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Ann Thorac Surg 1999;68:377-382
© 1999 The Society of Thoracic Surgeons
B in the cardioadaptive response to ischemia
a Division of Cardiothoracic Surgery, Department of Surgery, University of Washington School of Medicine, Seattle, Washington, USA
Address reprint requests to Dr Verrier, Division of Cardiothoracic Surgery, University of Washington, 1959 Pacific Ave NE, Box 356310, Seattle, WA 98195
e-mail: edver{at}u.washington.edu
Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.
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Abstract |
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B mediates IP.
Methods. Rabbits were randomly allocated to one of three groups: (1) 45 minutes of myocardial ischemia followed by 2 hours of reperfusion (I/R); (2) three cycles of 5-minute ischemia and 5 minutes of reperfusion followed by I/R (IP + I/R); or (3) IP in the presence of ProDTC, a specific NF-B inhibitor, followed by I/R (IPProDTC + I/R). Infarct size, indices of regional contractility, and NF-B activation were determined.
Results. In preconditioned rabbits (IP + I/R), infarct size was reduced 83% compared with both I/R alone and IPProDTC + I/R groups (p < 0.05). Throughout reperfusion, preconditioned myocardium showed enhanced regional contractile function compared with I/R andIPProDTC + I/R groups (p < 0.05). Gel shift analysis showed NF-B activation with IP that was blocked by ProDTC. I/R and IPProDTC + I/R groups showed NF-B activation with I/R that was absent in preconditioned animals.
Conclusions. The cytoprotective effects induced by IP require activation of NF-B.
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Introduction |
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Although the phenomenon of ischemic preconditioning is well recognized, the molecular mechanisms mediating this phenomenon are a matter of controversy. Initial rapid metabolic and ionic alterations have been identified in preconditioned myocardium mediated, in part, by adenosine and norepinephrine. This receptor-induced cardioprotection requires a G-protein-mediated signal, activation of K+ATP channels, and localization of protein kinase C to the plasma membrane [2–4]. Cytoprotection during ischemic preconditioning may also require new protein synthesis. Proteins shown to confer protection in IP include endogenous antioxidants and the stress proteins of the heat shock family; however, the intracellular mediators, transcription factors, and specific genes and their products have yet to be definitively identified [2–4].
Recent studies utilizing isolated buffer-perfused rat hearts, suggests that the transcription factor nuclear factor B (NF-B) is involved in ischemic preconditioning of the heart [5]. NF-B is a redox-sensitive transcription factor involved in transcription of proteins in response to mutagenic, oxidative, and hypoxic stress [6]. Under normal physiologic conditions, NF-B is held inactive in the cytoplasm by the inhibitory subunit, IB
[7]. Under conditions of oxidative stress, NF-B disassociates from IB, and translocates to the nucleus, where it initiates the transcription of proinflammatory, procoagulant, and vasoactive genes [8]. In contrast to the role NF-B activation has in the destructive events of inflammation, NF-B also mediates the expression of cytoprotective proteins that block apoptosis or inhibit inflammation in response to several types of cellular stress [9]. In a negative feedback manner, these cytoprotective proteins inhibit NF-B [10]. Because the cardioadaptive response to ischemia has been shown to involve both new protein synthesis as well as oxidative stress, we have postulated that the redox sensitive transcription factor NF-B is involved in IP.
In the present study, we demonstrate activation of NF-B during IP and inhibition of NF-B activation during ischemia and reperfusion after IP in rabbit hearts. Prolinedithiocarbamate (ProDTC) blocked NF-B activation during IP, and reversed the inhibition of NF-B activation and cytoprotection during I/R. These results suggest that NF-B plays an essential role in the myocardial adaptive response to ischemia.
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Material and methods |
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Experimental preparation
Rabbits weighing 3 to 4 kg were anesthetized with an initial intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). The rabbits were endotracheally intubated and maintained on inhaled halothane (1%–2%) anesthesia in 100% O2 using a small animal respiratory (Model 607; Harvard Apparatus Co, Inc, Dover, MA). Respiratory rate and tidal volume were adjusted to maintain arterial blood gases within normal physiologic range. The rabbits were kept warm using an electric warm blanket. Lactated Ringer injection was infused via ear vein at 5 cc/kg/h as a liquid support. A 20-gauge flexible catheter was placed in the left carotid artery to measure heart rate (HR), blood pressure (BP), and mean arterial pressure (MAP). The heart was exposed via median sternotomy. A 4.0 Proline suture was passed twice around a large anterolateral branch of the left main coronary artery supplying the majority of the left ventricle, and the ends of the suture were passed through a small length of polyethene tubing to form a snare. Ischemia and reperfusion were induced by tightening and releasing the snare. Snaring of the artery caused epicardial cyanosis and regional hypokinesis within 10 s. Reperfusion was confirmed by conspicuous blushing of the previous ischemic myocardium. Three sonometric piezoelectric crystals were embedded in the area at risk of the left ventricle (Sonometric Corp, Ontario, Canada). Two crystals were embedded in the subepicardium 5–7 mm apart, and the third crystal was placed on the endocardium, tangential to the two epicardial crystals. Differences in distance between the epicardial and endocardial crystal measured changes in wall thickness. Signals from the crystals were monitored and recorded.
Experimental protocol
The time course of preconditioning and I/R injury for this study is depicted in Figure 1. After a 20–30-minute stabilization period, regional myocardial function baseline values were recorded. Preconditioning was initiated performing three cycles of 5-minute regional ischemia followed by 5 minutes of reperfusion. After an additional 2-hour stabilization period without ischemia, hearts underwent 45 minutes of ischemia followed by 120 minutes of reperfusion (I/R). Ten minutes before IP, preconditioned (IP + I/R) animals (n = 6) were administered 1 mL phosphate-buffered saline (PBS) followed by an infusion of 30 mL PBS, to be run throughout IP only. In the preconditioned plus ProDTC group (IPProDTC + I/R), animals (n = 6) were administered 15 mg/kg of ProDTC (provided by Norbert Frank, PhD, German Cancer Research Center, Heidelberg, Germany) dissolved in 1 mL PBS, followed by an infusion of ProDTC dissolved in 30 mL PBS (15 mg/kg/h), to be run throughout IP only. In the nonpreconditioned (I/R only) group, the animals (n = 6) underwent prolonged I/R only, preceded by the same bolus and infusion of PBS as the preconditioned group. Functional measurements were made at 30 and 45 minutes of ischemia and at 15, 30, 60, and 120 minutes of reperfusion. After 120 minutes of reperfusion, hearts were rapidly excised, and the myocardial tissue was processed for calculation of infarct size. An additional 18 animals were utilized for detection of NF-B activation. Hearts (n = 3) from each experimental group were excised at two time points: immediately after IP, and at the end of the prolonged I/R period.
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Determination of infarct size
At the completion of the 120-minute reperfusion period, the coronary artery was reoccluded. Six milliliters of 20% Evan’s Blue dye (Sigma, St. Louis, MO) was injected into the left atrium, and allowed to circulate in order to stain all perfused tissue. The area supplied by the ligated vessel remained unstained, demarcating the myocardium at risk for infarction. After the heart was rapidly excised, the left ventricle (LV) was isolated from the rest of the heart, weighed, and then cut into 2-mm-thick transverse slices. The normal myocardium (stained blue) was separated from the area at risk (unstained). The area at risk was placed in a 37°C solution of 1% triphenyl-tetrazolium chloride (TTC) for 30 minutes. TTC stains the viable tissue brick red, leaving the necrotic zone pale. The TTC-red (noninfarcted) tissue was separated from the TTC-pale (necrotic) and each area was weighed. The left ventricular area at risk (LVAR) was calculated as the sum of the noninfarcted and necrotic tissue. The LVAR was then expressed as a percentage of the total weight of the left ventricle. The infarct size (IS) was calculated by dividing the weight of the TTC-pale tissue by the weight of the total area at risk.
Nuclear protein extractions
Hearts were excised as described in "Experimental Protocol." Nuclear protein extracts were prepared from tissue by the method of Deryckere and Gannon [11]. Aliquots of frozen tissue were mixed with liquid nitrogen and ground to powder using a mortar and pestle. Four milliliters of solution A (0.6% Nonidet P-40, 150 mM NaCl, 10 mmol/L Hepes, pH 7.9, 1 mM EDTA, and 0.5 mM PMSF) was added to the mortar. The contents of the mortar were placed in a Dounce tissue homogenizer (Kontes Co, Vineland, NJ), and the cells were lysed with five strokes of the pestle. After transfer to a 15-mL tube, debris was pelleted by centrifuging at 2000 rpm for 30 seconds. The supernatant containing intact nuclei was transferred to 50 mL Corex tubes, incubated on ice for 5 minutes, and centrifuged for 10 minutes at 5000 rpm. Nuclear pellets were then resuspended in 300 mL of solution B (25% glycerol, 20 mmol/L Hepes, pH 7.9, 420 mmol/L NaCl, 1.2 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, 2 mmol/L benzamidine, 0.5 mg/mL pepstatin, 0.5 mg/mL leupeptin, and 0.5 mg/mL aprotinin) and incubated on ice for 30 minutes. The mixture was then transferred to microcentrifuge tubes, and nuclei were pelleted by centrifugation at 14,000 rpm for 1 minute. Supernatants containing nuclear proteins were saved, aliquoted, and stored at -70°C. Protein quantitation was performed using the Bradford assay [12].
Electrophoretic mobility shift assays
An oligonucleotide containing the consensus sequence motif for NF-B binding, 5'-GTTGAGGGGACTTTCCCAGGC-3' (Promega, Madison, WI), was end-labeled with [
32P]ATP (Amersham, Arlington Heights, IL) using polynucleotide kinase. Twenty-microgram samples of nuclear protein extracts were incubated for 20 minutes at 25°C with the 32P-end-labeled, double-stranded oligonucleotide probe, subjected to electrophoresis in native 6% polyacrylamide gels, and autoradiographed. Cold competition was performed by adding 50-fold molar excess of specific unlabeled double-stranded probe to the reaction mixture for 20 minutes before the addition of the 32P-end-labeled oligonucleotide probe.
Statistical analysis
Mean values and standard error of the mean (SEM) are shown. The data analysis was performed using SPSS for Windows, version 6.1. Infarct size statistical differences between groups was analyzed utilizing independent samples t test. For each of the treatment groups, several parameters (expressed as continuous variables) were studied at the indicated time points. To determine if a statistically significant change had occurred compared with the baseline values, their parameters were compared with that value using paired t tests. For intergroup comparisons, the mean change (from baseline) for each of these parameters was analyzed at the specific time points using analysis of variance. Mann-Whitney analysis of variance was utilized for post hoc comparisons. Values of p 
0.05 were considered statistically significant.
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Results |
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B activation during IP and I/R, nuclear proteins from cells in the area at risk were isolated and analyzed by EMSA. Ischemic preconditioning alone resulted in NF-B activation (Fig 4, lane 2), which resolved within the 2-hour period preceding I/R (data not shown). ProDTC blocked NF-B activation during IP (Fig 4, lane 1). In preconditioned animals, NF-B activation was absent during I/R, whereas NF-B activation occurred during I/R in animals preconditioned in the presence of ProDTC (Fig 5, lane 4) in an equivalent amount to those receiving I/R alone. Both groups in which NF-B was activated during I/R (IPProDTC + I/R and I/R alone) showed increased necrosis and regional contractile dysfunction as compared with the preconditioned animals in which NF-B was inhibited.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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B inhibitor, suggesting that the redox-sensitive transcription factor NF-B may have an essential role in the cardioprotective effects of IP. Gel shift analysis of nuclear proteins show NF-B activation during IP, and inhibition of NF-B activation during IP by ProDTC. Furthermore, NF-B activation during I/R is blocked by IP. Ischemic preconditioning-associated inhibition of NF-B activation during I/R is reversed when IP is carried out in the presence of ProDTC. Thus, NF-B activation during IP appears to result in changes in the cell that prevent NF-B activation during I/R.
Tissue injury induced by I/R occurs secondary to an acute inflammatory reaction induced by activation of various types of cells within the heart. For example, endothelial cell (EC) activation is characterized by the expression of proinflammatory (eg, E-selectin, P-selectin, ICAM-1, and VCAM-1), procoagulant (tissue factor, plasminogen activator inhibitor-1), and vasoactive genes (eg, nitric oxide synthases) that result in microvascular thrombosis and neutrophil-mediated tissue necrosis characteristic of I/R. Inhibition of many of the proteins with specific monoclonal antibodies attenuates myocardial ischemia-reperfusion injury [1]. Transcription in EC of genes encoding proinflammatory, procoagulant, and vasoactive proteins is controlled, in part, by NF-B [13]. During oxidative stress, EC NF-B activation in the vasculature of the heart may contribute substantially to the destructive inflammatory reactions associated with myocardial I/R injury.
The seemingly paradoxical effects of NF-B during IP and I/R are explained by recent studies that suggest a role for NF-B for the expression of cytoprotective proteins [9]. NF-B activation in EC mediates the expression of proteins that block cell death (apoptosis). These include Bcl family members, Bcl-2, Bcl-XL, and A1; the zinc finger protein, A20; and the endogenous antioxidants manganese superoxide dismutase (mSOD) and heme-oxygenase-1 (HO). In addition to their antiapoptotic properties, these proteins also serve an antiinflammatory role in their ability to inhibit NF-B activation [9]. Thus, during IP, NF-B activation may result predominately in the accumulation of several cytoprotective proteins that protect the cardiomyocyte from subsequent inflammatory damage induced by I/R. In the setting of I/R without preconditioning, however, NF-B activation results predominately in the expression of proinflammatory genes (Fig 5). Our results suggest that acquisition of cytoprotection associated with NF-B activation during IP results in suppression of NF-B during I/R.
Ischemic preconditioning in our study also may be mediated by an increase in expression of heat shock proteins (HSP). HSPs are a family of molecular chaperones that block protein degradation and aggregation during periods of cellular environmental and physiologic stress. In preconditioned cells, the tolerance that evolves is temporally correlated with the appearance of the HSPs [14]. Overexpression of HSP70 confers myocardial protection, as observed by resistance to myocardial ischemic stress and reperfusion damage [15]. Recent evidence suggests that certain HSPs may inhibit NF-B. HSP70 blocks inducible nitric oxide synthase expression by binding NF-B in the cytosol, preventing its translocation to the nucleus [16]. In cultured rat hepatocytes and murine lung epithelium, stress protein induction also inhibits NF-B nuclear translocation and subsequent inducible nitric oxide synthase (iNOS) promoter activation [17]. Finally, exposure to prostanoids and hyperthermia not only leads to activation of the HSP transcription factor (HSF1) but also to inhibition of NF-B [18]. Therefore, the cytoprotective effect of the heat shock response may be amplified by rendering cells unresponsive to proinflammatory signals through inhibition of NF-B.
We utilized ProDTC to inhibit NF-B activation and to examine the role of NF-B in ischemic preconditioning. ProDTC is a derivative of a family of dithiocarbamates that also include pyrrolidine dithiocarbamate (PDTC). Pyrrolidine dithiocarbamate reversibly suppresses the release of the inhibitory protein, IB, from NF-B in the cytoplasm, thereby preventing NF-B activation [19]. We have demonstrated in vitro that ProDTC, like PDTC, inhibits oxidative stress-induced NF-B activation in cultured human umbilical vein endothelial cells, and we have demonstrated a reduction of infarct size in an in vivo model of I/R by inhibition of NF-B with dithiocarbamates (data not shown). Although the chemical basis for the inhibitory effect of dithiocarbamate derivatives on NF-B seems to be dependent on an oxygen radical scavenging effect, the exact mechanism of dithiocarbamate-mediated inhibition of NF-B remains to be elucidated.
PDTC was first suggested for use as an anti-AIDS therapy with the intention of blocking NF-B activation by oxygen radicals. A limiting pharmacological feature of PDTC has been its prominent toxicity in vivo, which may preclude the use of PDTC clinically. Replacement of the pyrrolidine derivative with an amino acid-based, six-membered ring to form ProDTC may substantially reduce toxicity in vivo [20]. Throughout the study, we observed no indication of adverse effects of ProDTC such as hemodynamic collapse, myocardial dysfunction, or acidosis. In this study, we did not examine potential toxicity of ProDTC, but the lack of significant differences in hemodynamic parameters between treated and control animals would support a conclusion that ProDTC toxicity did not confound our results.
In summary, utilizing an in vivo model of I/R, we show that inhibition of NF-B during IP reverses the cytoprotective effect of IP during subsequent I/R. Molecular analyses of preconditioned myocardium show that IP activates NF-B. In preconditioned myocardium, NF-B is not activated during I/R injury. Understanding the molecular mechanisms mediating IP may allow us to mimic this potent endogenous adaptive response through genetic or pharmacological means, thereby providing valuable cytoprotection in settings where I/R injury is encountered.
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Acknowledgments |
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B in preventing TNF-a induced cell death. Science 1996;274:782-784.B dependent mechanism. J Biol Chem 1996;271:18068-18073.B and IkBa. J Exp Med 1994;179:503-508.B activation. J Biol Chem 1996;271:17224-17232.
B degradation and NF-B nuclear translocation. Biochem Biophys Res Commun 1997;231:257-263.[Medline]
B activation in intact cells. J Exp Med 1992;175:1181-1194.This article has been cited by other articles:
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 S. Frantz, J. Bauersachs, and G. Ertl Post-infarct remodelling: contribution of wound healing and inflammation Cardiovasc Res, February 15, 2009; 81(3): 474 - 481. [Abstract] [Full Text] [PDF]
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