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Integrative Physiology
Myocardial Dysfunction With Coronary Microembolization
Signal Transduction Through a Sequence of Nitric Oxide, Tumor Necrosis
Factor-␣, and Sphingosine
Matthias Thielmann, Hilmar Dörge, Claus Martin, Sergej Belosjorow, Uwe Schwanke,
Anita van de Sand, Ina Konietzka, Astrid Büchert, Arne Krüger, Rainer Schulz, Gerd Heusch
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Abstract—Coronary microembolization results in progressive myocardial dysfunction, with causal involvement of tumor
necrosis factor-␣ (TNF-␣). TNF-␣ uses a signal transduction involving nitric oxide (NO) and/or sphingosine. Therefore,
we induced coronary microembolization in anesthetized dogs and studied the role and sequence of NO, TNF-␣, and
sphingosine for the evolving contractile dysfunction. Four sham-operated dogs served as controls (group 1). Eleven dogs
received placebo (group 2), 6 dogs received the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME,
group 3), and 6 dogs received the ceramidase inhibitor N-oleoylethanolamine (NOE, group 4) before microembolization
was induced by infusion of 3000 microspheres (42-␮m diameter) per milliliter inflow into the left circumflex coronary
artery. Posterior systolic wall thickening (PWT) remained unchanged in group 1 but decreased progressively in group
2 from 20.6⫾4.9% (mean⫾SD) at baseline to 4.1⫾3.7% at 8 hours after microembolization. Leukocyte count, TNF-␣,
and sphingosine contents were increased in the microembolized posterior myocardium. In group 3, PWT remained
unchanged (20.3⫾2.6% at baseline) with intracoronary administration of L-NAME (20.8⫾3.4%) and 17.7⫾2.3% at 8
hours after microembolization; TNF-␣ and sphingosine contents were not increased. In group 4, PWT also remained
unchanged (20.7⫾4.6% at baseline) with intravenous administration of NOE (19.5⫾5.7%) and 16.4⫾6.3% at 8 hours
after microembolization; TNF-␣, but not sphingosine content, was increased. In all groups, systemic hemodynamics,
anterior systolic wall thickening, and regional myocardial blood flow remained unchanged throughout the protocols. A
signal transduction cascade of NO, TNF-␣, and sphingosine is causally involved in the coronary microembolizationinduced progressive contractile dysfunction. (Circ Res. 2002;90:807-813.)
Key Words: coronary microembolization 䡲 tumor necrosis factor-␣ 䡲 nitric oxide 䡲 sphingosine
C
(NO) and sphingosine are part of the signal transduction of
TNF-␣ in ischemia/reperfusion injury and chronic heart
failure.7,8
Therefore, we used an experimental model of coronary
microembolization in anesthetized open-chest dogs5 and studied the role of NO and sphingosine by use of the NO synthase
(NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME)
and the ceramidase inhibitor N-oleoylethanolamine (NOE).
oronary microembolization in patients with spontaneous
or therapeutic atherosclerotic plaque rupture has recently
been identified as a potential cause of arrhythmias, contractile
dysfunction, and infarcts.1–3 In the experiment, coronary
microembolization induces progressive regional myocardial
contractile dysfunction with an unchanged or even slightly
increased myocardial blood flow.4 Such progressive myocardial contractile dysfunction is associated with an inflammatory response, characterized by increased leukocyte infiltration.5 Tumor necrosis factor-␣ (TNF-␣), which is increased
after coronary microembolization within the myocardium, is
causally involved in the progressive contractile dysfunction,
inasmuch as a recent study has reported that contractile
dysfunction after microembolization is prevented by pretreatment with TNF-␣ antibodies and that intracoronary infusion
of exogenous TNF-␣ induces contractile dysfunction in the
absence of microembolization.6 The detailed signal cascade
for myocardial dysfunction in the scenario of coronary
microembolization is still unknown, but both nitric oxide
Materials and Methods
Animal Care
The experimental protocols were approved by the bioethics committee of the district of Düsseldorf, Germany. Dogs were handled
according to the guidelines of the American Physiological Society.
Experimental Preparation
Twenty-seven mongrel dogs (28.6⫾3.8 kg body weight) were
anesthetized with an initial bolus of sodium thiamylal (15 mg/kg IV).
After endotracheal intubation, anesthesia was maintained by ventilation with an enflurane/N2O mixture. Left ventricular and aortic
Original received October 19, 2001; revision received January 17, 2002; accepted February 21, 2002.
From Abteilung für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen.
Correspondence to Prof Dr med Dr hc G. Heusch, Abteilung für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen,
Hufelandstraße 55, 45122 Essen, Germany. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000014451.75415.36
807
808
Circulation Research
April 19, 2002
pressures were measured with catheter-tipped manometers (PC 350,
Millar). For the measurement of regional myocardial blood flow, a
polytetrafluoroethylene (Teflon) catheter was placed into the left
atrium for microsphere injection, and another Teflon catheter was
inserted into the upper descending aorta for blood withdrawal.
Ultrasonic crystals were implanted in the anterior left ventricular
myocardium and in the posterolateral myocardium perfused by the
left circumflex coronary artery (LCx) for measurement of regional
wall thickness. The LCx was dissected 2 cm proximal to its first
branch. A 25-gauge cannula (Microlance, Becton-Dickinson GmbH)
was inserted into the LCx, distal to an electromagnetic flow probe,
for intracoronary injection of embolizing microspheres (42-␮m
diameter) and the administration of L-NAME. Regional myocardial
blood flow was determined with 4 differently colored (yellow, red,
blue, and violet) 15-␮m microspheres.9 For each measurement,
⬇5⫻106 to 10⫻106 microspheres suspended in 6 mL saline with
0.02% Tween 80 were injected into the left atrium, followed by a
flush of 6 mL saline. The withdrawal of arterial reference blood
samples was started 30 seconds before injection of the microspheres
and continued for 150 seconds at a rate of 5 mL/min.
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Experimental Protocols
Sham-operated dogs without coronary microembolization (n⫽4)
served as controls (group 1). Systemic hemodynamics, regional
myocardial blood flow, and systolic wall thickening were measured
at baseline and after 1, 4, and 8 hours. After measurements at 8
hours, the coronary blood flow response to an intracoronary bolus of
1 ␮g bradykinin into the LCx was assessed (41.1⫾8.3 versus
84.2⫾7.3 mL/min, P⬍0.05). Subsequently, the responses of coronary blood flow and systolic wall thickening to intracoronary
infusion of 3.0 nmol/min sphingosine into the LCx over 240 seconds
and to intracoronary infusion of 0.3 ␮mol/min S-nitroso-N-acetylDL-penicillamine (SNAP) into the left anterior descending coronary
artery over 4 hours were tested. Before and after the infusion of
sphingosine, transmural biopsies were taken for the measurement of
sphingosine content.
In group 2 (n⫽11), placebo-treated dogs were subjected to
coronary microembolization with infusion of microspheres (42-␮m
diameter, 3000 per mL/min coronary inflow; Dynal-Particles AS,
Dynal Biotech ASA) into the LCx. Systemic hemodynamics, regional myocardial blood flow, and systolic wall thickening were
measured at baseline and at 1, 4, and 8 hours after coronary
microembolization.
In group 3 (n⫽6), after baseline measurements, the NOS inhibitor
L-NAME (10 ␮g/kg per minute IC) was infused for 15 minutes
before myocardial blood flow was measured. The L-NAME infusion
was then continued for 8 hours. The effective inhibition of NOS was
validated at the end of each experiment by an unchanged coronary
blood flow in response to an intracoronary bolus of 1 ␮g bradykinin10,11 (50.7⫾13.1 versus 58.9⫾10.9 mL/min, P⫽NS). The remaining protocol was identical to that in group 2.
In group 4 (NOE group, n⫽6), after baseline measurements, the
ceramidase inhibitor NOE (74 ␮g/kg IV) was infused for 15 minutes,
followed by continuous intravenous infusion (2.4 ␮g/kg per minute)
for 8 hours. This dose of NOE was shown previously to abolish the
increase in myocardial sphingosine content in response to TNF-␣.12
The remaining protocol was identical to that in group 2.
Postmortem Analysis
The hearts were excised and sectioned from base to apex into 5
slices. Transmural biopsies were taken from the anterior and posterior walls and stored at ⫺70°C until further processing for the
measurement of TNF-␣ and sphingosine contents. Additional transmural biopsies of ⬇300 mg were taken from both walls and fixed
with formaldehyde for microscopical analysis of infarct size, apoptosis, and leukocyte infiltration. After triphenyltetrazolium chloride
(TTC) staining for macroscopic analysis of myocardial infarction,5
regional myocardial blood flow was analyzed as previously
described.9
Infarct Size and Inflammatory Cells
The formaldehyde-fixed specimens were embedded in paraffin and
sectioned into slices of 5-␮m thickness. Four sections each from the
anterior and posterior walls were stained with hematoxylin and eosin
and examined by using phase-contrast microscopy at ⫻100 magnification (DMSL, Leica).13 The area of necrosis was determined by
planimetry and expressed as percentage of the total analyzed area of
the anterior wall (2.8⫾0.4 cm2) and posterior wall (2.9⫾0.5 cm2),
respectively. Inflammatory cells were counted in 10 fields of view of
⬇190 000-␮m2 area each from each section.
TUNEL Staining
Myocardial apoptosis was detected by using the terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)
staining.14 Slices were incubated with TUNEL labeling mixture
(Roche-Diagnostics) and counterstained with bis-benzimide (HOE
33342, Sigma Chemical Co) for quantitative comparison between
nuclei with and without DNA-strand breaks and with TRITC-marked
phalloidin (Sigma) for distinction between myocyte nuclei and
nuclei of other cells in the myocardium. TUNEL-positive myocyte
nuclei were counted and expressed as percentage of the total number
of myocyte nuclei. A total of 42 861⫾11 141 cardiomyocyte nuclei
of the anterior myocardium versus 37 609⫾10 163 cardiomyocyte
nuclei of the posterior myocardium were analyzed in group 1;
36 672⫾10 164 versus 36 029⫾14 400 cardiomyocyte nuclei, respectively, were analyzed in group 2; 39 875⫾11 690 versus
35 902⫾11 787 cardiomyocyte nuclei, respectively, were analyzed
in group 3; and 33 462⫾12 478 versus 31 516⫾15 204 cardiomyocyte nuclei, respectively, were analyzed in group 4.
Myocardial TNF-␣ Content
Approximately 200 mg of the anterior and posterior myocardium
each were homogenized in a micro-dismembrator (B. Braun Biotech,
Melsungen, Germany) for 30 seconds. To each myocardial sample, 5
vol of cold isotonic homogenization buffer (in mmol/L: imidazole
acetate 50, magnesium acetate 10, KH2PO4 4, and EDTA 2, along
with 50 ␮mol/L N-acetylcysteine 50 and 12.5 ␮mol/L sulfur, pH 7.6)
was added. Samples were centrifuged at 2000g for 15 minutes at
4°C, and supernatants were then collected for measurement of
TNF-␣ content by using the WEHI-164 cytotoxic cell assay,15 which
correlates closely with the results obtained by ELISA.16
Myocardial Sphingosine Content
Myocardial sphingosine content was measured by using the method of
Merrill et al17 with tetradecylamine used as an internal standard.12
Tissue samples were extracted and derivatized with o-phthaldialdehyde.
The derivatives were separated by reverse phase high-performance
liquid chromatography, and fluorescence was measured (340-nm excitation, 455-nm emission).
Myocardial iNOS mRNA
Frozen myocardial biopsies were homogenized in 4 mol/L guanidinium thiocyanate containing 0.1% ␤-mercaptoethanol. Total RNA
was isolated by acid phenol-chloroform extraction18 and redissolved
in water. RNA concentration was determined by measurement of
optical density at 260 nm. By using oligo(dT)15 as a primer of reverse
transcriptase (AMV Reverse Transcriptase, Promega), 1 ␮g total
RNA was reverse-transcribed into cDNA. Quantification of inducible NOS (iNOS) cDNA was carried out by real-time polymerase
chain reaction (Gene Amp 5700 Sequence Detection System, Applied Biosystems) by using the SYBR Green PCR Master Mix
(Applied Biosystems).19,20 Sense and antisense primers for human
iNOS were as follows: 5⬘-CTTCAACCCCAAGGTTGTCTGCAT
and 3⬘-ATGTCATGAGCAAAGGCGCAGAAC (GenBank accession No. U05810). Serial dilutions of the human iNOS-specific
cDNA fragment were used as standards. Each sample was quantified
in triplicate.
Thielmann et al
TABLE 1.
Signal Transduction in Coronary Microembolization
809
Systemic Hemodynamics
Heart rate, bpm
dP/dtmax, mm Hg/s
LVPpeak, mm Hg
LVPed, mm Hg
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AOPmean, mm Hg
Group
Baseline
Treatment
1 Hour
4 Hours
8 Hours
1
120⫾8
䡠䡠䡠
119⫾8
118⫾4
118⫾8
2
115⫾13
118⫾12
117⫾9
3
127⫾12
䡠䡠䡠
125⫾11
118⫾13
126⫾11
126⫾17
123⫾10
4
123⫾10
124⫾8
126⫾8
125⫾6
121⫾12
1
1490⫾148
䡠䡠䡠
1474⫾146
1485⫾133
1491⫾171
2
1298⫾107
1284⫾101
1292⫾90
1407⫾106
䡠䡠䡠
1411⫾103
1303⫾130
3
1425⫾98
1424⫾95
1436⫾142
4
1373⫾123
1340⫾174
1349⫾160
1386⫾140
1377⫾136
1
112⫾10
䡠䡠䡠
111⫾13
112⫾11
113⫾11
2
104⫾12
105⫾11
105⫾9
3
106⫾6
䡠䡠䡠
107⫾5
105⫾12
114⫾8
118⫾7
118⫾5
4
117⫾9
117⫾13
118⫾13
115⫾12
112⫾13
1
8⫾3
䡠䡠䡠
9⫾3
7⫾2
7⫾2
2
8⫾3
9⫾2
10⫾2
3
6⫾1
䡠䡠䡠
6⫾1
10⫾3
8⫾2
8⫾2
7⫾1
4
9⫾2
10⫾1
10⫾2
10⫾2
10⫾2
1
87⫾11
䡠䡠䡠
85⫾12
86⫾9
87⫾8
2
87⫾11
85⫾10
83⫾8
84⫾6
䡠䡠䡠
85⫾6
87⫾10
3
93⫾7
96⫾7
97⫾7
4
95⫾7
94⫾9
93⫾9
88⫾14
86⫾14
dP/dtmax indicates maximum left ventricular dP/dt; LVPpeak, left ventricular peak pressure; LVPed, left
ventricular end-diastolic pressure; and AOPmean, mean aortic pressure. Group 1 is sham group; group
2, placebo; group 3, L-NAME; and group 4, NOE. Data are mean⫾SD.
Statistical Analysis
Regional Myocardial Function
Data are reported as mean⫾SD. Hemodynamics, regional myocardial blood flow, and function were compared by 2-way ANOVA
(time and group) for repeated measurements. When a significant
overall effect was detected, Fisher least significant difference tests
were performed to compare single mean values. The number of
leukocytes, infarct size, number of apoptotic cardiomyocytes,
TNF-␣, and sphingosine contents were compared between the
anterior and posterior myocardial wall by paired t test. Responses to
intracoronary bradykinin, sphingosine, and SNAP were analyzed by
paired t test. A value of P⬍0.05 was taken to indicate a significant
difference.
Coronary blood flow at baseline was not different between
groups, and the number of embolizing microspheres was
165 000⫾42 000, 150 000⫾18 000, and 162 000⫾36 000 in
groups 2, 3, and 4, respectively.
Anterior systolic wall thickening was unaltered throughout
the protocol in all groups and was not different between
groups. In group 1, posterior systolic wall thickening was
stable throughout the protocol, whereas in group 2, posterior
systolic wall thickening decreased progressively (Table 3). In
group 3, posterior systolic wall thickening remained unchanged after the intracoronary infusion of L-NAME. At 1
hour after coronary microembolization, posterior systolic
wall thickening was slightly reduced from 20.8⫾3.4% to
16.9⫾2.2% but remained stable at this level until 8 hours
after microembolization. In group 4, posterior systolic wall
thickening remained unchanged after the intravenous infusion
of NOE. Again, at 1 hour after coronary microembolization,
posterior systolic wall thickening was slightly reduced but
remained stable at this level until 8 hours after
microembolization.
Systemic Hemodynamics
TTC Staining
Results
Heart rate,
peak, and
unchanged
groups and
maximum left ventricular dP/dt, left ventricular
end-diastolic and mean aortic pressures were
throughout the experimental protocol in all 4
were not different between groups (Table 1).
Regional Myocardial Blood Flow
Anterior and posterior subendocardial and transmural myocardial blood flows did not change significantly throughout
the protocol within the groups and were not different between
groups (Table 2).
TTC staining did not detect any infarction in the anterior and
posterior walls in all 4 groups.
Histology
There was only minimal myocardial infarction in the anterior
wall in all 4 groups, which was confined to the site of crystal
implantation (Table 4). Whereas there was no infarction in
the posterior wall in group 1, there was a transmurally
homogeneous distribution of small necrotic foci with an
aggregate infarct size of ⬇2% in the microembolized posterior wall of groups 2, 3, and 4 (Table 4). Whereas there were
810
Circulation Research
TABLE 2.
April 19, 2002
Regional Myocardial Blood Flow
⫺1
ASBF, mL 䡠 min
⫺1
䡠g
ATBF, mL 䡠 min⫺1 䡠 g⫺1
PSBF, mL 䡠 min⫺1 䡠 g⫺1
PTBF, mL 䡠 min⫺1 䡠 g⫺1
Group
Baseline
Treatment
1 Hour
4 Hours
8 Hours
1
1.25⫾0.50
䡠䡠䡠
1.36⫾0.66
1.16⫾0.32
1.26⫾0.54
2
1.22⫾0.25
䡠䡠䡠
1.18⫾0.31
1.22⫾0.51
1.10⫾0.32
1.03⫾0.22
1.68⫾0.51
1.69⫾0.37
1.50⫾0.51
3
䡠䡠䡠
4
䡠䡠䡠
1.24⫾0.47
1.03⫾0.22
0.93⫾0.27
1.07⫾0.28
0.87⫾0.20
1
䡠䡠䡠
1.30⫾0.66
1.18⫾0.37
1.27⫾0.54
2
1.05⫾0.17
䡠䡠䡠
1.19⫾0.28
1.13⫾0.49
1.02⫾0.22
0.96⫾0.19
1.59⫾0.38
1.55⫾0.40
1.34⫾0.47
3
䡠䡠䡠
4
䡠䡠䡠
1.28⫾0.42
0.98⫾0.25
0.93⫾0.28
1.03⫾0.23
0.98⫾0.25
1
䡠䡠䡠
1.33⫾0.53
1.17⫾0.30
1.27⫾0.47
2
1.18⫾0.27
䡠䡠䡠
1.13⫾0.30
1.25⫾0.47
1.12⫾0.48
1.12⫾0.25
1.61⫾0.37
1.50⫾0.33
1.35⫾0.52
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3
䡠䡠䡠
4
䡠䡠䡠
1.25⫾0.42
1.06⫾0.23
0.95⫾0.33
0.95⫾0.35
0.84⫾0.19
1
䡠䡠䡠
1.31⫾0.61
1.15⫾0.32
1.27⫾0.49
2
1.11⫾0.32
䡠䡠䡠
1.04⫾0.23
1.21⫾0.48
1.05⫾0.26
1.07⫾0.25
1.47⫾0.34
1.43⫾0.37
1.28⫾0.46
3
䡠䡠䡠
4
1.00⫾0.29
0.97⫾0.26
0.95⫾0.27
0.90⫾0.14
䡠䡠䡠
ASBF indicates anterior subendocardial blood flow; ATBF, anterior transmural blood flow; PSBF, posterior
subendocardial blood flow; and PTBF, posterior transmural blood flow. Group 1 is sham group; group 2, placebo;
group 3, L-NAME; and group 4, NOE. Data are mean⫾SD.
no apoptotic cardiomyocyte nuclei in group 1, there was
significant apoptosis in the microembolized posterior wall in
groups 2, 3, and 4, which was mainly located within and
around patchy microinfarctions.
The number of inflammatory cells infiltrating the posterior
wall was significantly increased over that in the anterior wall
in groups 2, 3, and 4 (Figure 1).
Myocardial TNF-␣ and Sphingosine Contents
In groups 2 and 4, but not in groups 1 and 3, myocardial
TNF-␣ content was increased in the microembolized posterior wall (Figure 2). In group 2, but not in groups 1, 3, and 4,
sphingosine content was increased in the posterior wall
(Figure 3).
Myocardial iNOS mRNA
Myocardial iNOS mRNA expression in the posterior wall was
not different from that in the anterior wall in group 1 (1.86⫾0.41
versus 2.05⫾0.90 fg/␮g total RNA, respectively; P⫽NS), group
TABLE 3.
AWT, %
PWT, %
2 (0.74⫾0.23 versus 1.07⫾0.64 fg/␮g, P⫽NS), group 3
(1.99⫾0.69 versus 2.52⫾0.34 fg/␮g, P⫽NS), and group 4
(0.67⫾0.55 versus1.25⫾0.88 fg/␮g, P⫽NS).
Responses to Intracoronary Sphingosine
and SNAP
Intracoronary infusion of sphingosine into the LCx in group
1 decreased posterior systolic wall thickening from
20.1⫾6.3% to ⫺3.2⫾4.5% after 240 seconds (P⬍0.05),
whereas coronary blood flow (LCx) remained unchanged
(45.5⫾15.6 versus 49.1⫾15.0 mL/min, P⫽NS). Myocardial
sphingosine content of the posterior wall was increased from
226⫾38 to 366⫾110 pmol/g wet weight (P⬍0.05). After
termination of the sphingosine infusion, posterior systolic
wall thickening returned to baseline.
Subsequent intracoronary infusion of SNAP into the left
anterior descending coronary artery in group 1 increased
coronary blood flow from 42.1⫾14.2 to 75.8⫾23.9 mL/min
Regional Myocardial Function
Group
Baseline
1
27.3⫾9.0
2
26.2⫾9.3
3
Treatment
1 Hour
4 Hours
8 Hours
䡠䡠䡠
28.3⫾8.5
27.4⫾8.2
28.1⫾8.7
29.0⫾9.2
25.5⫾9.3
24.7⫾7.1
28.4⫾3.7
䡠䡠䡠
28.3⫾3.4
28.0⫾4.7
26.8⫾5.4
27.9⫾5.0
4
23.1⫾5.6
22.3⫾6.3
25.6⫾8.0
23.4⫾6.5
24.4⫾5.5
1
21.4⫾4.4
䡠䡠䡠
20.7⫾2.9
22.3⫾3.4
22.1⫾4.3
2
20.6⫾4.9
13.8⫾4.9*†
3
20.3⫾2.6
䡠䡠䡠
20.8⫾3.4
16.9⫾2.2
16.6⫾3.0‡
17.7⫾2.3‡
4
20.7⫾4.6
19.5⫾5.7
16.4⫾6.7
16.3⫾7.4‡
16.4⫾6.3‡
8.8⫾4.5*#†
4.1⫾3.7*#†
AWT indicates anterior systolic wall thickening; PWT, posterior systolic wall thickening. Group 1 is sham group;
group 2, placebo; group 3, L-NAME; and group 4, NOE. Data are mean⫾SD. *P⬍0.05 vs baseline; #P⬍0.05 vs
preceding value; †P⬍0.05 vs group 1; and ‡P⬍0.05 vs group 2.
Thielmann et al
TABLE 4.
Signal Transduction in Coronary Microembolization
811
Histology
Group
Anterior Wall
Posterior Wall
Infarct Size (% analyzed
1
0.00⫾0.00
0.00⫾0.00
area)
2
0.20⫾0.27
1.96⫾2.22*
3
0.02⫾0.05
2.08⫾1.48*
4
0.26⫾0.22
2.31⫾2.27*
Apoptosis (% cardiomyocyte
1
0.00⫾0.00
0.00⫾0.00
nuclei)
2
0.01⫾0.01
0.35⫾0.27*
3
0.00⫾0.00
0.31⫾0.22*
4
0.00⫾0.00
0.23⫾0.18*
Group 1 is sham group; group 2, placebo; group 3, L-NAME; and group 4,
NOE. Data are mean⫾SD. *P⬍0.05 vs anterior wall.
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(P⬍0.05). Anterior systolic wall thickening was increased
from 23.9⫾1.2% to a maximum of 30.3⫾2.4% (P⬍0.05)
within the first hour of infusion and then declined back to
baseline and was not different from baseline after 4 hours of
infusion (23.8⫾1.6%, P⫽NS).
Discussion
The clinical importance and frequency of coronary microembolization have become increasingly recognized recently.1–3
Pathological evidence for coronary microembolization
emerged from autopsy studies of patients with acute coronary
syndromes who died from sudden cardiac death.21–23 Evidence has also been obtained from the protection devices
used during coronary interventions.24,25
In accordance with results of prior experimental studies,
which showed loss of regional myocardial function in proportion to the number of injected microspheres,4,26 we have
recently demonstrated progressive contractile dysfunction in
the presence of unchanged regional myocardial blood flow
(perfusion-contraction mismatch), associated with a local
inflammatory response5 and a causal involvement of TNF-␣.6
The aim of the present study was to examine in more detail
the signal transduction underlying the progressive myocardial
contractile dysfunction after coronary microembolization.
Figure 1. Number of infiltrating leukocytes in the anterior and
posterior walls 8 hours after coronary microembolization. The
number of inflammatory cells was significantly increased in the
embolized posterior wall compared with that in the untreated
anterior wall. Group 1 is the sham-operated group; group 2, placebo group; group 3, L-NAME group; and group 4, NOE group.
Data are mean⫾SD. *P⬍0.05 vs anterior wall.
Figure 2. Myocardial content of TNF-␣ in the anterior and posterior walls after 8 hours. The TNF-␣ content in groups 2 and 4
was increased in the posterior wall compared with that in the
anterior wall. Data are mean⫾SD. *P⬍0.05 vs anterior wall.
In the placebo group, we confirmed progressive contractile
dysfunction, patchy microinfarctions, an inflammatory response, and increased myocardial TNF-␣ content. The slight
amount of apoptotic cardiomyocyte nuclei, which is unlikely
to account for the observed dysfunction, is consistent with
prior studies.27 In addition, sphingosine contents were increased, consistent with prior studies that found increased
sphingosine content in myocardium on TNF- ␣
stimulation.12,28,29
Intracoronary pretreatment with L-NAME completely prevented the progressive myocardial contractile dysfunction,
and TNF-␣ and sphingosine contents were no longer increased, indicating that NO acted upstream from TNF-␣ and
sphingosine. NO has previously been reported to act downstream from TNF-␣ and to mediate its negative inotropic
effect.30 However, there is also evidence for a role of NO
upstream from TNF-␣. In endotoxemic rats, inhibition of
NOS with NG-monomethyl-L-arginine reduced TNF-␣ levels.31 Conversely, TNF-␣ synthesis was upregulated by NO
through a cGMP-dependent pathway in the failing heart.32
cGMP also upregulated TNF-␣ synthesis in rat peritoneal
macrophages,33 and NO-releasing agents enhanced cytokineinduced TNF-␣ synthesis in human mononuclear cells.34
NOE disrupts the sphingomyelinase pathway by blocking
the enzyme ceramidase, which catalyzes the conversion of
Figure 3. Myocardial content of sphingosine in the anterior and
posterior walls after 8 hours. Only in group 2 was the sphingosine content increased in the posterior wall. Data are
mean⫾SD. *P⬍0.05 vs anterior wall.
812
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April 19, 2002
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ceramide to sphingosine.35,36 Mammalian cardiomyocytes
produce sphingosine and use it as a mediator of TNF-␣–
induced negative inotropism.12,37 The negative inotropic action of sphingosine12,38 has been attributed to inhibition of
sarcoplasmic calcium release7,39 and decreased myocyte calcium transients.40 NOE completely abrogated the negative
inotropic effect of TNF-␣ in isolated adult feline myocytes12
and human atrial trabeculae.41 Inhibition of myocardial sphingosine synthesis by NOE also abolished the progressive
contractile dysfunction after coronary microembolization in
the present study. Although myocardial TNF-␣ content was
still increased after microembolization in NOE-treated dogs,
sphingosine content was unchanged, and contractile dysfunction was prevented, indicating that sphingosine clearly acts
downstream from TNF-␣.
Thus, the present study demonstrates the causal involvement of NO, TNF-␣, and sphingosine as mediators, and it
identifies a sequence of NO, TNF-␣, and sphingosine in the
signal transduction of myocardial contractile dysfunction
after coronary microembolization.
However, the cellular and biochemical sources of NO,
TNF-␣, and sphingosine were not identified in the present
study. The unchanged myocardial iNOS mRNA expression
suggests endothelial NOS (eNOS) as the source of NO. The
cellular source of TNF-␣ at the mRNA level was identified by
in situ hybridization in viable myocytes surrounding the
microinfarcts in a recent study using the same experimental
model.6 The sphingomyelinase pathway is a ubiquitous signaling system.42
Our findings imply not a simplistic monocausal sequence
of mediators, with increased NO, in turn, increasing TNF-␣
and sphingosine levels; in fact, the interaction of these
mediators may be more complex and involve intermediate
steps and feedback loops, which we did not determine.
Although we have measured increased contents of TNF-␣
and sphingosine and have induced contractile dysfunction in
the absence of coronary microembolization by exogenous
TNF-␣6 and sphingosine and although we have also prevented the contractile dysfunction resulting from microembolization by TNF-␣ antibodies6 and ceramidase inhibition,
the situation is more complex, particularly for NO. We did
not measure myocardial NO content, and the unchanged
iNOS mRNA suggests that NO production was probably not
substantially increased. Also, the NO donor SNAP did not
induce contractile dysfunction in the absence of coronary
microembolization. Finally, given the otherwise established
anti-inflammatory properties of endogenous NO,43,44 our data
are consistent with the idea that the suppression of basal
and/or stimulated eNOS-derived NO acts to remove a restraining influence on adenosine release,45 and the increased
adenosine levels may then suppress TNF-␣,46,47 sphingosine,
and, finally, contractile dysfunction. Indeed, an involvement
of adenosine is suggested by the tendency of regional
myocardial blood flow to increase over time in the L-NAME
group. This hypothesis needs to be tested in future experiments. Apart from potential intermediate signal steps, such as
adenosine, there may be feedback loops; eg, sphingosine may
activate eNOS and increase NO production.48
Thus, the present study established a sequence of a signal
transduction through NO, TNF-␣, and sphingosine and may
help to identify more specific targets for drug therapy against
inflammation and the associated myocardial dysfunction in
patients who experience coronary microembolization.
Microembolization by inert5 microspheres certainly underestimates the inflammatory response to true atherosclerotic
plaque material with its thrombogenic, vasoconstrictor, and
inflammatory potential. On the other hand, the acute openchest preparation certainly sensitizes for the release and effect
of inflammatory cytokines.49
The size of the individual microinfarctions in the present
study corresponds with that in human autopsy studies.21–23
Also, the aggregate infarct size of ⬇2% is probably realistic
with respect to the enzyme elevations observed in patients
with coronary interventions.50,51 Finally, the present study
was limited to a time frame of 8 hours; future studies will
have to look at a more chronic outcome. Therefore, to what
extent the present model in anesthetized dogs truly reflects
the clinical scenario and to what extent the present data on the
signal transduction of coronary microembolization can be
extrapolated to humans remain to be defined.
Acknowledgments
The present study was supported by a grant from the Medical Faculty
of Essen (Interdisziplinäre Forschungsförderung der Universität
Essen [IFORES] grant 107509-0) and the Pinguin Foundation.
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Myocardial Dysfunction With Coronary Microembolization: Signal Transduction Through
a Sequence of Nitric Oxide, Tumor Necrosis Factor- α, and Sphingosine
Matthias Thielmann, Hilmar Dörge, Claus Martin, Sergej Belosjorow, Uwe Schwanke, Anita
van de Sand, Ina Konietzka, Astrid Büchert, Arne Krüger, Rainer Schulz and Gerd Heusch
Circ Res. 2002;90:807-813; originally published online March 7, 2002;
doi: 10.1161/01.RES.0000014451.75415.36
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