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Interact CardioVasc Thorac Surg 2006;5:531-535. doi:10.1510/icvts.2006.130765 © 2006 European Association of Cardio-Thoracic Surgery
Impact of antioxidative treatment on nuclear factor kappa-B regulation during myocardial ischemia–reperfusion
a Department of Cardiothoracic Surgery, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany Received 9 February 2006; received in revised form 30 June 2006; accepted 3 July 2006
*Corresponding author. Tel.: +49 221 478 6043; fax: +49 221 478 5906.
Nuclear factor kappa-B (NF  B), a transcription factor, plays a role in numerous pathological states such as myocardial ischemia–reperfusion (I/R), apoptosis, and ischemic preconditioning. As both myocardial ischemia and reperfusion (by reactive oxygen intermediates) can activate NFB, we investigated the impact of the antioxidant N-acetylcysteine (NAC) on NFB-regulation in patients subjected to cardioplegic arrest (CA) on cardiopulmonary bypass (CPB). Seventeen coronary artery surgery patients (66±9[S.D.] years) subjected to cardiopulmonary bypass (CPB) and cardioplegic arrest were randomized in a double-blind fashion to receive either NAC (100 mg/kg into CPB prime followed by infusion at 20 mg/kg/h; n=9) or placebo (n=8). Transmural LV biopsies were collected prior to CPB (baseline) and at CPB-end and immuno-cytochemically stained against active NFB and phosphorylated IB (activates NFB). At the end of CPB both NFB and IB were unchanged in endothelial cells of controls compared to baseline (45.6±7.6 vs. 49.9±7.1 and 36.8±6.1 vs. 47.5±8.6 counts per viewfield (cpv), P>0.05, respectively). In NAC, NFB and IB in endothelial cells were significantly decreased at CPB-end (19.8±1.7 vs. 39.1±4.1 cpv, P<0.001, and 22.1±1.9 vs. 38.3±4.4 cpv, P=0.006). In cardiomyocytes, however, there were no changes observed in either group. Antioxidative treatment with NAC decreases NFB-activity follwing I/R in endothelial cells. We conclude that NFB-activity post I/R is mediated by free radicals rather than ischemia alone.
Key Words: Nuclear factor kappa-B; Oxidative stress; Ischemia–reperfusion; Cardiopulmonary bypass; Cardioplegic arrest; Antioxidants
Oxidative stress has been identified as the main cause of myocardial ischemia/reperfusion (I/R) injury. Consequently, a considerable number of experimental and clinical studies sought to evaluate the effects of antioxidants on myocardial protection during I/R. Despite inconsistent findings probably due to different species, experimental designs, and different antioxidants used in these studies, antioxidative treatment is now accepted as a potent beneficial strategy in myocardial protection. Studies using the antioxidant N-acetylcysteine have accumulated data suggesting beneficial effects on oxidative stress-related organ injuries in general [1] and, particularly, in myocardium subjected to cardiopulmonary bypass (CPB) and cardioplegic arrest (CA) [2,3]. While reduction of direct tissue damage mediated by reactive–oxygen-derived species (ROS) represents one mechanism of antioxidant action, additional effects of antioxidative treatment on the subcellular and molecular level can be expected, as several intracellular regulatory pathways are redox-sensitive.
Nuclear factor kappa-B (NF
NF
NF
A potential role for NF
The purpose of our study was twofold: first to investigate NF
2.1. Patients Following approval by the University of Cologne Human Ethics Committee, written, informed consent was obtained from each patient during the preoperative interview. Seventeen patients scheduled for elective or urgent coronary artery bypass surgery were randomized into either the NAC group (n=9) or the placebo group (n=8) according to a computer-generated allocation list. NAC (Fluimucil®, Kerpen, Germany) and placebo were supplied in identical-looking glass vials containing either 5 g NAC per 50 ml or isotonic sodium chloride solution. Patients of the NAC group received 100 mg NAC per kg body weight into the CPB prime followed by intravenous infusion at 20 mg NAC per kg body weight per minute until the end of CPB [11]. Patients of the placebo group received equivalent amounts of placebo. Patients were subjected to CPB at 32–34 °C, the aorta was cross-clamped, and myocardial revascularization was performed during cardioplegic arrest using single-shot antegrade cold (4 °C) crystalloid Bretschneider cardioplegia (Custodiol®, Dr. Köhler Chemie, Germany). Intraoperative characteristics are shown in Table 1.
2.2. LV Biopsies Prior to CPB initiation, we collected a transmural biopsy from a fat-free area of the LV anterior wall using a 14 G biopsy needle (Gallini®, Modena, Italy). A second LV biopsy was taken at the end of the extracorporeal circulation prior to weaning from CPB. All LV biopsies were placed in 4% paraformaldehyde for 4 h, then rinsed in 0.1 M phosphate-buffered saline (PBS) for 24 h followed by storage for 12 h in PBS solution with 18% sucrose for cryoprotection and frozen at –80 °C. Prior to immunohistochemical examination, 7 µm slices from the biopsies were placed in a bathing solution of 3% H2O2 and methanol for 20 min, then cells were lysed with 0.25% Triton-X 100 in 0.5 M ammoniumchloride. Thereafter, specimens were treated with 5% bovine serum (BSA) solution in 0.05 M TBS. Prior to each step the sections were rinsed three times in 0.05 M TBS buffer. For NF B and IB staining we used a rabbit polyclonal anti-phospho-NFB p65 (Ser536)-antibody (1:500) and a monoclonal anti-phosphor–IB (Ser32/36)-antibody (1:250) (Cell Signaling Tech, Beverly, MA, USA) and a secondary goat anti-rabbit or a goat anti-mouse antibody (1:400, DAKO, Germany). Tissue sections were incubated with the primary antibody over night at 4 °C. A streptavidin-horseradish peroxidase complex was then applied as a detection system (1:150) for 1 h. Finally, staining was developed for 10–20 min with 3,3-diaminobenzidine tetrahydrochloride (DAB) in 0.1 M PBS. Negative controls were done in the absence of the primary antibody and were negative.
2.4. I B and NFB immunostaining in cardiomyocytes we measured the gray values of 30 cardiomyocytes from six randomly selected areas (the investigator was blinded to the treatment). Cardiomyocytes were selectively surrounded to avoid endothelial measurements. The staining intensity was reported as the mean of measured cardiomyocyte gray value minus background gray value. The background gray value was measured at a cell-free area of the slice on at least three different randomly selected points for every picture. For staining intensity detection a Zeiss Axiophot microscope coupled to a 3-chip CCD-camera was used and the analysis was performed using the Optimas 6.01 image analysis program installed on a Pentium PC.
2.5. I level of 5% by using the two-tailed Student t-test for paired samples, as implemented in the software package SASS for Windows, version 10.0.
3.1. I B- and NFB activity in cardiomyocytes
Fig. 1 depicts the IB- and NFB activities pre CPB and at the end of CPB in cardiomyocytes for placebo and NAC patients (TV densitometry, gray units). Activity for both enzymes was unchanged at the end of CPB.
3.2. I B- and NFB activity in coronary endothelium
IB- and NFB activities pre CPB and at the end of CPB in coronary endothelium are shown in Fig. 2. While the number of myocardial capillaries stained for IB (a) and NFB (b) remained unchanged in the placebo group, there was a significant reduction for both IB-positive (a) and NFB-positive (b) capillaries in the NAC group at the end of CPB.
Fig. 3 shows immunohistochemical staining against activated NF B (p65 subunit) pre CPB and at the end of CPB for both placebo and NAC patients representative for the measurements shown in Figs. 1 and 2.
Our data show that NF B activity was not affected in human myocardium subjected to cardioplegic arrest and reperfusion neither in cardiomyocytes nor in coronary endothelium. However, antioxidative treatment with N-acetylcysteine significantly reduced NFB activity in coronary endothelium as compared to no change in hearts of patients subjected to placebo.
4.1. NF B activation by myocardial ischemia and reperfusion [5,12]. In addition, Valen et al. reported increased NFB acitivity in human myocardium subjected to cardioplegia and reperfusion [6]. In the present study, however, we did not find NFB activation following cardioplegic arrest and reperfusion. To reconcile these seemingly contradictory findings we have to take into account the following considerations:
In isolated hearts NF B can be activated by reactive oxygen intermediates [13] generated especially during the early phase of reperfusion (oxidative burst). In previous studies we and others have shown the potent and beneficial effects of N-acetylcysteine (NAC) on cardioplegic arrest- and cardiopulmonary bypass-related oxidative stress [2,3,11]. Thus, we sought to investigate the impact of antioxidative treatment using NAC on the redox-sensitive NFB regulation during cardioplegic arrest and reperfusion. The significant reduction of NFB activity at the end of CPB in the NAC group demonstrates the effective antioxidative capacity of NAC and also indicates that the myocardial NFB acitivity found pre CPB might result from surgery and/or anesthesia-related oxidative stress even before CPB is initiated.
In summary, we did not find significant NF
We found, however, a baseline activity of NF
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Abstract
1. Introduction
