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J Thorac Cardiovasc Surg 2002;124:933-941
© 2002 The American Association for Thoracic Surgery
Cardiopulmonary Support and Physiology (CSP) |
B
complex and attenuation of nuclear factor B
From the Divisions of Cardiothoracic Surgery and Biochemical Genetics, Tokyo Medical and Dental University Graduate School of Medicine, Tokyo, Japan.
Received for publication Sept 24, 2001. Revisions requested Oct 25, 2001, revisions received Nov 9, 2001. Accepted for publication Dec 7, 2001. Address for reprints: Makoto Sunamori, MD, Professor and Chief, Division of Cardiothoracic Surgery, Tokyo Medical and Dental University Graduate School of Medicine, Bunkyo-ku yushima 1-5-45, Tokyo 113-8519, Japan (E-mail: sunamori.tsrg{at}tmd.ac.jp).
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Abstract |
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B
, thereby inhibiting degradation and attenuating activation of nuclear factor B and cellular injury in rat myocardium. B levels and detection of heat shock protein 70-inhibitory B complexes. Myocardium from the remaining 10 rats in each group was exposed to 30 minutes of ischemia and 30 minutes of reperfusion (n = 5) to evaluate nuclear factor B activity or to 24 hours of reperfusion (n = 5) to evaluate infarct size. B was activated in the control ischemia group and attenuated in the lipopolysaccharide group (P < .05). Heat shock protein 70 levels were increased in the lipopolysaccharide group (P < .05), but inhibitory B levels were similar in both groups. Heat shock protein 70-inhibitory B complexes were detected only in the lipopolysaccharide group. Colocalization of the 2 proteins was observed in the lipopolysaccharide group. B and attenuates activation of nuclear factor B and myocardial infarct size. Our results suggest that attenuation of nuclear factor B through a mechanism forming heat shock protein 70-inhibitory B complexes might protect the myocardium from ischemia-reperfusion injury. |
Introduction |
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1 Many methods to prevent myocardial injury caused by ischemia and reperfusion have been examined. Early (immediate) or delayed (24-72 hours later) preconditioning of the heart is a promising procedure, resulting in cardiac protection by means of different phenomena and mechanisms. 2,3 It is reported that ischemic preconditioning 2 and pharmacologic pretreatment 4 showed protection in the early phase, whereas ischemic preconditioning, 3 heat stimuli, 5 monophosphoryl lipid A, 6 and interleukin 1 pretreatment 7 have been reported to reduce ischemia-reperfusion injury and to prevent hemodynamic depression after ischemia in the delayed phase.
Lipopolysaccharide (LPS) pretreatment is also known as delayed preconditioning and is used to enhance cellular tolerance to ischemia in various organs, including the liver, 8 kidney, 9 brain, 10 and heart. 11,12 In LPS-treated hearts it was shown that LPS increased levels of heat shock protein 70 (HSP70), 11 and investigators have shown that myocardial protection reduces infarct size 11 and maintains cardiac function, 12 which is associated with the HSP70. HSP70 functions as a chaperone, folding and transporting newly synthesized proteins and degrading damaged proteins. 13 When expression of HSP70 is induced in cells exposed to stress, HSP70 often rescues cells from death through its chaperone function. In the myocardium HSP70 is induced 24 to 72 hours after LPS pretreatment 11 and ischemic preconditioning. 14 However, there is no evidence that HSP70 directly attenuates ischemia-reperfusion injury in the myocardium.
In the process of myocardial infarction, the transcription factor nuclear factor B (NF-B) plays a role in the regulation of cellular function. 15,16 NF-B is involved in inflammatory processes and promotes transcriptions of multiple depressive cytokines. NF-B is a ubiquitous, inducible transcription factor that exists as a latent cytoplasmic form complexed primarily with p50 and p65 bound to inhibitory B (IB) proteins. 15,16 IB proteins consist of several subtypes, but IB is thought to play an essential role. 15,16 Once IB is phosphorylated 16 by any stimuli, it is disassociated from the most common form of the NF-B, and IB permits translocation into the cardiomyocyte nucleus with transcriptional regulation of multiple depressive cytokines, such as tumor necrosis factor. NF-B translocates from the cytosol to the nucleus in a process called NF-B activation.
We hypothesized that increased levels of HSP70, which forms complexes with IB and inhibits its degradation, lead to attenuation of NF-B activation and reduction in myocardial infarct size. We tested our hypothesis in hearts of LPS-treated rats.
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Materials and methods |
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Experimental procedure
Male Sprague-Dawley rats (body weight, 250-300 g) were acclimated in a quarantine room and maintained on a standard pellet diet for 10 days without any stresses. LPS (Escherichia coli, O-127) was purchased from Sigma Chemical Company (St Louis, Mo). The experimental procedure is shown in Figure 1. We established 3 experimental groups: the sham operation group (intraperitoneal saline solution, 2 mL; n = 5); the control group (intraperitoneal saline solution, 2 mL; n = 15); and the LPS group (intraperitoneal LPS, 3 mg/kg; n = 15). At 48 hours after LPS administration, 5 rats each in the control and LPS groups were anesthetized with ketamine (40 mg/kg administered intraperitoneally), and hearts were rapidly excised without ischemia and arrested in cold phosphate-buffered saline solution (PBS). The apex of the left ventricle was cut and prepared in OCT compound (Tissue-Tek; Sakura, Tokyo, Japan) for immunohistochemistry. The remaining left ventricle was frozen in liquid nitrogen for evaluation of the HSP70 and IB protein levels and for detection of HSP70-IB complexes. The remaining 10 rats in the control and LPS groups and 5 rats in the sham group were used in ischemia-reperfusion experiments. Each of the rats was anesthetized with ketamine, intubated, and ventilated. After left thoracotomy, the left anterior descending coronary artery was occluded for 30 minutes and reperfused either for 30 minutes for evaluation of NF-B activation (all groups, n = 5 in each) or for 24 hours for assessment of infarct size (control and LPS groups, n = 5 in each).
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B complexes are present in myocytes, we used neonatal rat cardiomyocytes. The cardiomyocytes were cultured and stimulated with LPS for 48 hours to detect HSP70-IB complexes.
Evaluation of infarct size
Infarct size was evaluated as described previously. 17 After 24 hours of reperfusion and after the 30-minute period of ischemia, hearts were excised and arrested in ice-cold PBS. The left anterior descending coronary artery was ligated, and 1% Evans blue dye solution was injected through the ascending aorta to determine the risk and nonrisk areas. The heart was cut into 2-mm-thick slices, and the second slice from the ligation was stained by means of incubation at 37°C for 20 minutes in 1% triphenyl-tetrazolium-chloride in PBS (pH 7.4). Slices were then scanned with a computerized scanner, and total area, nonrisk area, and infarct area were measured with Scion Image software (Microsoft Corporation, Redmond, Wash).
Preparation of nuclear extracts and electrophoretic mobility shift assays
After 30 minutes of reperfusion and 30 minutes of ischemia, hearts were excised. Nuclear proteins were prepared with a modification of the method described by Dignam and colleagues 18 and Manning and coworkers. 19 In brief, the left ventricle of the risk area was homogenized in 5 mL of buffer A (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 10 mmol/L; MgCl2, 1.5 mmol/L; KCl, 10 mmol/L; dithiothreitol, 1 mmol/L; and phenylmethylsulfonyl fluoride, 1 mmol/L) with a Dounce homogenizer (Bellco Glass, Inc, Vineland, NJ). The samples were centrifuged at 900g for 10 minutes. The pellets were dissolved in 5 mL of buffer A and 0.1% Nonidet P-40 and recentrifuged at 200g for 10 minutes to eliminate unbroken cells. The supernatant was collected and recentrifuged at 900g for 10 minutes. The pellets were washed once with buffer A and purified by using the modified sucrose-gradient method. 20 The pellets were dissolved in 50 µL of buffer B (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 20 mmol/L; glycerol, 25% vol/vol; NaCl2, 420 mmol/L; MgCl2, 1.5 mmol/L; ethylenediamine tetra-acetic acid, 0.2 mmol/L; dithiothreitol, 1 mmol/L; and phenylmethylsulfonyl fluoride, 1 mmol/L), incubated for 45 minutes on ice, and centrifuged at 20,000g for 20 minutes. The supernatant was collected, and the protein concentrations were measured with the bicinchoninic acid method (Amersham Corp, Arlington Heights, Ill).
An electrophoretic mobility shift assay was performed to evaluate NF-B activity, as described previously. 19 Twenty micrograms of each nuclear protein sample was incubated with an end-labeled double-stranded oligonucleotide probe, which had the NF-B consensus sequence 5'-AGT TGA GGG GAG TTT CCC AGG C-3' (Promega Corp, Madison, Wis); [
-32P] adenosine triphosphatase (3000 Ci/mmol, Amersham); and T4 polynucleotide kinase (Amersham) according to the manufacturers' protocols. The DNA protein complexes were separated on 4% nondenaturing polyacrylamide gels, and the gels were vacuum dried and exposed to Image-Plate (Fuji Film, Tokyo, Japan) overnight at room temperature. Specific band intensities were quantified with the FLA 3000 fluoroimage analyzer (Fuji Film) and analyzed with the ImageGauge analyzing system (Version 3.12, Fuji Film).
Western blotting and immunoprecipitation
HSP70 and IB protein levels were examined by means of Western blotting. In brief, a small part of the excised frozen left ventricle was thawed and homogenized in 1.5 mL of buffer A with a Dounce homogenizer. After centrifugation (10,000g for 30 minutes), the supernatants were collected, and the protein concentrations were measured by using the bicinchoninic acid method. Forty micrograms of each sample was separated by means of electrophoresis on a 10% denaturing sodium dodecyl sulfate gel. After electrophoresis, the proteins were transferred electrophoretically to polyvinilidene difluoride membrane (Bio-Rad Laboratories Inc, Hercules, Calif) overnight at 4°C. The membrane was incubated in 2% nonfat dry milk and 0.2% Tween-20 in PBS followed by incubation with specific anti-IB antibody (anti-rabbit polyclonal, C21, Santa Cruz, Calif) or anti-HSP70 antibody (anti-mouse monoclonal, W27; Santa Cruz). After extensive rinsing with Tween-PBS, blots were incubated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibody and developed with the enhanced chemiluminescence system (ECL kit, Amersham). The developed films were scanned with a computer scanner, and the intensities of the bands were measured with Scion Image software (Microsoft).
HSP70-IB complexes were detected by means of immunoprecipitation and Western blotting. In brief, 200 µg of cytosolic extracts was incubated with 20 µL of Protein G-Sepharose (Amersham). After centrifugation (10,000g for 5 seconds), 5 µg of anti-HSP70 antibody was added to the total supernatants. After a 1-hour incubation at 4°C, 20 µL of Protein G-Sepharose was added to each sample. After centrifugation (2000g for 5 minutes), the pellets were collected and washed 5 times with Buffer A. The precipitates were examined by means of Western blotting with anti-IB antibodies. The blots were developed with the ECL system (Amersham).
Immunohistochemistry
Immunofluorescent staining of IB with rhodamine-conjugated anti-rabbit IgG antibody (23828, Polysciences, Inc, Warrington, Pa) was performed as described previously. 21 After staining, immunofluorescent staining of HSP70 with FITC-conjugated anti-mouse IgG antibody (23799, Polysciences) was performed. Immunofluorescent images were obtained with a ZEISS LSM510 laser-scanning confocal microscope (Carl Zeiss Corp, Stuttgart, Germany).
Statistical analysis
Results are expressed as means ± SEM. Data were analyzed by means of analysis of variance. If the analysis of variance showed an overall difference, post hoc comparisons were performed with the Bonferroni-Dunn test for paired or unpaired data, as appropriate.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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B activationB activation after 30 minutes of reperfusion are shown in Figure 4. NF-B bands in the control group were significantly more intense than those in the sham-operated group (2.35 ± 0.17 for the control group and 1.00 ± 0.10 for the sham-operated group, P = .0001). NF-B bands in the LPS group were significantly less intense than those in the control group (1.86 ± 0.21 for the LPS group, P = .0039).
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B protein levelsB protein levels in the cytosol were examined by means of Western blotting (Figure 5). HSP70 levels in the LPS group were higher than those in the control group (1.00 ± 0.07 for the control group and 1.25 ± 0.08 for the LPS group, P = .0469). However, IB levels were similar in both groups (1.00 ± 0.10 for the control group and 0.98 ± 0.17 for the LPS group).
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B immunocomplexesB complexes are visible as IB bands. HSP70-IB complexes were present at detectable levels only in the LPS group (Figure 6, bottom).
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B densities were similar in both the control and LPS groups (Figure 7, A and C), but HSP70 was stained more strongly in samples from the LPS group than in samples from the control group (Figure 7, B and D). A confocal image of a sample from the LPS group is shown (Figure 7, E). Localization of IB (red; Figure 7, C) and HSP70 (green; Figure 7, D) was identical, suggesting the presence of complexes, as shown in yellow in the merged image (Figure 7, E, arrows).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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B activation in rat hearts and cultured myocytes. HSP70 expression was induced by means of LPS pretreatment, and HSP70-IB complexes were formed. These complexes subsequently attenuated NF-B activation. These changes appeared to be associated with a reduction of infarct size in rat hearts after ischemia and reperfusion.
Regarding the method of measuring the risk area of the heart, we used Evans blue dye (molecular weight 960.8 d), according to the method of Yamashita and colleagues. 17 It is criticized that dyes with a small molecule, like Evans blue, used for measuring the risk area might perfuse into the nonperfused area. However, zinc cadmium sulfide microspheres (1- to 10-µm diameter) used for this purpose resulted in a similar risk area to ours. 22
Resistance of cells to various forms of injury is induced by LPS, and this resistance has been studied in the liver, 8 kidney, 9 brain, 10 and myocardium. 11,12 In myocardium LPS pretreatment was reported to reduce infarct size in relation to HSP70 levels 9 and to improve cardiac function after ischemia and reperfusion. 12 Various mechanisms for the protective effects of LPS pretreatment have been proposed: (1) alteration of inflammatory mediators from monocytes and macrophages 23; (2) downregulation of the expression of endothelial cell adhesion receptors, which could account for decreased tissue sequestration of neutrophils 24; (3) effects of the NO system 25; and (4) increased myocardial capillary density. 26 However, the precise mechanism underlying HSP70-associated reductions in infarct size or improvement of cardiac function is not yet well understood.
It is generally agreed that expression of HSP70 increases 24 to 72 hours after cells are exposed to various stimuli, including LPS, 10 heat shock, 5 and ischemic preconditioning. 14 Our previous data showed HSP70 was detected most strongly at 48 hours compared with at 24 or 72 hours by LPS administration (data were not shown). We decided to use the condition of 48 hours. LPS was administrated intraperitoneally at 3 mg/kg, which is among the amounts used by other investigators. 12,27 The levels of HSP70 after LPS pretreatment in our study were consistent with those of other studies.
HSP70 has a chaperone function, folding newly synthesized proteins, transporting them, and degrading damaged proteins. 13 Of these functions, degradation of damaged proteins is particularly important. HSP70 is known to affect the ubiquitin-dependent proteolysis system. 28 Most proteins in the cytosol exist in a soluble form, with hydrophilic amino acids on the outside and hydrophobic amino acids in the core. When these proteins are damaged by various stresses, the molecular structure is changed to expose the hydrophobic residues to the exterior of the protein. The damaged proteins begin to aggregate to form insoluble pellets, and the proteins are subsequently degraded by proteolysis. 28 During this process, HSP70 binds to the hydrophobic surface of the damaged proteins to prevent aggregation. In the reperfused ischemic myocardium, many damaged proteins might be affected by the chaperoning function.
The NF-B activation consists of 2 processes, translocation to the nucleus and binding to DNA, and both of the 2 activation processes are controlled by redox regulation. 15,16 Dissociated IB is polyubiquitinated by ubiquitin ligase and degraded through 26S proteosome proteolysis. 16 Thus, NF-B activation can be inhibited through 2 possible pathways: the reduction of various reactive oxygen species and the prevention of the IB degradation process. Theoretically, HSP70 might affect both pathways; however, to our knowledge, there have been no reports of attenuation of NF-B activation by HSP70. Therefore, we focused our analysis on the pathway in which HSP70 directly affects IB.
In the present study IB protein levels were not significantly different in hearts from control and LPS group animals at 48 hours after administration of LPS. Whether IB levels are increased at 48 hours after LPS administration remains controversial, 29 but our results indicate that the myocardium had attenuated NF-B activation and increased tolerance to ischemia without increased IB levels. These results suggest that mechanisms other than an increase in IB expression might exist. Thus, we hypothesized that HSP70 forms a complex with IB, attenuating NF-B activity, and our experiment revealed formation of HSP70-IB complexes.
How the HSP70-IB complex acts during the process of NF-B activation is still unclear. HSP70 might bind IB to prevent its phosphorylation by IB kinase, or HSP70 might inhibit IB kinase directly. It is also possible that the HSP70-IB complex inhibits the IB kinase indirectly. Although we observed formation of complexes between IB and HSP70, it is still unclear whether HSP70 forms a complex with IB/NF-B dimers or with IB protein during degradation. It is also unclear whether HSP70 affects IB through unknown proteins derived from pathways triggered by extracellular stimuli. Immunoprecipitation with anti-HSP70 antibody and anti-NF-B antibody, Western blotting with anti-phosphorylated IB antibody, and Western blotting with anti-ubiquitin antibody should be investigated to confirm that in vivo inhibition of NF-B translocation during myocardial ischemia-reperfusion injury is secondary to HSP70 interference with IB.
This is the first report of IB stabilization because formation of complexes with HSP70 attenuates NF-B activation. We proposed that there is cytoprotective effect of LPS by delayed preconditioning and a certain mechanism of the protection. As for LPS cytotoxity, the signaling of LPS into the cardiomyocyte and into the myocardial resident macrophage has been well worked out. LPS is typically bound by a circulating LPS binding protein, which typically docks on toll-like receptor 4. The intracellular extension of toll-like receptor 4 binds to an adaptor protein (MYD88), which then links to interleukin 1 receptor-associated kinase, which can activate tumor necrosis factor receptor associated factor. Through a subsequent series of kinase activations, IB is phosphorylated, and NF-B is activated. 30 Compared with studies of LPS cytotoxity, the mechanism of LPS cytoprotection is not elucidated enough. Direct administration of LPS is not allowed clinically, but pharmacologic preconditioning, such as that with monophosphoryl lipid A, is known to induce delayed preconditioning effects, such as LPS. 6 Our results suggest that attenuation of NF-B by the mechanism of HSP70-IB complexes plays a clinically relevant role in the reduction of infarct size.
In conclusion, we show that HSP70 induced by LPS pretreatment forms complexes with IB and inhibits IB degradation and NF-B translocation into the nucleus, resulting in a reduction in myocardial infarct size.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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B as a frequent target for immunosuppressive and anti-inflamatory molecules. Adv Immunol. 1997;95:111-37.B activity. Annu Rev Immunol. 2000;18:621-63.[Medline]B is activated during acute inflamation in vivo in association with elevated endothelial cell adhesion molecule gene expression and leukocyte recruitment. J Inflamm. 1995;45:283-96.[Medline]B- protein promote tolerance to endotoxin. Am J Physiol. 1998;275:H1084-91.B through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J Biol Chem. 1999;274:7611-4.This article has been cited by other articles:
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  J. McGuinness, J. Byrne, C. Condron, J. McCarthy, D. Bouchier-Hayes, and J. M. Redmond Pretreatment with {omega}-3 fatty acid infusion to prevent leukocyte-endothelial injury responses seen in cardiac surgery J. Thorac. Cardiovasc. Surg., July 1, 2008; 136(1): 135 - 141. [Abstract] [Full Text] [PDF]
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 Y. Chen and R. W. Currie Small interfering RNA knocks down heat shock factor-1 (HSF-1) and exacerbates pro-inflammatory activation of NF-{kappa}B and AP-1 in vascular smooth muscle cells Cardiovasc Res, January 1, 2006; 69(1): 66 - 75. [Abstract] [Full Text] [PDF]
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 B. Xu, G.-h. Dong, H. Liu, Y.-q. Wang, H.-w. Wu, and H. Jing Recombinant Human Erythropoietin Pretreatment Attenuates Myocardial Infarct Size: A Possible Mechanism Involves Heat Shock Protein 70 and Attenuation of Nuclear Factor-kappaB Ann. Clin. Lab. Sci., April 1, 2005; 35(2): 161 - 168. [Abstract] [Full Text] [PDF]
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 J. Zhu, A. A. Quyyumi, H. Wu, G. Csako, D. Rott, A. Zalles-Ganley, J. Ogunmakinwa, J. Halcox, and S. E. Epstein Increased Serum Levels of Heat Shock Protein 70 Are Associated With Low Risk of Coronary Artery Disease Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 1055 - 1059. [Abstract] [Full Text] [PDF]
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