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Eur J Cardiothorac Surg 2004;26:974-980
© 2004 Elsevier Science NL
Division of Cardiovascular Surgery, First Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received 22 January 2004; received in revised form 31 May 2004; accepted 16 June 2004.
* Corresponding author. Tel.: +81-6-6879-3154; fax: +81-6-6879-3159. (E-mail: sawa{at}surg1.med.osaka-u.ac.jp).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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mRNA expression (190±97 vs. 4805±3017, P=0.024) as well as TNF production (0 vs. 9.6±2.5ng/ml, P<0.05) and also inhibited p38 MAPK activation. Its administration improved recovery of cardiac contractile function during reperfusion: LVDP (130±18 vs. 82±21mmHg (P=0.002)), max/min dP/dt (2812±328/–2283±216 vs. 1520±424/–1325±237mmHg/s, P=0.003). CPK leakage was significantly reduced in FR167653 treated hearts versus untreated hearts (54±6 vs. 0.5±0.1, P<0.05) and reduction of coronary flow was improved (110±13 vs. 77±11%) 1h after beginning of reperfusion (P<0.05). Moreover, FR administration attenuated the number of TUNEL positive cardiomyocytes (3±1 vs. 9±2%). Conclusion: These data demonstrated positive inotropic and antiapoptotic effects of a newly synthesized compound (FR167653) of cytokine inhibitors and its inhibitory effect on myocardial TNF production and p38 MAPK activation in ischemic-reperfused rat heart. This suggested that cytokine inhibition is significant as a method for myocardial protection against ischemia-reperfusion injury. |
1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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, IL-1ß, IL-6, IL-8, etc. These cause local and systemic reactions, such as systemic organ dysfunction, acute respiratory distress syndrome and postoperative bleeding [1–4]. Inhibition of proinflammatory cytokine production in ischemia-reperfusion injured myocardium is one of the primary tasks in cardiovascular surgery. Sufficient inhibition of its negative effects on myocardium might significantly improve the results of open heart surgery and heart transplantation. Recently several studies have proved an important role of mitogen activated protein kinases (MAPKs) [5–10] in ischemic injury. In the heart, enhanced activation of p38 MAPK is associated with ischemia-reperfusion injury. Activation of this kinase [6,7] by several stimuli during ischemia and reperfusion leads to apoptosis [8] and expression of proinflammatory cytokines [10], which, in turn, results in the depression of myocardial function [11–13] and cell loss. Moreover, the negative inotropic effect of activated p38MAPK is mediated by decreasing myofilament response to calcium [14] and inhibition of p38MAPK during ischemia-reperfusion prevents apoptotic cell loss.
A newly synthesized organic compound, FR167653 (FR) (Fujisawa Pharmaceutical Co. Ltd, Osaka, Japan), has been characterized as a strong suppressant of TNF and IL-1ß expression [15], promising a useful clinical application. Moreover, its protective effect was experimentally proved in lung [15] and liver models. However, no data related to the inhibition of p38 MAPK and myocardial TNF expression by FR 167653 has been shown in ischemic-reperfused heart.
Therefore, we hypothesized that administration of FR 167653 could decrease cytokine expression [16] and attenuate depression of myocardial function caused by ischemia-reperfusion. In the present study, we evaluated different aspects of the myocardial protective effect of FR 167653 in ischemia-reperfusion injury in rat myocardium.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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2.1. Experimental design
Thirty-two Lewis rats (200 g) were divided into four groups (eight rats in each group): the control group (received vehicle pretreatment), the FR group (administered FR 167653 1mg/kg intraperetoneal (IP) 1h before ischemia and 1mg/l in perfusion Krebs solution), the 2FR group (2.0mg/kg IP, 2.0mg/l) and the 0.5FR group (0.5mg/kg IP, 0.5mg/l) respectively. All drug solution was diluted using deionized sterile water (Sigma®) and filtered. The Rats were anesthetized with pentobarbital 50mg/kg IP and heparinized (1000U/kg, intravenous). After 5min of anesthesia, the hearts were rapidly excised from the chest and were mounted onto the Langendorff perfusion system and retrograde perfused via the aorta with modified normothermic (37°C) Krebs–Henseleit buffer solution (120mM NaCl; 4.5mM KCl; 2.5mM CaCl2; 1.2mM KH2PO4; 1.2mM MgSO4; 25mM NaHCO3; 10mM glucose, pH 7.4) at the constant pressure of 100cm H2O, saturated with 95% O2 and 5% CO2. A pulmonary arteriotomy and left atrial resection were performed before inserting a water-filled latex balloon through the left atrium into the left ventricle. The balloon pressure was then adjusted to 10mmHg of the left ventricular end-diastolic pressure. Several indices of myocardial function such as left ventricular development pressure (LVDP), the derivative of LV pressure (max and min dP/dt, reflecting both systolic and diastolic function), heart rate (HR) and coronary flow (CF) were measured at the end of a 20min stabilization period. After this, the heart was arrested by stopping perfusion and kept at 37°C in an organ bath of the previously described degassed solution for 30min followed by 180min of reperfusion, during which time cardiac function and coronary flow was measured continuously using the Unique Acquisition software, amplifier and flowmeter (Unique Medical, Japan). Creatine phosphokinase (CPK) level was checked in all collected effluent samples using immunoinhibition method.
2.2. Evaluation of apoptotic activity in reperfused myocardium
The other 48 rats, 24 from both the control and the FR group, were perfused for 180min. Hearts samples (three hearts (n=3) per every time point) were taken before ischemia, during ischemia and at 10, 20, 30, 60, 120, and 180min after reperfusion was started to evaluate the time-course of ferment activation. Hematoxylin–Eosin and Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin In Situ Nick-End Labeling (TUNEL Intergen® Kit) staining according to the manufacturer's instruction were performed to evaluate apoptotic signs in ischemia-reprfusion injured myocardium.
2.3. Western blot analysis of p38MAPK activity
Western blot analysis for detection of p38 MAPKinase (Thr180/Tyr182) phosphorylation status (PhosphoPlus® p38 MAPKinase antibody kit, Cell Signaling® Technology) was performed with every heart sample that helped us to clarify the time dependent p38MAPK activation status. Cells or tissue extracts were prepared by tissue homogenization using Cell Lysis Buffer (Cell Signaling®). Protein concentration was measured (DC Protein Assay kit, BioRad®) and protein extracts were separated by SDS polyacrilamyde gel (4–20% gradient gels, Invitrogen®) and transferred to PVDF membrane (Pall FluoroTrans®) followed by the staining with primary antibodies. We used phosho-p38 MAP Kinase (Thr180/Tyr182) and p38 MAP Kinase rabbit polyclonal IgG antibody, affinity purified (1:1000) that does not appreciably cross-react with other IR activated corresponding phosphorilated forms of either p42/44 MAPK or SAPK/JNK; anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:2000) and Phototope®-HRP Western detection Kit. As a control, we used phosphorelated/nonphosphorelated C-6 glyoma cell extracts and as the loading control anti ßactin antibody (Sigma). Biotinylated protein marker detection pack (Cell Signaling® Technology) was used to detect molecular weight markers on Western blot. The blot was visualized with Phototop®-HRP Western Blot Detection System (Cell Signaling®) and quantification was performed using Kodak D1 software. Results are presented as mean intensity±SD in three individual samples those were taken at every time point in each group.
2.4. Immunohistochemical analysis
The primary rabbit antibodies (1:50), as described, above were used for Immunohistochemical (IHC) analysis of rat myocardium tissue. Tissue slides with paraformaldehyde fixed and paraffin embedded myocardium were washed several times with cold PBS solution followed by blocking solution (PBS containing 5% Albumin (Sigma), 0.1% Triton X-100, 0.01% sodium acid). 4,6-diamidino-2 phenylindole (DAPI) nucleus staining and donkey anti-rabbit IgG Alexa Flour 594 (Molecular probes) as secondary antibody were used for IHC analysis with laser scanning confocal microscope (LSM-510, Carl Zeiss).
2.5. Evaluation of myocardial TNF expression and production
Total RNA was isolated from heart samples using RNeasy® Mini kit (Qiagen®). All RNA samples had an A260/A280 ratio 1.9–2.1 at pH>7.5. TNF mRNA expression level was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) (Super ScriptTM First-strand Synthesis System for RT-PCR, Gibco BRL) and Taq Man® Universal PCR Master Mix (Applied Biosystems®) at ABI Prism® 7700 real time sequence detection system using specific rat TNF primer and probe. Results were expressed relative to the amount of GAPDH mRNA present in each specimen and presented as mean±SD in 10 individual samples in each group.
TNF level in coronary flow leakage samples at different time points before during and after ischemia was checked using rat TNF ELISA kit (Endogene®) to clarify myocardial TNF production time course. Results presented as mean±SD from three individual coronary flow samples at every time point.
All values were expressed as the mean±SD. Statistical differences were evaluated by unpaired Student's t-test for two groups or using ANOVA for multiple comparisons. A P value <0.05 was considered statistically significant.
The experiments were performed in adherence with ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ published in the National Institutes of Health (NIH Pub N. 85-23, revised 1985).
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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3.3. Creatine phosphokinase leakage and FR treatment
CPK leakage was significantly higher in the coronary flow samples taken during the reperfusion period in the control group (54±6IU/ml) compared to that in the FR (0.5±0.1IU/ml) or 0.5FR group (1.2±0.3IU/ml), P<0.05.
3.4. P38MAP kinase inhibition effect of FR 167653
Activation of p38 MAPK was checked by analysis of phosphorylation Thr180/Tyr182 p38MAPK status. Significantly higher activity of p38 MAPK was detected in the control group (Fig. 3A) with a maximum at 20–60min after reperfusion; in contrast, there was an absence of such high activity in the FR167653 treated group (Fig. 3B). Immunohistochemical examination of rat myocardium subjected to ischemia-reperfusion with anti-phospho p38 MAPK antibodies displayed significantly higher activity of the p38 MAPK in the control group versus the FR 167653 treated group (Fig. 3D).
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mRNA expression and production by FR 167653 administration mRNA expression level assayed by real time RT-PCR analysis in the FR group myocardial tissue was markedly lower than in the control group one hour after reperfusion (190±97 vs. 4805±3017, P=0.024), (Fig. 4A). We did not find any significant difference in TNF level checked by ELISA in the samples perfusat taken before ischemia, however production of TNF was significantly increased during reperfusion with a maximum after 90–120min in untreated versus FR treated myocardium (Fig. 4B).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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expression and production during reperfusion, and reduction of CPK leakage during reperfusion in FR 167653 treated hearts. These results suggested that FR167653 has a significant potential to attenuate ischemic-reperfusion injury in myocardium.
Myocardial apoptosis occurs after ischemia-reperfusion injury [8,21–23] and its prevention has recently gained much attention as a new target for myocardial protection from ischemia-reperfusion injury [7–10]. We found that administration of FR 167653 in the rat heart ischemia-reperfusion injured model resulted in marked decrease in number of apoptotic cells after 150–180min of reperfusion. This suggested that the attenuation of apoptosis by FR 167653 administration may be related to inhibition of p38 MAPK that might deactivate downstream kinases in the p38 MAPK pathway triggering apoptosis [14,19]. Cell death as a result of ischemia-reperfusion injury is a key element in the development of heart failure. Inhibition of apoptosis initiation may help us to prevent further impairment of myocardial function due to cardiomyocytes loss [19,20]. Also, inhibition of myocardial TNF production by FR167653 administration may be one of the methods to attenuate contraction dysfunction [12,17,18] and apoptosis caused by death signals from the TNF receptor [24].
As the core of the cytokine network, TNF plays an important role [24] and induces not only the production of other inflammatory cytokines but also migration and adherence of neutrophils to endothelial cells followed by endothelial cell injury [21].
Moreover, there has been recent focus on TNF as a direct mediator of myocardial ischemic damage [1–5,24]. The hemodynamic effects of TNF are characterized by decreased myocardial contractile efficiency [17], reduced ejection fraction, hypotension, and decreased systemic vascular resistance [1–3,11]. In this study we found that FR 167653 pretreatment decreased ischemia-reperfusion induced myocardial TNF production even in the absence of blood circulation.
Also, the inhibition of cytokine production by blood cells potentially could decrease endothelial cell injury in in vivo models and minimize negative TNF hemodynamic effects. While this could not specifically be assessed in this experiment because the hearts were perfused with saline rather than blood, we suspect that FR 167653 would prevent TNF production by blood cells, thereby preventing endothelial injury caused by cytokine production of blood cells in vivo.
We speculate that the administration of FR 167653 inhibits p38MAPK which leads to decreasing expression of TNF mRNA resulting in less myocardial TNF production and it may also has a potential to relieve contractile dysfunction caused by local myocardial TNF action, even without neutrophils [21] or other blood cells.
Moreover, in vitro assay demonstrated that enhanced p38 MAPK activation negatively regulates cardiomyocyte contractility, whereas inhibition of p38 MAPK activity leads to a positive inotropic effect. The negative inotropic effect of p38 MAPK might be mediated by decreasing myofilament response to Ca2+ [14].
Interestingly, administration of FR 167653 just during the reperfusion period without preischemic administration did not show any significant influence on cardiac function recovery or on p38 MAPK activation status. No significant difference between the groups was observed in this case, data not shown. It is possible, however, that if the FR is added during the ischemic period, rather than just during reperfusion as in this experiment, there would be a positive inotropic effect. The absence of such effect may be due to the pharmacodynamics of FR such that there is delayed intracellular delivery or it may be related to the nature of the phosphorylation of p38MAPK which was probably initiated during ischemic period.
In summary, treatment with FR167653 decreased p38MAPK activation and cytokine production caused by ischemia-reperfusion, improved myocardial contractile function in the postischemic period and reduced apoptosis. These results suggest that it has potential significance for clinical application in cardiac surgery as a therapeutic intervention, especially in the case of cardio-pulmonary bypass.
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Acknowledgments |
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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processing by a metalloproteinase inhibitor. Nature 1994;370:558-561.[CrossRef][Medline]
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