; myocardial energy metabolism; tumor
necrosis factor-
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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TUMOR NECROSIS
FACTOR (TNF)- is a proinflammatory cytokine important in the
pathophysiology of chronic heart failure, myocarditis, cardiomyopathy,
cardiac allocraft rejection, myocardial reperfusion injury, myocardial
infarction, and remodeling. Several studies have revealed negative
inotropic effects of TNF- in the isolated heart model, in isolated
myocardial muscle strips, and cultured cardiac myocytes as well as in
in situ animal hearts. Cardiac myocytes have been identified as the
source and target of TNF-. TNF- binds to TNF receptors I and II,
cell surface receptors that mediate virtually all known effects on
myocytes (for a review, see Ref. 11).
Sphingolipids are structural elements of membranes, and there is a
growing amount of knowledge about their role in regulating numerous
cell functions (for a review, see Ref. 9). Previously, it
has been reported that the TNF--mediated negative inotropic effect
is dependent on a sphingosine-signaling pathway (15). Cytokines can regulate sphingolipid metabolism by modulating
sphingomyelinase and ceramidase activity. TNF- leads to degradation
of cellular sphingomyelin with the formation of ceramide and its base
sphingosine. Both have been demonstrated to be able to deteriorate
myocardial force generation, at least in part due to alterations of
cellular calcium handling (9).
TNF- also seems to play a role in deteriorating mechanical function
through nitric oxide (NO) signaling (3). However, it is
unclear which NO synthase (NOS) isoform is responsible for these rapid
TNF- effects on force generation. Although TNF- is known to
induce NOS II expression, the rapid effect on force generation does not
imply transcriptional regulation of NOS. On the other hand, TNF- was
demonstrated to impact on myocyte calcium handling, raising the
possibility that its NO-dependent effects are mediated through
constitutive NOS isoforms (NOS I and NOS III).
Experiments using isolated blood-perfused canine heart preparations
demonstrate an increase in oxygen consumption in the presence of high
concentrations of human TNF- (14). These authors report an early moderate negative inotropic effect and a significant coronary
vasodilatation after TNF- administration.
Given the recognition that 1) TNF- alters
Ca2+ handling, 2) myocardium and endothelium
expresses constitutive NOS, 3) NO plays a role in the
regulation of myocardial oxygen consumption (19), and
4) sphingolipid signaling controls endothelial NOS (eNOS) activity and mitochondrial function (2, 4), the present study investigated TNF--mediated myocardial contraction economy and
its candidate regulatory pathways.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Chemicals.
n-Oleoylethanolamine (NOE),
NG-nitro-L-arginine methyl ester
(L-NAME), D-sphingosine, and 2,3-butanedione
monoxime (BDM) were purchased from Sigma-Aldrich (Deisenhofen,
Germany). Recombinant rat TNF- was purchased from R&D Systems
(Wiesbaden, Germany).
Muscle preparation and measurement of mechanical parameters and
oxygen consumption.
The animals used in this study were handled in accordance with the
guidelines of the animal care committee of the University of Wuerzburg
and adhere to the American Physiological Society "Guiding Principles
in the Care and Use of Animals." Immediately after the anesthesic was
administered to male Wistar rats, a cardiectomy was performed, and the
heart was submerged in oxygenated cardioplegic solution (modified Krebs
buffer), which contained (in mmol/l) 152 Na+, 3.6 K+, 135 Cl
, 25 HCO
1 · mm2)
was recorded. Calculation of the myocardial oxygen consumption (MO2) was done by a computer program
(for details, see Ref. 13).
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Experimental protocols.
Muscle strips were stimulated with 1-5 Hz (60-300 beats/min).
After a steady state was reached,
MO2 and mechanical data were
recorded for each stimulation frequency (for original registration, see
Fig. 2). Muscle fibers that showed a
significant loss in force development compared with the initial 1-Hz
value were excluded.
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was used. TNF- was
dissolved in PBS and added to the Krebs buffer perfusate. The final
concentration of 5 ng/ml, in perfusate, was chosen as stable, and a
significant effect was found in preliminary experiments (see
RESULTS). After baseline data were collected, TNF- was
added, and the measurements were repeated after 10 min of incubation.
In second set of experiments, muscle strips were incubated with either
L-NAME (500 µM) or NOE (5 µM) for at least 1 h to
inhibit NOS enzymes or ceramidase activity, respectively. After
baseline data of the L-NAME- and NOE-treated muscle strips
were collected, TNF- was added, and the measurements were repeated
after 10 min of incubation.
In a third set of experiments, exogenous D-sphingosine (1 µM) was added, and its effects were studied after a period of 15 min.
NOE and L-NAME were dissolved in DMSO. The DMSO
concentration in the perfusate was under 0.1% and showed no
significant effect (data not shown).
Calculations.
FTI (in
N · s · min1 · mm2)
is defined as the area between peak systolic force and diastolic force
during the stimulation interval. It represents an equivalent of work in
isometrically contracting myocardium and is a major determinant of
MO2 (13). FTI was plotted against MO2. The
MO2-axis intercept of the
MO2-FTI relation (i.e.,
extrapolation to zero FTI) represents oxygen consumption for
excitation-contraction coupling and basal metabolism. The slope of the
MO2-FTI regression line reflects
chemomechanical force transduction by cross-bridge cycling and relates
mechanical output to chemical energy input (17). Its
reciprocal indicates the contractile economy.
1 · mm2).
Analysis of oxygen data (ml
[O2] · mm3 · min1)
was performed using Muscle Research System software from
Scientific Instruments.
Statistical analysis. Statistics were done using WinSTAT (Beneke & Schwippert), implying Excel spreadsheets. The regression line was calculated from all data points. Values for slope and y-axis intercepts were compared between groups and tested for significant differences. Statistical significance was determined for paired data by the Wilcoxon signed-rank test. For unpaired data, a Mann-Whitney U-test was applied. Values of P < 0.05 were considered significant. Data are expressed as means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of TNF- perfusion.
Muscle strips treated with TNF- developed an immediate (<10 min)
reduction in systolic force development (Fig.
3). Hereby, the force-frequency relation
was not altered. Five picograms per milliliter of TNF- was
the lowest concentration able to exhibit a depressant effect on force
development. For 5 ng/ml, the TNF--induced force reduction was
39 ± 3% of the initial value (n = 11).
Concerning the mechanical effect, no significant dose dependency could
be demonstrated.
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O2-FTI regression was highly
linear, both before and after TNF-. TNF- showed a dose-dependent
effect on the specific oxygen demand, expressed as
MO2/FTI (Fig.
4). This was paralleled by a significant
increase in the slope of the MO2-FTI
regression line. A significant deterioration of myocardial economy was
first observed for 50 pg/ml TNF-. TNF- (5 ng/ml) impaired the
economy of active force generation, as expressed by the slope of the
MO2-FTI regression line (0.050 vs.
0.082 ml
[O2] · N1 · s1 · m1,
n = 11, P < 0.05), whereas the basal
MO2 remained stable (0.045 vs. 0.044 ml
[O2] · mm3 · min1,
n = 11, P < 0.05; Fig.
5).
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Effect of L-NAME preincubation.
L-NAME reduced systolic force development by 31 ± 6%. Simultaneously, the basal MO2
increased from 0.047 to 0.056 ml
[O2] · mm3 · min1
(n = 10, P < 0.05; Fig.
6). Comparable to control conditions, TNF- exhibited a negative effect on the economy of force generation (0.057 vs. 0.083 ml [O2] · N1 · s1 · m1,
n = 12, P < 0.05) of
L-NAME-incubated muscle strips, whereas the basal
MO2 remained unchanged (0.045 vs.
0.045 ml
[O2] · mm3 · min1;
Fig. 7). TNF- had no additional
significant effect on the force development of
L-NAME-incubated muscle fibers (Fig. 3).
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Effect of NOE preincubation.
Throughout the stimulation rate, pretreatment with NOE led to a mean
reduction of 45 ± 4% in developed force. TNF- produced an additional significant systolic force reduction (31 ± 5%)
compared with NOE values (Fig. 3). After TNF- administration, the
basal MO2 was not different from
control conditions (0.042 ml
[O2] · mm3 · min1,
n = 11, P < 0.05). Simultaneously, the
slope of the MO2-FTI regression line
(0.093 ml
[O2] · N1 · s1 · m1,
r2 = 0.90, n = 11)
increased but was not significantly different from control conditions
(Fig. 8).
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Effect of exogenous sphingosine.
D-Sphingosine exhibited a negative effect on force
generation. Administration of 1 µM D-sphingosine led to a
significant reduction of systolic force, whereas the force-frequency
relation was not altered (Fig. 3). Furthermore, sphingosine induced a
significant negative effect on the economy of force generation (0.059 vs. 0.157 ml[O2] · N1 · s1 · m1,
n = 8, P < 0.05), whereas the basal
MO2 remained unchanged (0.046 vs.
0.039 ml
[O2] · mm3 · min1;
Fig. 9).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, TNF- altered the myocardial economy of
chemomechanical energy transduction by a sphingosine-dependent mechanism. This effect could be inhibited by pretreatment of muscle fibers with the ceramidase inhibitor NOE and was mimicked by exogenous sphingosine.
Impaired myocardial economy and reduction of force development by
TNF-.
Characteristic hemodynamic effects of TNF- are a decrease of
contractile efficiency and reduced ejection fraction, hypotension, decreased systemic vascular resistance, and biventricular dilatation (11). Reduction of myocardial force development can be
attributed to an altered calcium metabolism of myocytes. TNF-
disrupts L-type Ca2+ channel-induced calcium influx and
thereby depresses the calcium transient and systolic function
(8). These effects are due to sphingosine- and/or
NO-mediated signaling. Calcium regulatory proteins such as sarcolemmal
Ca2+-ATPase or sarcoplasmatic reticulum
Ca2+- ATPase show energy dependency. Their
impairment can cause a decrease in contractile performance
(12). Alterations of myocardial energy-dependent processes
after administration of TNF- might therefore contribute to impaired
force development.
immediately (<5 min) impaired
myocardial function: a reduction of systolic force generation was
accompanied by an altered energy status. The results demonstrate that
the elevation of oxygen demand by TNF-, previously reported by
Miyano et al. (14), is due to an impaired economy of
active force generation. The slope of the
MO2-FTI regression line
significantly increased compared with control conditions, indicating a
higher specific oxygen demand of the contracting myocardium after TNF administration, indicating a deterioration in chemomechanical conversion.
NO-mediated action. NO derived from the endothelial and cardiac NOS is able to modulate both cardiac contractility (5, 18) and oxygen consumption (19). In vivo experiments showed that oxygen consumption was increased after inhibition of NOS. Furthermore, several in vitro studies found an inhibitory effect of NO on oxygen consumption by a direct action on mitochondrial metabolism. This is at least partly due to the inhibitory effect of NO on mitochondrial cytochrome c oxidase (19). On the other hand, studies concerning NO and cardiac respiration found contradictory results. Both in vitro and in vivo studies are connected with confounding effects: NO regulates regional and systemic vascular tone. Systemic blood pressure alters cardiac workload and provokes feedback answers by the autonomous nervous system, capable of NO signaling. Changes of local myocardial perfusion can alter energy supply and myocardial metabolism (6, 7). Furthermore, studies on single myocytes or isolated mitochondria have their own restrictions that might have lead to some contradictory findings: studies on single myocytes cannot asses the load dependency of myocardial energetics, as myocardial energy metabolism is regulated by the energy demand in different contractile status. Isolated mitochondria are no longer subjected to exogenous NO or the modulating changes of the cellular high-energy phosphate demand and metabolism. Therefore, some of the discrepancies should be related to the different models used.
In our experiments, blocking NOS resulted in an elevated basal MO2. Thus we are in line with those
investigators who found an attenuating effect of endogenous NO on
cardiac mitochondrial respiration that is paralleled by a reduced
mechanical performance (18, 19).
On L-NAME-pretreated muscle strips, TNF- still exhibited
similar energetic effects but displayed no additional reduction in
systolic force. This could implicate that the negative inotropic effect
of TNF- is at least in part NO mediated. Evidence for a fast
TNF--induced NO-dependent reduction in force development was
provided by Finkel et al. (3). Cain et al.
(1) reported abrogation of TNF- effects on
contractility by inhibiting both NOS and ceramidase.
The data presented here suggest that deterioration in myocardial
economy is not NO dependent, because L-NAME pretreatment did not alter the TNF- effect on chemomechanical conversion. We
argue that if the energetic effect was mediated by NO formation, it
would rather influence basal MO2 by
an interference with mitochondrial respiration, creatine kinase
reaction, or changes in substrate utilization, which were not observed
in our study.
Sphingolipid-mediated effects.
TNF- generates elevated intracellular sphingosine levels through TNF
receptor I signaling (15, 20). The mechanical effect of
TNF- could be mimicked by exogenous sphingosine (15), a potent inhibitor of sarcoplasmatic calcium release (10,
21). The ceramidase inhibitor NOE has been previously shown to
abrogate the negative inotropic effect after TNF- administration. In
line with the findings in our study, NOE itself exhibits dose-dependent attenuating effects on myocardial force generation (15).
Furthermore, NOE leads to an increase in ceramide content due to its
inhibited conversion to sphingosine (15). TNF- should
further increase ceramide levels by its stimulating effect on
sphingomyelinase. The observed persistence of the TNF--mediated
decrease in systolic force development after NOE treatment in our study
could be explained if the reduced mechanical performance was due to
increased ceramide levels. Therefore, the results would be in line with
the previous notion: that TNF- reduces force development by sphingosine.
stimulation of cardiac myocytes (16). Thus we
conclude that the energetic effect of TNF- is sphingosine related.
However, the molecular mechanism underlying the impaired economy of
active force generation remains unclear.
Unaltered oxygen consumption at basal conditions, in contrast to an
overproportional increase in oxygen consumption at increasing mechanical work, indicates that production of energy-rich phosphates is
preserved and that the problem lies in uneconomic energy utilization.
TNF- effects on mechanics and energetics are based on alterations in
intracellular calcium homeostasis. Proposed mechanisms include a direct
influence of TNF- on calcium channels and/or calcium desensitization
of the myofilaments (3, 5). Our results raise the
possibility that TNF- could also lead to reduced force generation by
an impaired economy of chemomechanical energy transduction and an
increased energy cost for calcium cycling. It is conceivable, given
that ceramide is capable of a calcium-independent immediate activation
of eNOS (4), and ceramide among other sphingolipids can
induce alterations in intracellular calcium handling, that
calcium-sensitive NOS activation is linked to sphingosine pathways.
Limitations.
The concentration of 5 ng/ml TNF- used for most experiments was
almost 10 times higher than plasma levels observed in
pathophysiological relevant states. However, the fact that cardiac
myocytes themselves generate TNF- suggests that tissue
concentrations are much higher than those in plasma. Furthermore, in
vivo TNF- binds to soluble receptors that modulate free TNF-
concentrations as well as its action on cell surface receptors. Our
results demonstrate that even lower concentrations of 50 pg/ml have a
significant influence on myocardial economy.
leads to a fast
deterioration in myocardial economy. This impairment in energy
metabolism is associated with no change in basal
MO2, but a reduced economy of
chemomechanical energy transduction, indicating an uneffective energy
utilization. The observed effect is sphingosine dependent as the
ceramidase inhibitor NOE is able to abrogate and exogenous sphingosine
is able to mimic the energetic effect.
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ACKNOWLEDGEMENTS |
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We thank Verena Popp and Helga Wagner for perfect technical assistance and R. D. Sigrid Kuhlencordt for critically reviewing the manuscript.
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FOOTNOTES |
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The study was supported by grants from the Deutsche Stiftung für Herzforschung (to A. W. Bonz) and by Deutsche Forschungsgemeinschaft Grant SFB-355 (to A. W. Bonz and G. Ertl).
Address for reprint requests and other correspondence: A. W. Bonz, Medizinische Universitätsklinik, Josef-Schneider-Str. 2, 97080 Würzburg, Germany (E-mail: bonz_a{at}klinik.uni-wuerzburg.de).
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. Section 1734 solely to indicate this fact.
First published January 30, 2003;10.1152/ajpheart.00888.2002
Received 15 October 2002; accepted in final form 24 January 2003.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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