Myocardial Dysfunction With Coronary Microembolization
Signal Transduction Through a Sequence of Nitric Oxide, Tumor Necrosis Factor-α, and Sphingosine
Coronary microembolization results in progressive myocardial dysfunction, with causal involvement of tumor necrosis factor-α (TNF-α). TNF-α 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-α, 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-μm 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-α, 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-α 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-α, 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-α, and sphingosine is causally involved in the coronary microembolization-induced progressive contractile dysfunction.
Coronary 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-α (TNF-α), 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-α antibodies and that intracoronary infusion of exogenous TNF-α 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 (NO) and sphingosine are part of the signal transduction of TNF-α 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).
Materials and Methods
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.
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 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-μm diameter) and the administration of L-NAME. Regional myocardial blood flow was determined with 4 differently colored (yellow, red, blue, and violet) 15-μm 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.
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 μg 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 μmol/min S-nitroso-N-acetyl-dl-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-μm 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 μg/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 μg 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 μg/kg IV) was infused for 15 minutes, followed by continuous intravenous infusion (2.4 μg/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-α.12 The remaining protocol was identical to that in group 2.
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-α 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-μm 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-μm2 area each from each section.
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-α 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 μmol/L N-acetylcysteine 50 and 12.5 μmol/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-α 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% β-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 μg 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.
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-α, 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.
Heart rate, maximum left ventricular dP/dt, left ventricular peak, and end-diastolic and mean aortic pressures were unchanged throughout the experimental protocol in all 4 groups and 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).
Regional Myocardial Function
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.
TTC staining did not detect any infarction in the anterior and posterior walls in all 4 groups.
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 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-α and Sphingosine Contents
In groups 2 and 4, but not in groups 1 and 3, myocardial TNF-α 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/μg total RNA, respectively; P=NS), group 2 (0.74±0.23 versus 1.07±0.64 fg/μg, P=NS), group 3 (1.99±0.69 versus 2.52±0.34 fg/μg, P=NS), and group 4 (0.67±0.55 versus1.25±0.88 fg/μg, 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 (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).
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-α.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.
In the placebo group, we confirmed progressive contractile dysfunction, patchy microinfarctions, an inflammatory response, and increased myocardial TNF-α 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-α stimulation.12,28,29⇓⇓
Intracoronary pretreatment with L-NAME completely prevented the progressive myocardial contractile dysfunction, and TNF-α and sphingosine contents were no longer increased, indicating that NO acted upstream from TNF-α and sphingosine. NO has previously been reported to act downstream from TNF-α and to mediate its negative inotropic effect.30 However, there is also evidence for a role of NO upstream from TNF-α. In endotoxemic rats, inhibition of NOS with NG-monomethyl-l-arginine reduced TNF-α levels.31 Conversely, TNF-α synthesis was upregulated by NO through a cGMP-dependent pathway in the failing heart.32 cGMP also upregulated TNF-α synthesis in rat peritoneal macrophages,33 and NO-releasing agents enhanced cytokine-induced TNF-α synthesis in human mononuclear cells.34
NOE disrupts the sphingomyelinase pathway by blocking the enzyme ceramidase, which catalyzes the conversion of ceramide to sphingosine.35,36⇓ Mammalian cardiomyocytes produce sphingosine and use it as a mediator of TNF-α–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-α 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-α 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-α.
Thus, the present study demonstrates the causal involvement of NO, TNF-α, and sphingosine as mediators, and it identifies a sequence of NO, TNF-α, and sphingosine in the signal transduction of myocardial contractile dysfunction after coronary microembolization.
However, the cellular and biochemical sources of NO, TNF-α, 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-α 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-α 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-α and sphingosine and have induced contractile dysfunction in the absence of coronary microembolization by exogenous TNF-α6 and sphingosine and although we have also prevented the contractile dysfunction resulting from microembolization by TNF-α 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-α,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-α, 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 open-chest 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.
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.
Original received October 19, 2001; revision received January 17, 2002; accepted February 21, 2002.
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