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Cardiovascular Research 2004 63(1):51-59; doi:10.1016/j.cardiores.2004.03.002
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Inhibition of I{kappa}B phosphorylation in cardiomyocytes attenuates myocardial ischemia/reperfusion injury

Yasuyuki Onaia, Jun-ichi Suzukia, Tsunekazu Kakutaa, Yasuhiro Maejimaa, Go Haraguchia, Hiroshi Fukasawab, Susumu Mutob, Akiko Itaib and Mitsuaki Isobe*,a

aDepartment of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan
bMedical Molecular Design, Inc., 5-24-5 Hongo, Bunkyo, Tokyo, Japan

*Corresponding author. Tel.: +81-3-5803-5951; fax: +81-3-5803-0238. Email address: isobemi.cvm{at}tmd.ac.jp

Received 20 November 2003; revised 26 February 2004; accepted 1 March 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Reperfusion injury is related closely to inflammatory reactions such as activation of inflammatory cells and expression of cytotoxic cytokines. We investigated the efficacy of I{kappa}B phosphorylation blockade in a rat myocardial ischemia/reperfusion injury model. Methods and results: IMD-0354 inhibited phosphorylation of I{kappa}B{alpha} and nuclear translocation of nuclear factor-kappa B (NF-{kappa}B) induced by tumor necrosis factor-{alpha} (TNF-{alpha}) in cultured cardiomyocytes. TNF-{alpha}-induced production of interleukin-1β and monocyte chemoattractant protein-1 from cultured cardiomyocytes was reduced significantly by IMD-0354. Transient left coronary artery occlusion (30 min) and reperfusion (24 h) were carried out in Sprague–Dawley rats. IMD-0354 (1, 5, 10 mg/kg) was injected intraperitoneally 5 min before the start of reperfusion. Treatment with IMD-0354 resulted in a significant dose-dependent reduction of the infarction area/area at risk ratio (vehicle, 47.0±3.4%; 10 mg/kg of IMD-0354, 19.4±4.0%; P<0.01) and the preservation of fractional shortening ratio (vehicle, 25.0±1.5%; 10 mg/kg of IMD-0354, 42.3±1.7%; P<0.01). Histological analysis showed that accumulation of polymorphonuclear neutrophils in the area at risk was decreased significantly. Conclusions: Inhibition of nuclear translocation of NF-{kappa}B by I{kappa}B{alpha} phosphorylation blockade could provide an effective approach to attenuation of ischemia/reperfusion injury. The cardioprotective effects of IMD-0354 include not only reduction of harmful neutrophil accumulation in myocardium but also inhibition of harmful cytokine and chemokine production by cardiomyocytes.

KEYWORDS Myocardial infarction; Reperfusion injury; Nuclear factor-kappaB; Cytokine; Chemokine; Cardiomyocyte


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Ischemia, which is caused by coronary artery occlusion, followed by reperfusion evokes significant myocardial damage leading to cardiac dysfunction. Recently, evidence was given that nuclear factor-kappa B (NF-{kappa}B) is activated by ischemia and reperfusion [1], including that in human hearts subjected to cardioplegia and reperfusion during open heart surgery [2]. Activation of NF-{kappa}B induces gene programs that lead to transactivation of adhesion molecules, cytokines, and chemokines promoting the inflammatory status involved in myocardial reperfusion injury. NF-{kappa}B is a dimer of the Rel family members and the most common active form is composed of p50 or p52 and p65. In resting cells, NF-{kappa}B is inactive and segregated in the cytoplasm bound to an inhibitory protein known as the inhibitor of NF-{kappa}B (I{kappa}B). NF-{kappa}B activation requires phosphorylation of I{kappa}B by I{kappa}B kinase (IKK) complex. The phosphorylated I{kappa}B is then ubiquitinated and degraded by proteasomes. Subsequently, the unbound NF-{kappa}B is translocated to the nucleus and binds to the promoter or enhancer of specific genes, including those involved in inflammatory reactions [3]. In vivo experiments in postischemic myocardium showed that the activation of NF-{kappa}B was biphasic. Two peaks of activation occurred, one after 15 min and the other after 3 h of reperfusion. Presumably, the primary activation was caused by reactive oxygen intermediates, and the secondary activation was caused by proinflammatory cytokines produced by the first activation [4]. Proinflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-{alpha} (TNF-{alpha}), are regulated by NF-{kappa}B activation and are known to be the stimuli that cause the activation of I{kappa}B kinase. Since NF-{kappa}B is the main factor in the positive feedback loop of inflammation, inhibition of its activation may be an effective therapy for myocardial reperfusion injury from the standpoint of preventing inflammation. The purpose of this study was to examine the effect of I{kappa}B phosphorylation blocker IMD-0354 on myocardial reperfusion injury and to investigate its pharmacological mechanism in the heart.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Surgical procedures
Eight- to 10-week-old male Sprague–Dawley rats (250 to 300 g weight) were anesthetized with 40 to 60 mg/kg sodium pentobarbital intraperitoneally (i.p.) and intubated orally with a polyethylene tube for artificial respiration (SN-480-7, Shinano, Japan). After thoracotomy in the fourth intercostal space, the pericardium was gently removed to expose the heart. Myocardial ischemia was produced by ligation of the left anterior descending coronary artery (LAD) for 30 min, and this was followed by 24 h of reperfusion. The chest was then closed, and the animals were allowed to recover in a warm and clean cage. All animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The protocol was approved by the Animal Research Committee of the Tokyo Medical and Dental University.

2.2. Reagents
IMD-0354 (N-(3,5-Bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide) (Fig. 1) was kindly provided by the Institute of Medical Molecular Design (Tokyo, Japan). The drug was dissolved in 0.5% carboxymethylcellulose (CMC) vehicle (Sigma, Tokyo, Japan) immediately before use. Drug-free vehicle (0.5% CMC solution) was used as a control.


Figure 1
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  		Fig. 1 Chemical structure of IMD-0354.

 
2.3. Treatment protocols
The animals were assigned randomly into one of five treatment groups, as follows: i.p. IMD-0354 (1, 5, or 10 mg/kg; n=9, each) injection in a single bolus 5 min before the start of reperfusion; i.p. vehicle injection in a single bolus 5 min before the start of reperfusion (n=9); sham-operation (thoracotomy with LAD isolation but without ligation).

2.4. Measurement of area at risk and infarct sizes
At the end of 24 h of reperfusion, the LAD was re-ligated, and Evans blue dye was infused intravenously to determine the nonischemic zone (area not at risk, ANAR). Hearts were then sliced transversely into 4 slices. Slices were incubated as described previously [5] in 2.0% triphenyl tetrazolium chloride (Sigma) to identify the viable and necrotic areas in the ischemic myocardium (AAR). Each slice was weighed and photographed, and each area was determined with computer assisted planimetry (Scion Image β4.0.2) by blinded observers. The volume of all areas was estimated with the following formula: volume of area=(A1xWt1)+(A2xWt2)+(A3xWt3)+(A4xWt4), where each A is the percentage of the area of each respective section and each Wt is the weight of each respective section.

2.5. Measurement of rat heart cardiac function
Transthoracic echocardiography was carried out with an ultrasound machine (Nemio, Toshiba, Japan), and a 7-MHz annular array transducer. Hearts were imaged in the two-dimensional mode in short-axis views at the level of the papillary muscles. The left ventricular end-diastolic dimension (LVDd) and end-systolic dimension (LVDs), and the ratio of fractional shortenings (%FS) (=[(LVDd–LVDs)/LVDd]x100) were calculated from the M-mode recordings. Each dimension was presented as the average of measurements of three consecutive beats.

2.6. Estimation of myocardial neutrophil infiltration and myeloperoxidase (MPO) activity in cardiac tissue
To determine the extent of polymorphonuclear neutrophil (PMN) infiltration, the midventricular cardiac sections were stained with Gill No. 3 hematoxylin and eosin. Five hearts from each group were examined. The PMNs in AAR per high power field were counted in five random fields, and the counts were averaged. MPO activity was determined in the ischemic and nonischemic cardiac tissue as described previously [6].

2.7. Immunohistochemistry
To perform immunohistochemistry of reperfused myocardium, rat hearts were cut at the level of the papillary muscles and frozen in OCT compound (Sakura Finetek, Tokyo, Japan). Each section (5 µm) was incubated with primary antibodies for 2 h at room temperature and then with histofine simple stain rat (Nichirei, Tokyo, Japan). Finally, each section was reacted with AEC matrix solution for 5 to 20 min. Immunostained type- and class-matched nonimmune IgGs were used as the negative controls for each antibody.

2.8. Measurement of serum troponin T and blood chemistry
After 1 and 24 h of reperfusion, a 3-ml blood sample was obtained from the inferior vena cava. The concentration of serum troponin T (TnT) was determined by electrochemiluminescence immunoassay (ECLIA) with an ECLusys 2010 system (Roche Diagnostics K.K., Tokyo, Japan). Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine were measured with an automatic analyzer (TBA-120FR, Toshiba, Japan).

2.9. Reporter assay
Human hepatoma cells (HepG2) were cultured in Eagle's minimum essential medium with 10% fetal bovine serum and 1% non-essential amino acids for 24 h in a 24-well plate. The cells were transfected with 160 ng of pFLAG-CMV-IKKβ(S177E/S181E) vector, 480 ng of pNF-{kappa}B-Luc plasmid, and 160 ng of pCMV-βGal vector. Transfection was carried out with Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 24 h of transfection, the media were exchanged, and cells were allowed to recover for 4 h. Transfected cells were cultured for 2 h in the presence or absence of IMD-0354 (0.25 or 0.5 µM). The cells were then washed with PBS and incubated with 80 µl of Passive Lysis Buffer (Promega, Madison, WI) for 30 min. Luciferase activity was determined with PicaGene LT2.0 (Toyo Ink, Tokyo, Japan) according to the manufacturer's instructions, activity was expressed as relative light units and normalized for β-galactosidase activity.

2.10. Isolation and treatment of cardiomyocytes
Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were isolated, subjected to percoll gradient centrifugation, and cultured in vitro as described previously [7]. Homogeneity of the cell culture was estimated by antibody staining for {alpha}-sarcomeric actin (M0874, DAKO, Glostrup, Denmark). Cultures had at least 95% cardiomyocytes. The cardiomyocytes were incubated in Eagle's minimum essential medium (Sigma, St. Louis, MO) supplemented with 5% calf serum (JRH Biosciences, Lenexa, KS) and were serum-starved for 24 h. After the growth medium was changed, cells were cultured in the presence or absence of IMD-0354 (0.1 or 1.0 µM) while exposed to TNF-{alpha} (25 ng/ml) for 1 and 24 h, respectively. In vitro dosage of IMD0354 was referred to the blood concentration curve (data not shown).

2.11. Preparation of protein extractions
Heart sections were homogenized in an extraction buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% TritonX-100, 2 mM EGTA, 10 mM EDTA, 100 mM NaF, 1 mM Na4P2O7, 2 mM Na3VO4, 100 µg/ml PMSF, and protease inhibitor. After centrifugation, the second layer of supernatant was collected. For the preparation of cellular protein, as described before [8], cardiomyocytes were lysed with lysis buffer containing 10 mM Tris–HCl, 5 mM MgCl2, 10 mM KCl, 0.3 mM EGTA, 0.5 mM DTT, 0.3 M sucrose, 1 mM PMSF, protease inhibitor, and 0.5% NP-40, and the mixture was centrifuged. The supernatant was saved for cytosolic protein analysis. The pellet was lysed with high-salt Tris-buffer containing 20 mM Tris–HCl, 5 mM MgCl2, 320 mM KCl, 0.2 mM EGTA, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor, and the mixture was centrifuged. The supernatant was saved for nuclear protein analysis. Protein concentration of each sample was measured with the Bio-Rad Protein assay kit (Bio-Rad, Milan, Italy). The protein concentrations of samples were equal in all experiments.

2.12. Western blot analysis
Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with monoclonal antibodies to phospho-I{kappa}B-{alpha} (Cell Signaling, Beverly, MA), NF-{kappa}B p65 (Santa Cruz Biotechnology, Santa Cruz, CA), ICAM-1 (R&D Systems, Minneapolis, MN), and P-selectin (JT, Tokyo, Japan) at 4C overnight. The membranes were incubated with secondary antibody (Amersham Biosciences, Piscataway, NJ) for 2 h and developed with ECL reagent (Amersham Biosciences). Enhanced chemiluminescence was detected with an LAS-1000 (Fujifilm, Tokyo, Japan).

2.13. Cell counting and ELISA
Viable cardiomyocytes were counted with Cell Counting Kit-8 (Wako, Tokyo, Japan) after treatment with IMD-0354 (1.0 µM, 24 h). The total amounts of IL-1β and MCP-1 protein in the supernatants of the same number of cultured neonatal cardiomyocytes were determined by ELISA (BioSource, Camarillo, CA) according to the manufacturer's instructions.

2.14. Statistical analysis
Results are presented as mean±S.E.M. All data were analyzed by analysis of variance (ANOVA) followed by a Scheffe's test for multiple comparisons. A P value of <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. IMD-0354 prevents activation of NF-{kappa}B
To investigate the pharmacological characteristics of IMD-0354-mediated inhibition of phosphorylation of I{kappa}B, we performed a NF-{kappa}B-IKKβ reporter assay with the constitutively active mutant IKKβ. IMD-0354 inhibited the activated expression of NF-{kappa}B in a dose-dependent manner in HepG2 cells that were transfected with pFLAG-CMV-IKKβ(S177E/S181E) vector (Fig. 2A). I{kappa}B{alpha} degradation was confirmed by Western blot analysis for cytosolic phospho-I{kappa}B{alpha}. The levels of cytosolic phospho-I{kappa}B{alpha} in cardiomyocytes induced by TNF-{alpha} stimulation were decreased by IMD-0354 in a dose-dependent manner. The kinetics of NF-{kappa}B translocation were consistent with the degree of phosphorylation of cytosolic I{kappa}B{alpha}, and translocation was blocked markedly by treatment with IMD-0354, even at a concentration of 0.1 µM (Fig. 2B). IMD-0354 also prevented the nuclear translocation of NF-{kappa}B in the heart in vivo after 3 h of reperfusion (data not shown). Taken together, results showed that the pharmacological mechanism of IMD-0354 was that it inhibited IKKβ, resulting in the blockade of I{kappa}B{alpha} phosphorylation.


Figure 2
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  		Fig. 2 Pharmacological mechanism of IMD-0354. (A) NF-{kappa}B-IKKβ reporter assay. Treatment with IMD-0354 dose-dependently inhibited the activation of NF-{kappa}B induced by the transfection of IKKβ constitutive active (CA) mutant. Luciferase activity was expressed as relative light units (RLU), and normalized for β-galactosidase activity. *P<0.01 compared with cells not treated with IMD-0354 (n=3 each). (B) Representative Western blot results for cytosolic phosho-I{kappa}B and p65 NF-{kappa}B, and nuclear p65 NF-{kappa}B. Cardiomyocytes were cultured in the presence or absence of IMD-0354 (0.1 or 1.0 µM) during exposure to TNF-{alpha} (25 ng/ml) for 1 h. Phosphorylation of cytosolic I{kappa}B and nuclear translocation of p65 NF-{kappa}B are inhibited by IMD-0354 in a dose-dependent manner.

 
3.2. Reduction of IL-1β and MCP-1 production from cultured cardiomyocytes
TNF-{alpha} stimulation induced marked increases in IL-1β and MCP-1 protein production from cultured cardiomyocytes as compared with nonstimulated cells. IMD-0354 caused a significant reduction of both IL-1β (–77%) and MCP-1 (–76%) production in a concentration-dependent manner compared with vehicle-treated cells (Fig. 3A and B).


Figure 3
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  		Fig. 3 Production of IL-1β (A) and MCP-1 (B) by cultured cardiomyocytes in the presence (n=4) or absence of IMD-0354 (0.1 or 1.0 µM) (n=4 each) during exposure to TNF-{alpha} (25 ng/ml) for 24 h as estimated by ELISA. *P=0.001 and **P<0.001 compared with cells not treated with IMD-0354 (n=3).

 
3.3. Reduction of I/R-induced myocardial infarction
The mean values for AAR/LV were similar in all treatment groups. However, infarct/AAR values were reduced significantly by the administration of IMD-0354 in a dose-dependent manner relative to the vehicle-treated control groups (infarct/AAR values; 1 mg/kg of IMD-0354, 36.4±3.4%; 5 mg/kg of IMD-0354, 22.6±1.8%; 10 mg/kg of IMD-0354, 19.4±4.0%; vehicle, 47.0±3.4%) (Fig. 4A to C). A maximum reduction of infarct size of was achieved with the highest dose of IMD-0354 (10 mg/kg). The serum levels of TnT after 1 h of reperfusion were increased remarkably in vehicle-treated rats (15.1±2.9 ng/ml), although those of the sham-operated group were below the detection limit of the assay (<0.10 ng/ml, n=4). In contrast, administration of IMD-0354 resulted in a dose-related attenuation in the serum levels of TnT. The effect was significant when rats were treated with 10 mg/kg of IMD-0354 (6.2±1.3 ng/ml) compared with the vehicle-treatment (Fig. 4D). A similar effect of IMD-0354 was confirmed at the end of 24 h of reperfusion.


Figure 4
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  		Fig. 4 Effect of IMD-0354 on I/R-induced myocardial infarction. (A to C) Statistical analyses of infarction size and AAR in hearts from the vehicle- and IMD-0354-treated rats (n=9 each). (D) Serum TnT was estimated by ECLIA after 1 and 24 h of reperfusion (n=6 each). *P<0.01 and **P<0.05 compared with vehicle-treated rat. (IMD1=1 mg/kg of IMD-0354, IMD5=5 mg/kg of IMD-0354, IMD10=10 mg/kg of IMD-0354. All doses of IMD-0354 were given i.p.).

 
3.4. Prevention of myocardial dysfunction after infarction
A representative M-mode echocardiogram of a vehicle-treated rat after 24 h of reperfusion with abnormal wall motion in the anterolateral region of the left ventricle is shown in Fig. 5A. However, treatment with IMD-0354 (10 mg/kg) improved the regional wall motion markedly (Fig. 5B). The %FS of the IMD-0354-treated group was improved significantly compared with the vehicle-treated group (5 mg/kg of IMD-0354, 39.7±2.1%; 10 mg/kg of IMD-0354, 42.3±1.7%; vehicle, 25.0±1.5%; n=9 each) as shown in Fig. 5C. Treatment with 1 mg/kg of IMD-0354 caused moderate improvement of cardiac function, but the improvement was not statistically significant.


Figure 5
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  		Fig. 5 M-mode echocardiogram obtained from left ventricular (LV) short-axis-view before the operation and after 24 h of reperfusion. (A) Representative M-mode-echocardiogram of a vehicle-treated rat after 24 h of reperfusion. (B) Representative M-mode-echocardiogram of IMD-0354-treated rat after 24 h of reperfusion. Left anterior wall motion is improved significantly. (C) Serial change of %FS in each group. *P<0.01 and **P<0.001 compared with rat given vehicle. (IMD1=1 mg/kg of IMD-0354, IMD5=5 mg/kg of IMD-0354, IMD10=10 mg/kg of IMD-0354; n=9 each. All doses of IMD-0354 were given i.p.).

 
3.5. Reduction of myocardial PMN accumulation
Significantly fewer PMNs per high power field were found in the AAR of IMD-0354-treated hearts compared to the vehicle-treated hearts (10 mg/kg of IMD-0354, 4.4±0.5; vehicle, 33.7±2.3) (Fig. 6A). MPO activity was increased in the AAR myocardium of the vehicle-treated hearts. However, administration of IMD-0354 resulted in a significant reduction of MPO activity (10 mg/kg of IMD-0354, 4.8±0.8; vehicle, 10.4±1.1 U/100 mg heart) (Fig. 6B).


Figure 6
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  		Fig. 6 Effect of IMD-0354 on myocardial PMN accumulation. (A) Average numbers of infiltrating PMNs per x400 field (hpf) in the myocardium collected from vehicle- and IMD-0354-treated rats (n=6 each). PMNs were counted in five fields of each heart and the counts were averaged. *P<0.01 compared with rat given vehicle. (B) Myocardial MPO activity in the AAR and the ANAR of cardiac tissue samples obtained from vehicle- and IMD-0354-treated rats (n=6 each). *P<0.01 compared with rat given vehicle. IMD10=10 mg/kg of IMD-0354 i.p.

 
3.6. Decrease of expression of adhesion molecules
The expression of P-selectin was increased on the endothelium of the coronary artery (Fig. 7A) and small vessels in the AAR of vehicle-treated hearts. Simultaneously, the expression of ICAM-1 was increased mainly on the small venules (Fig. 7C). Reduced staining for P-selectin (Fig. 7B) and for ICAM-1 (Fig. 7D) was found in the AAR of hearts obtained from rats treated with 10 mg/kg of IMD-0354. A similar effect of IMD-0354 on the expression of adhesion molecules was confirmed by Western blot analysis, as shown in Fig. 7E and F.


Figure 7
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  		Fig. 7 Effects of IMD-0354 on the expression of adhesion molecules. Representative photographs of immunohistochemical findings of P-selectin (A, B) and ICAM-1 (C, D) expression in hearts obtained from vehicle- and IMD-0354-treated rats. Arrowheads indicate the expression of P-selectin on the coronary endothelium and arrows indicate the expression of ICAM-1 on the small venules. All photographs are of the same magnification (x400). Representative Western blots of P-selectin (E) and ICAM-1 (F) expression in the AAR and the ANAR of hearts obtained from vehicle- and IMD-0354-treated rats. Both P-selectin and ICAM-1 expressions are increased in the AAR site, and treatment with IMD-0354 significantly diminishes their expression. IMD10=10 mg/kg of IMD-0354 i.p.

 
3.7. IMD-0354 shows no general and cellular toxicity
After daily intraperitoneal injection of 10 mg/kg of IMD-0354 for 4 weeks, there was no significant change in body weight and blood chemistry for AST, ALT, ALP, BUN, and creatinine compared with the control group, as shown in Table 1. There also was no decrease in the number of cardiomyocytes when cells were treated with IMD-0354 (1.0 µM) in vitro compared with nontreated cells (Fig. 8).


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  		Table 1 Four-week study of IMD-0354 toxicity in rats

 

Figure 8
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  		Fig. 8 In vitro cellular toxicity assay of IMD-0354. Counts of cardiomyocytes were carried out after culturing in the presence or absence of IMD-0354 (1.0 µM) for 24 h. NS indicates not significant between two groups. O.D.=optical density.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
We have shown that blockade of I{kappa}B{alpha} phosphorylation to prevent nuclear translocation of NF-{kappa}B is effective in decreasing the myocardial infarction area while preserving cardiac function. The mechanism of this treatment includes the reduction of the transactivated cytokine, chemokine, and adhesion molecules necessary for the development of reperfusion injury. A previous study found that NF-{kappa}B decoy transfection into myocardium with hemagglutinating virus of Japan-liposomes reduced the myocardial infarction area by inhibiting expression of cytokines and adhesion molecules from aortic endothelial cells [9]. It is noteworthy that IMD-0354 is administered easily, requires no transfection vectors, and has a direct potent effect on cardiomyocytes.

In the pathway of NF-{kappa}B activation, phosphorylation of I{kappa}B by I{kappa}B kinase is essential. NF-{kappa}B activation is blocked completely after induction with various stimuli in I{kappa}B kinase (IKK1 and IKK2) double knockout mice [10]. The mechanism of IMD-0354 action was shown by reporter assay and Western blot analysis to be the blocking of cytosolic I{kappa}B phosphorylation, which resulted in the inhibition of nuclear translocation of p65 NF-{kappa}B. The effect was confirmed in other cells (data not shown), in addition to cultured cardiomyocytes. The antitranslocation effect of IMD-0354 was dose-dependent, similar to its in vivo effectiveness in reducing the infarction area. Previous works showed that inhibition of I{kappa}B degradation with 20S proteasome inhibitor [11], 15D-PGJ2 (15-deoxy{Delta}12,14-prostaglandin J2) [12] and parthenolide [13] prevented the myocardial reperfusion injury and that overexpression of I{kappa}B prevented postburn myocardial dysfunction [14]. Direct inhibition of I{kappa}B degradation by IMD-0354 could be a more useful and potent way to treat NF-{kappa}B-mediated cardiac injury.

PMN accumulation is the major factor in an acute inflammatory response in reperfused myocardium [15]. Infiltrating PMNs are key mediators of cytotoxic substances [16] and cause capillary plugging that impairs the restoration of coronary flow [17]. Adhesion molecules, such as the selectin families and the immunoglobulin superfamily, are indispensable for accumulation of PMNs. Actually, previous studies that targeted these molecules succeeded in preventing myocardial reperfusion injury [18–20]. We have shown that in vivo administration of IMD-0354 decreased the expression of P-selectin and ICAM-1 markedly in reperfused myocardium and caused a significant reduction of PMN accumulation. The expression of both P-selectin and ICAM-1 is regulated by NF-{kappa}B activation. Each molecule has a {kappa}B binding domain in its promoter site [21,22], and the inflammatory cytokines induce the transcription of each molecule [23].

MCP-1 is known as a chemotactic factor and also has a NF-{kappa}B binding domain in its gene arrangement. Studies have been reported of the role of MCP-1 in human coronary disease [24,25], and it has been found that administration of antibody against MCP-1 significantly decreases the reperfusion injury in a rat model [26]. In reperfused myocardium, monocytes and macrophages are recruited by MCP-1 and function along with PMNs as the main sources of inflammatory cytokines [27]. Furthermore, it has been reported that MCP-1 enhances the expression of ICAM-1 in cardiomyocytes [28] and that IL-1β and MCP-1 work together to enhance their own production [29]. Taken together, MCP-1 plays essential and multiple roles in the recruitment of inflammatory cells promoting myocardial reperfusion injury. Therefore, inhibition of NF-{kappa}B activation to suppress MCP-1 expression is an ideal way to prevent harmful inflammatory cell accumulation in reperfused myocardium.

Various stimuli, including reoxygenation, upregulate the expression of proinflammatory cytokines. Proinflammatory cytokines, such as IL-1β and TNF-{alpha}, produced by infiltrated inflammatory cells activate other cells by transactivating NF-{kappa}B. Most cells express proinflammatory cytokine receptors and have I{kappa}B kinase in their cytoplasm [23,30]. They respond to proinflammatory cytokines through NF-{kappa}B activation that leads to or enhances further expression of proinflammatory cytokines. In the present study, we showed that TNF-{alpha} stimulation activated the expression of IL-1β from cultured cardiomyocytes and that treatment with IMD-0354 inhibited the upregulated IL-1β production significantly. In myocardial inflammatory processes followed by ischemia/reperfusion, cardiomyocytes play an active role in maintaining the inflammatory states, not only by responding to proinflammatory cytokines but also by releasing several cytokines spontaneously and reacting to stimulation with other cytokines. Therefore, myocardium is in the middle of an inflammatory positive feedback loop when hearts are reperfused. Inhibition of I{kappa}B phosphorylation to block the translocation of NF-{kappa}B has a great potential for breaking the vicious feedback loop of inflammation.

Although anti-leukocyte therapy in animal models of myocardial reperfusion injury showed significant effect in several studies, there still remains no clinical evidence. Several reasons for this discrepancy might be based on the study limitations. It is possible that the myocardial reperfusion is not completely equal between human and animal models in the several conditions such as the duration of myocardial ischemia, the existence of myocardial preconditioning, the degree of coronary atherosclerosis, and the difference of collateral circulations. Additional investigations about anti-leukocyte therapy are required in order to clarify the difference between experimental and clinical results.

In summary, inhibition of I{kappa}B phosphorylation to prevent nuclear translocation of NF-{kappa}B attenuates myocardial reperfusion injury and preserves cardiac function after myocardial infarction. Because NF-{kappa}B plays central and multiple roles in the biological response to ischemia/reperfusion as a mediator of many inflammatory signal transductions, an inhibitor of NF-{kappa}B can work as a multifaceted suppressor of inflammation. Overall, inhibition of I{kappa}B phosphorylation in cardiomyocytes could be an effective therapy for treating patients to decrease the risk of reperfusion injury during myocardial infarction.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This study was supported by grants from the Organization of Pharmaceutical Safety and Research, Grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology. The authors wish to thank Noriko Tamura for technical assistance.


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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