Impact of antioxidative treatment on nuclear factor kappa-B regulation during myocardial ischemia–reperfusion
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《血管的通路杂志》
a Department of Cardiothoracic Surgery, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany
b Institute I for Anatomy, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany
c Department of Molecular and Cellular Medicine, German Sports University, Cologne, Germany
Abstract
Nuclear factor kappa-B (NFB), 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
1. Introduction
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 (NFB), a redox-sensitive transcription factor, regulates a battery of genes and has been associated with the pathophysiology of myocardial ischemia–reperfusion injury, ischemic preconditioning, and unstable angina [4].
NFB is sequestered within the cytosol by an inhibitory protein (IB; inhibitor of NFB) that masks the nuclear localization signal present within the NFB protein sequence [4]. Degradation of IB by the so-called IKK complex liberates NFB (p65 subunit), which subsequently translocates to the nucleus and binds to specific elements (kB-sites) within the promoters of responsive genes to activate their transcription [4].
NFB activation by myocardial ischemia and reperfusion has been reported, including the human heart subjected to cardioplegia and reperfusion during open heart surgery [5,6]. An important role for NFB during reperfusion indirectly results from its effect on genes regulating inhibition of leukocyte adhesion, cytokines, and chemokines, which in turn protects the heart against reperfusion injury [7].
A potential role for NFB has also been suggested in ischemic preconditioning, as NFB is activated during the preconditioning episodes and pharmacological inhibition of NFB abolishes the associated cardioprotective effects [8]. Furthermore, evidence has accumulated that NFB is involved in apoptosis regulation by acting as a survival factor [4]. As cardioplegic arrest and cardiopulmonary bypass have been shown to be associated with myocardial apoptosis induction in both cardiomyocytes and coronary endothelium, NFB regulation in this context remains to be investigated. Although NFB activation in human hearts subjected to cardioplegia and reperfusion during open heart surgery has been reported [4], data on myocardial distribution and cell types involved are missing.
The purpose of our study was twofold: first to investigate NFB activation in human myocardium subjected to cardioplegic arrest and reperfusion using immunohistochemistry in order to discriminate different cell types and quantify the extent of activity, and second, to determine the impact of antioxidative treatment using N-acetylcysteine (NAC) on myocardial NFB regulation. NAC is a nonspecific antioxidant that interacts with oxygen radicals and results in NAC-disulfide as a main end product [9]. In addition to its function as an oxygen radical scavenger, NAC also inhibits oxygen radical generation by polymorphonuclear leucocytes in vitro and in vivo [10].
2. Methods
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. Khler 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.
2.3. Immunocytochemistry
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 NFB 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. IB- and NFB activity in cardiomyocytes
All LV biopsy slices were incubated and stored under identical conditions. For quantitative intensity analysis of IB 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. IB- and NFB activity in coronary endothelial cells
Quantitative analysis was performed on five randomly selected fields (66.125 μm2 per frame) of LV biopsy cross-sections. All immunohistochemically stained capillaries per field were counted with a Zeiss Axiophot transmission light microscope with a 40X oil immersion objective and expressed as the number per square millimeter.
2.6. Statistical Analysis
All data are presented as mean±S.D. Data were analyzed for statistical significance on an 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. Results
3.1. IB- 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. IB- 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 NFB (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.
4. Discussion
Our data show that NFB 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. NFB acitivation during myocardial ischemia and reperfusion
Several studies in rats have shown NFB 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 NFB activation starts shortly after ischemia initiation and is augmented by reperfusion [5]. However, in hearts subjected to in vivo infarction, NFB-activation is biphasic, peaking at 15 min and at 3 h of reperfusion. As we sampled the second biopsy at about 25–30 min reperfusion, the absence of increased NFB-activity in both cardiomyocytes and coronary endothelium in patients of the placebo group could result from the time point of sample collection. However, in the clinical setting it is not possible to collect multiple LV biopsies for up to 3 h after aortic cross-clamp release. In addition, in our study we used cold cardioplegic solution to arrest the heart and moderate hypothermia during ischemia, which could also explain for the different findings compared to the study of Valen et al. [6] Furthermore, Valen et al. found NFB activation and expression of several NFB-regulated genes more pronounced in patients with unstable angina [6]. All patients in our study had stable angina and were electively subjected to cardiac surgery, which could additionally account for the lack of NFB activation.
4.2. NFB activation
NFB 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 NFB activation in human myocardium subjected to cardioplegic arrest and reperfusion neither in cardiomyocytes nor in coronary endothelium. This difference compared to other studies could be due to different conditions during cardioplegic arrest and different patient populations investigated. However, considering our data, we do not support the general hypothesis of NFB activation during ischemia in human myocardium.
We found, however, a baseline activity of NFB which was significantly reduced by means of N-acetylcysteine administration. We attribute this effect to the antioxidative capacity of N-acetylcysteine. As NFB activation has been suggested to aggravate myocardial ischemia/reperfusion injury [14], and inhibition of NFB activation during reperfusion has been shown to reduce infarct size [15], ROS-scavenging appears to represent a promising adjunct to myocardial preservation techniques. Future studies are required to further elucidate the time course of NFB regulation and to determine the optimum time for anti-oxidant administration to gain its full potential benefit.
References
Tepel M, van der Giet M, Schwarzfeld C, Laufer U, Liermann D, Zidek W. Prevention of radiographic-contrast-agent-induced reductions in renal function by acetylcysteine. N Engl J Med 2000; 343:180–184.
Fischer UM, Cox CS Jr, Allen SJ, Stewart RH, Mehlhorn U, Laine GA. The antioxidant N-acetylcysteine preserves myocardial function and diminishes oxidative stress after cardioplegic arrest. J Thorac Cardiovasc Surg 2003; 126:1483–1488.
Fischer UM, Tossios P, Huebner A, Geissler HJ, Bloch W, Mehlhorn U. Myocardial apoptosis prevention by radical scavenging in patients undergoing cardiac surgery. J Thorac Cardiovasc Surg 2004; 128:103–108.
Valen G, Yan ZQ, Hansson GK. Nuclear factor kappa-B and the heart. J Am Coll Cardiol 2001; 38:307–314.
Li C, Browder W, Kao RL. Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart. Am J Physiol 1999; 276:H543–H552.
Valen G, Paulsson G, Vaage J. Induction of inflammatory mediators during reperfusion of the human heart. Ann Thorac Surg 2001; 71:226–232.
Gumina RJ, Newman PJ, Kenny D, Warltier DC, Gross GJ. The leukocyte cell adhesion cascade and its role in myocardial ischemia-reperfusion injury. Basic Res Cardiol 1997; 92:201–213.
Maulik N, Sato M, Price BD, Das DK. An essential role of NFkappaB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett 1998; 429:365–369.
Flanagan RJ, Meredith TJ. Use of N-acetylcysteine in clinical toxicology. Am J Med 1991; 91:131S–139S.
Bernard GR, Lucht WD, Niedermeyer ME, Snapper JR, Ogletree ML, Brigham KL. Effect of N-acetylcysteine on the pulmonary response to endotoxin in the awake sheep and upon in vitro granulocyte function. J Clin Invest 1984; 73:1772–1784.
Andersen LW, Thiis J, Kharazmi A, Rygg I. The role of N-acetylcystein administration on the oxidative response of neutrophils during cardiopulmonary bypass. Perfusion 1995; 10:21–26.
Chandrasekar B, Freeman GL. Induction of nuclear factor kappaB and activation protein 1 in postischemic myocardium. FEBS Lett 1997; 401:30–34.
Li N, Karin M. Is NF-kappaB the sensor of oxidative stress Faseb J 1999; 13:1137–1143.
Valen G. Signal transduction through nuclear factor kappa B in ischemia-reperfusion and heart failure. Basic Res Cardiol 2004; 99:1–7.
Morishita R, Sugimoto T, Aoki M, Kida I, Tomita N, Moriguchi A, Maeda K, Sawa Y, Kaneda Y, Higaki J, Ogihara T. In vivo transfection of cis element ‘decoy’ against nuclear factor-kappaB binding site prevents myocardial infarction. Nat Med 1997; 3:894–899.(Uwe M. Fischer, Albert An)
b Institute I for Anatomy, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany
c Department of Molecular and Cellular Medicine, German Sports University, Cologne, Germany
Abstract
Nuclear factor kappa-B (NFB), 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
1. Introduction
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 (NFB), a redox-sensitive transcription factor, regulates a battery of genes and has been associated with the pathophysiology of myocardial ischemia–reperfusion injury, ischemic preconditioning, and unstable angina [4].
NFB is sequestered within the cytosol by an inhibitory protein (IB; inhibitor of NFB) that masks the nuclear localization signal present within the NFB protein sequence [4]. Degradation of IB by the so-called IKK complex liberates NFB (p65 subunit), which subsequently translocates to the nucleus and binds to specific elements (kB-sites) within the promoters of responsive genes to activate their transcription [4].
NFB activation by myocardial ischemia and reperfusion has been reported, including the human heart subjected to cardioplegia and reperfusion during open heart surgery [5,6]. An important role for NFB during reperfusion indirectly results from its effect on genes regulating inhibition of leukocyte adhesion, cytokines, and chemokines, which in turn protects the heart against reperfusion injury [7].
A potential role for NFB has also been suggested in ischemic preconditioning, as NFB is activated during the preconditioning episodes and pharmacological inhibition of NFB abolishes the associated cardioprotective effects [8]. Furthermore, evidence has accumulated that NFB is involved in apoptosis regulation by acting as a survival factor [4]. As cardioplegic arrest and cardiopulmonary bypass have been shown to be associated with myocardial apoptosis induction in both cardiomyocytes and coronary endothelium, NFB regulation in this context remains to be investigated. Although NFB activation in human hearts subjected to cardioplegia and reperfusion during open heart surgery has been reported [4], data on myocardial distribution and cell types involved are missing.
The purpose of our study was twofold: first to investigate NFB activation in human myocardium subjected to cardioplegic arrest and reperfusion using immunohistochemistry in order to discriminate different cell types and quantify the extent of activity, and second, to determine the impact of antioxidative treatment using N-acetylcysteine (NAC) on myocardial NFB regulation. NAC is a nonspecific antioxidant that interacts with oxygen radicals and results in NAC-disulfide as a main end product [9]. In addition to its function as an oxygen radical scavenger, NAC also inhibits oxygen radical generation by polymorphonuclear leucocytes in vitro and in vivo [10].
2. Methods
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. Khler 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.
2.3. Immunocytochemistry
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 NFB 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. IB- and NFB activity in cardiomyocytes
All LV biopsy slices were incubated and stored under identical conditions. For quantitative intensity analysis of IB 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. IB- and NFB activity in coronary endothelial cells
Quantitative analysis was performed on five randomly selected fields (66.125 μm2 per frame) of LV biopsy cross-sections. All immunohistochemically stained capillaries per field were counted with a Zeiss Axiophot transmission light microscope with a 40X oil immersion objective and expressed as the number per square millimeter.
2.6. Statistical Analysis
All data are presented as mean±S.D. Data were analyzed for statistical significance on an 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. Results
3.1. IB- 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. IB- 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 NFB (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.
4. Discussion
Our data show that NFB 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. NFB acitivation during myocardial ischemia and reperfusion
Several studies in rats have shown NFB 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 NFB activation starts shortly after ischemia initiation and is augmented by reperfusion [5]. However, in hearts subjected to in vivo infarction, NFB-activation is biphasic, peaking at 15 min and at 3 h of reperfusion. As we sampled the second biopsy at about 25–30 min reperfusion, the absence of increased NFB-activity in both cardiomyocytes and coronary endothelium in patients of the placebo group could result from the time point of sample collection. However, in the clinical setting it is not possible to collect multiple LV biopsies for up to 3 h after aortic cross-clamp release. In addition, in our study we used cold cardioplegic solution to arrest the heart and moderate hypothermia during ischemia, which could also explain for the different findings compared to the study of Valen et al. [6] Furthermore, Valen et al. found NFB activation and expression of several NFB-regulated genes more pronounced in patients with unstable angina [6]. All patients in our study had stable angina and were electively subjected to cardiac surgery, which could additionally account for the lack of NFB activation.
4.2. NFB activation
NFB 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 NFB activation in human myocardium subjected to cardioplegic arrest and reperfusion neither in cardiomyocytes nor in coronary endothelium. This difference compared to other studies could be due to different conditions during cardioplegic arrest and different patient populations investigated. However, considering our data, we do not support the general hypothesis of NFB activation during ischemia in human myocardium.
We found, however, a baseline activity of NFB which was significantly reduced by means of N-acetylcysteine administration. We attribute this effect to the antioxidative capacity of N-acetylcysteine. As NFB activation has been suggested to aggravate myocardial ischemia/reperfusion injury [14], and inhibition of NFB activation during reperfusion has been shown to reduce infarct size [15], ROS-scavenging appears to represent a promising adjunct to myocardial preservation techniques. Future studies are required to further elucidate the time course of NFB regulation and to determine the optimum time for anti-oxidant administration to gain its full potential benefit.
References
Tepel M, van der Giet M, Schwarzfeld C, Laufer U, Liermann D, Zidek W. Prevention of radiographic-contrast-agent-induced reductions in renal function by acetylcysteine. N Engl J Med 2000; 343:180–184.
Fischer UM, Cox CS Jr, Allen SJ, Stewart RH, Mehlhorn U, Laine GA. The antioxidant N-acetylcysteine preserves myocardial function and diminishes oxidative stress after cardioplegic arrest. J Thorac Cardiovasc Surg 2003; 126:1483–1488.
Fischer UM, Tossios P, Huebner A, Geissler HJ, Bloch W, Mehlhorn U. Myocardial apoptosis prevention by radical scavenging in patients undergoing cardiac surgery. J Thorac Cardiovasc Surg 2004; 128:103–108.
Valen G, Yan ZQ, Hansson GK. Nuclear factor kappa-B and the heart. J Am Coll Cardiol 2001; 38:307–314.
Li C, Browder W, Kao RL. Early activation of transcription factor NF-kappaB during ischemia in perfused rat heart. Am J Physiol 1999; 276:H543–H552.
Valen G, Paulsson G, Vaage J. Induction of inflammatory mediators during reperfusion of the human heart. Ann Thorac Surg 2001; 71:226–232.
Gumina RJ, Newman PJ, Kenny D, Warltier DC, Gross GJ. The leukocyte cell adhesion cascade and its role in myocardial ischemia-reperfusion injury. Basic Res Cardiol 1997; 92:201–213.
Maulik N, Sato M, Price BD, Das DK. An essential role of NFkappaB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett 1998; 429:365–369.
Flanagan RJ, Meredith TJ. Use of N-acetylcysteine in clinical toxicology. Am J Med 1991; 91:131S–139S.
Bernard GR, Lucht WD, Niedermeyer ME, Snapper JR, Ogletree ML, Brigham KL. Effect of N-acetylcysteine on the pulmonary response to endotoxin in the awake sheep and upon in vitro granulocyte function. J Clin Invest 1984; 73:1772–1784.
Andersen LW, Thiis J, Kharazmi A, Rygg I. The role of N-acetylcystein administration on the oxidative response of neutrophils during cardiopulmonary bypass. Perfusion 1995; 10:21–26.
Chandrasekar B, Freeman GL. Induction of nuclear factor kappaB and activation protein 1 in postischemic myocardium. FEBS Lett 1997; 401:30–34.
Li N, Karin M. Is NF-kappaB the sensor of oxidative stress Faseb J 1999; 13:1137–1143.
Valen G. Signal transduction through nuclear factor kappa B in ischemia-reperfusion and heart failure. Basic Res Cardiol 2004; 99:1–7.
Morishita R, Sugimoto T, Aoki M, Kida I, Tomita N, Moriguchi A, Maeda K, Sawa Y, Kaneda Y, Higaki J, Ogihara T. In vivo transfection of cis element ‘decoy’ against nuclear factor-kappaB binding site prevents myocardial infarction. Nat Med 1997; 3:894–899.(Uwe M. Fischer, Albert An)