Neutralization of Interleukin-18 Ameliorates Ischemia/Reperfusion-induced Myocardial Injury
2009; Elsevier BV; Volume: 284; Issue: 12 Linguagem: Inglês
10.1074/jbc.m808824200
ISSN1083-351X
AutoresKaliyamurthi Venkatachalam, Sumanth D. Prabhu, V. Seenu Reddy, William H. Boylston, Anthony J. Valente, Bysani Chandrasekar,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoIschemia/reperfusion (I/R) injury is characterized by the induction of oxidative stress and proinflammatory cytokine expression. Recently demonstrating that oxidative stress and TNF-α each stimulate interleukin (IL)-18 expression in cardiomyocytes, we hypothesized that I/R also induces IL-18 expression and thus exacerbates inflammation and tissue damage. Neutralization of IL-18 signaling should therefore diminish tissue injury following I/R. I/R studies were performed using a chronically instrumented closed chest mouse model. Male C57BL/6 mice underwent 30 min of ischemia by LAD coronary artery ligation followed by various periods of reperfusion. Sham-operated or ischemia-only mice served as controls. A subset of animals was treated with IL-18-neutralizing antibodies 1 h prior to LAD ligation. Ischemic LV tissue was used for analysis. Our results demonstrate that, compared with sham operation and ischemia alone, I/R significantly increased (i) oxidative stress (increased MDA/4-HNE levels), (ii) neutrophil infiltration (increased MPO activity), (iii) NF-κB DNA binding activity (p50, p65), and (iv) increased expression of IL-18Rβ, but not IL-18Rα or IL-18BP transcripts. Administration of IL-18-neutralizing antibodies significantly reduced I/R injury measured by reduced infarct size (versus control IgG). In isolated adult mouse cardiomyocytes, simulated ischemia/reperfusion enhanced oxidative stress and biologically active IL-18 expression via IKK-dependent NF-κB activation. These results indicate that IL-18 plays a critical role in I/R injury and thus represents a promising therapeutic target. Ischemia/reperfusion (I/R) injury is characterized by the induction of oxidative stress and proinflammatory cytokine expression. Recently demonstrating that oxidative stress and TNF-α each stimulate interleukin (IL)-18 expression in cardiomyocytes, we hypothesized that I/R also induces IL-18 expression and thus exacerbates inflammation and tissue damage. Neutralization of IL-18 signaling should therefore diminish tissue injury following I/R. I/R studies were performed using a chronically instrumented closed chest mouse model. Male C57BL/6 mice underwent 30 min of ischemia by LAD coronary artery ligation followed by various periods of reperfusion. Sham-operated or ischemia-only mice served as controls. A subset of animals was treated with IL-18-neutralizing antibodies 1 h prior to LAD ligation. Ischemic LV tissue was used for analysis. Our results demonstrate that, compared with sham operation and ischemia alone, I/R significantly increased (i) oxidative stress (increased MDA/4-HNE levels), (ii) neutrophil infiltration (increased MPO activity), (iii) NF-κB DNA binding activity (p50, p65), and (iv) increased expression of IL-18Rβ, but not IL-18Rα or IL-18BP transcripts. Administration of IL-18-neutralizing antibodies significantly reduced I/R injury measured by reduced infarct size (versus control IgG). In isolated adult mouse cardiomyocytes, simulated ischemia/reperfusion enhanced oxidative stress and biologically active IL-18 expression via IKK-dependent NF-κB activation. These results indicate that IL-18 plays a critical role in I/R injury and thus represents a promising therapeutic target. For both men and women, ischemic heart disease is one of the leading causes of death in the United States today, and its pathobiology has been attributed to many factors, including proinflammatory cytokines. Interleukin (IL) 2The abbreviations used are: IL, interleukin; IL-18BP, interleukin-18 binding protein; IL-18R, interleukin-18 receptor; AN, area of necrosis; AR, area at risk; c/EBP, CAAT/enhancer-binding protein; dnIKKβ, dominant negative IKKβ; dnIκBα, dominant negative IκBα; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; IFN, interferon; IκB, inhibitory κB; IKK, IκB kinase; I/R, ischemia/reperfusion; sI/R, simulated I/R; LAD, left anterior descending; LPS, lipopolysaccharide; LV, left ventricle; MDA, malondialdehyde; 4-HNE, 4-hydroxyalkenals; MOI, multiplicity of infection; MPO, myeloperoxidase; NF-κB, nuclear factor κB; PDTC, pyrrolidine dithiocarbamate; RT, reverse transcription; qPCR, quantitative polymerase chain reaction; TNF, tumor necrosis factor; TTC, 2,3,5-triphenyltetrazolium chloride; PBS, phosphate-buffered saline; nt, nucleotides; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; rRNA, ribosomal RNA; JNK, c-Jun N-terminal kinase; ROS, reactive oxygen species. 2The abbreviations used are: IL, interleukin; IL-18BP, interleukin-18 binding protein; IL-18R, interleukin-18 receptor; AN, area of necrosis; AR, area at risk; c/EBP, CAAT/enhancer-binding protein; dnIKKβ, dominant negative IKKβ; dnIκBα, dominant negative IκBα; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; IFN, interferon; IκB, inhibitory κB; IKK, IκB kinase; I/R, ischemia/reperfusion; sI/R, simulated I/R; LAD, left anterior descending; LPS, lipopolysaccharide; LV, left ventricle; MDA, malondialdehyde; 4-HNE, 4-hydroxyalkenals; MOI, multiplicity of infection; MPO, myeloperoxidase; NF-κB, nuclear factor κB; PDTC, pyrrolidine dithiocarbamate; RT, reverse transcription; qPCR, quantitative polymerase chain reaction; TNF, tumor necrosis factor; TTC, 2,3,5-triphenyltetrazolium chloride; PBS, phosphate-buffered saline; nt, nucleotides; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; rRNA, ribosomal RNA; JNK, c-Jun N-terminal kinase; ROS, reactive oxygen species.-18 is a pleiotropic cytokine belonging to the IL-1 family (1Okamura H. Tsutsi H. Komatsu T. Yutsudo M. Hakura A. Tanimoto T. Torigoe K. Okura T. Nukada Y. Hattori K. Akita K. Namba M. Tanabe F. Konishi K. Fukuda S. Kurimoto M. Nature. 1995; 378: 88-91Crossref PubMed Scopus (2382) Google Scholar, 2Gracie J.A. Robertson S.E. McInnes I.B. J. Leukocyte Biol. 2003; 73: 213-224Crossref PubMed Scopus (607) Google Scholar, 3Dinarello C.A. Novick D. Puren A.J. Fantuzzi G. Shapiro L. Muhl H. Yoon D.Y. Reznikov L.L. Kim S.H. Rubinstein M. J. Leukocyte Biol. 1998; 63: 658-664Crossref PubMed Scopus (331) Google Scholar, 4Wang M. Markel T.A. Meldrum D.R. Shock. 2008; 30: 3-10Crossref PubMed Scopus (162) Google Scholar, 5Arend W.P. Palmer G. Gabay C. Immunol. Rev. 2008; 223: 20-38Crossref PubMed Scopus (634) Google Scholar), whose expression is up-regulated in numerous immune, infectious, and inflammatory conditions (1Okamura H. Tsutsi H. Komatsu T. Yutsudo M. Hakura A. Tanimoto T. Torigoe K. Okura T. Nukada Y. Hattori K. Akita K. Namba M. Tanabe F. Konishi K. 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Circulating IL-18 levels have been shown to be independent predictors of coronary events in humans, with increased basal levels of IL-18 observed in individuals who later developed coronary events (7Hernesniemi J.A. Karhunen P.J. Rontu R. Ilveskoski E. Kajander O. Goebeler S. Viiri L.E. Pessi T. Hurme M. Lehtimaki T. Atherosclerosis. 2008; 196: 643-649Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Increased circulating IL-18 levels have also been measured during heart failure as well as in stroke patients (8Yuen C.M. Chiu C.A. Chang L.T. Liou C.W. Lu C.H. Youssef A.A. Yip H.K. Circ. J. 2007; 71: 1691-1696Crossref PubMed Scopus (45) Google Scholar, 9Hulthe J. McPheat W. Samnegard A. Tornvall P. Hamsten A. Eriksson P. Atherosclerosis. 2006; 188: 450-454Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).IL-18 signals via the IL-18 receptor, a heterodimer consisting of a ligand binding α-subunit and a signal-transducing β-subunit (10Born T.L. 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Interestingly, transgenic mice overexpressing human IL-18BP exhibit reduced inflammation and disease severity (13Fantuzzi G. Banda N.K. Guthridge C. Vondracek A. Kim S.H. Siegmund B. Azam T. Sennello J.A. Dinarello C.A. Arend W.P. J. Leukocyte Biol. 2003; 74: 889-896Crossref PubMed Scopus (43) Google Scholar).Cytotoxic free radicals and proinflammatory cytokines are produced during ischemia/reperfusion (I/R) injury. Using isolated adult rat cardiomyocytes, we have recently described NF-κB-dependent IL-18 and IL-18Rβ expression following hydrogen peroxide and TNF-α treatment in vitro (14Chandrasekar B. Colston J.T. de la Rosa S.D. Rao P.P. Freeman G.L. Biochem. Biophys. Res. Commun. 2003; 303: 1152-1158Crossref PubMed Scopus (80) Google Scholar). Moreover, although IL-18BP gene expression under basal conditions in these cells is undetectable, TNF-α and H2O2 each induce IL-18BP mRNA in a delayed but persistent manner (14Chandrasekar B. Colston J.T. de la Rosa S.D. Rao P.P. Freeman G.L. Biochem. Biophys. Res. Commun. 2003; 303: 1152-1158Crossref PubMed Scopus (80) Google Scholar), which suggests tight regulation of IL-18 bioavailability. However, the precise role of IL-18 in I/R injury and its regulation in vivo are incompletely understood, and the signal transduction pathways involved in its induction in myocardial constituent cells in vitro have not been identified.The purpose of this study was to (i) determine the temporal expression of IL-18, IL-18 receptors, and IL-18BP during I/R in vivo, (ii) define the causal role of IL-18 in myocardial I/R injury in vivo, and (iii) examine the signaling pathways involved in simulated I/R (sI/R)-mediated IL-18 induction in isolated adult mouse cardiomyocytes in vitro. Our results demonstrate that (i) IL-18 is induced in vivo in postischemic myocardium, (ii) its neutralization blunts I/R-induced tissue injury, and (iii) I/R up-regulates IL-18Rβ and IL-18BP but not IL-18Rα expression. Further, sI/R enhances biologically active IL-18 expression in primary adult mouse cardiomyocytes via IKK-dependent IκB degradation and NF-κB activation. Together, these results indicate that IL-18 plays a critical role in ischemia/reperfusion injury and thus is a potential therapeutic target for ischemic heart disease.EXPERIMENTAL PROCEDURESAnimals-All animal studies conformed to National Institutes of Health guidelines (48National Research Council Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D. C.1996Google Scholar), and were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center (San Antonio, TX). I/R studies were performed using a chronically instrumented closed chest mouse model (15Nossuli T.O. Frangogiannis N.G. Knuefermann P. Lakshminarayanan V. Dewald O. Evans A.J. Peschon J. Mann D.L. Michael L.H. Entman M.L. Am. J. Physiol. 2001; 281: H2549-H2558PubMed Google Scholar, 16Nossuli T.O. Lakshminarayanan V. Baumgarten G. Taffet G.E. Ballantyne C.M. Michael L.H. Entman M.L. Am. J. Physiol. 2000; 278: H1049-H1055PubMed Google Scholar, 17Colston J.T. de la Rosa S.D. Koehler M. Gonzales K. Mestril R. Freeman G.L. Bailey S.R. Chandrasekar B. Am. J. Physiol. 2007; 293: H1839-H1846Crossref PubMed Scopus (94) Google Scholar). In brief, male C57BL/6 mice weighing ∼25–30 g and aged 3–4 months were anesthetized by urethane (1000 mg/kg intraperitoneally) and etomidate (25 mg/kg intraperitoneally) and mechanically ventilated with a rodent ventilator set at 150 breaths/min (100% oxygen). Mice were placed on a heated, temperature-controlled operating table for small animals (Vestavia Scientific). Using microscopic dissection (surgical microscope system; Applied Fiberoptics), the chest was opened along the left side of the sternum by cutting through the ribs to approximately midsternum, and the chest walls were retracted with 6-0 sutures. The pericardium was then gently dissected to allow visualization of coronary artery anatomy. An 8-0 Surgipro monofilament polypropylene suture with the U-shaped tapered needle was passed under the LAD. The needle was then cut from the suture, and the two ends of the 8-0 suture were then threaded through a 0.5-mm piece of PE-10 tubing that was previously soaked in 100% alcohol overnight, forming a loose snare around the LAD coronary artery. Each end of the suture was then exteriorized through each side of the chest wall. The chest was then closed with four interrupted stitches utilizing 6-0 sutures. The ends of the exteriorized 8-0 suture were then tucked under the skin, which was then also closed with 6-0 sutures. At 1 week after instrumentation, the animals were reanesthetized and randomly assigned to sham or I/R groups. The 8-0 suture, which had been previously exteriorized, was cleared of all debris from the skin and chest and carefully taped to heavy metal picks. Ischemia of LAD coronary artery was accomplished by gently pulling the heavy metal picks apart until an S-T segment elevation appeared on the electrocardiogram. The electrocardiogram was constantly monitored throughout the entire ischemic interval to ensure persistent ischemia. After 30 min, reperfusion was accomplished by pushing the metal picks toward the animal and cutting the suture close to the chest wall. Reperfusion was confirmed by resolution of the S-T segment elevation, which usually occurred very quickly. An additional four animals underwent only ischemia (30 min of ischemia and no reperfusion). Sham-operated animals were prepared identically without undergoing the I/R protocol. At the indicated time points (Fig. 1A), the hearts were rapidly excised and rinsed in ice-cold physiological saline. The right ventricle and atria were trimmed away, and the left ventricle was divided into ischemic and nonischemic zones and snap frozen in liquid N2.Adult Mouse Cardiomyocytes-Calcium-tolerant adult mouse ventricular myocytes were isolated by modified Langendorff perfusion and collagenase digestion, adapting the methodology described by the Alliance for Cellular Signaling (18Sambrano G.R. Fraser I. Han H. Ni Y. O'Connell T. Yan Z. Stull J.T. Nature. 2002; 420: 712-714Crossref PubMed Scopus (73) Google Scholar) (available on the World Wide Web). In brief, mice were given heparin (1000 units/kg intraperitoneally) and then deeply anesthetized with intramuscular ketamine (43.5 mg/kg), acepromazine (1.5 mg/kg), and xylazine (1.7 mg/kg). Median sternotomy was performed, and the heart was rapidly excised and rinsed with physiologic saline. The aortic lumen was isolated and tied to an 18-gauge cannula, and the heart was perfused with oxygenated (95% O2, 5% CO2), Ca2+-free modified Tyrode's bicarbonate buffer (126 mm NaCl, 4.4 mm KCl, 1 mm MgCl2, 18 mm NaHCO3, 11 mm glucose, 4 mm HEPES, 10 mm 2,3-butanedione monoxime, 30 mm taurine, pH 7.35) at 37 °C for 5 min. Following this, the heart was perfused with 50 ml of digestion buffer (modified Tyrode's bicarbonate buffer with 0.25 mg/ml Liberase Blendzyme type 1 (Roche Applied Science), 0.14 mg/ml trypsin (Sigma), and CaCl2 2.5 μm) in a recirculating fashion for 12–15 min. The heart was then removed, and the left ventricle (LV) was separated and dissected with small, blunt forceps in 2–3 ml of digestion buffer. The minced tissue suspension was gently agitated by repeated pipette aspiration and transferred into myocyte stopping buffer 1 (modified Tyrode's bicarbonate buffer with 10% fetal calf serum and 12.5 μm CaCl2) in a 50-ml conical tube and allowed to sediment for 10 min. The supernatant was transferred to another tube and centrifuged at 90 × g for 2 min. The sediment/pellet from both tubes were combined and resuspended in myocyte stopping buffer 2 (modified Tyrode's bicarbonate buffer with 5% fetal calf serum and 12.5 μm CaCl2) in a 100-mm culture dish. Small aliquots of CaCl2 were then added in a graded fashion at 4-min intervals to sequentially increase the Ca2+ concentration to 500 μm (five total steps). The suspension was then placed in a 50-ml conical tube and allowed to sediment for 10 min at 22 °C. As above, the supernatant was transferred to another tube and gently centrifuged at 90 × g for 2 min, and myocytes contained in both the sediment and the pellet were combined and resuspended in minimal essential medium (pH 7.35–7.45; catalogue number M1018; Sigma) containing 1.2 mm Ca2+, 12 mm NaHCO3, 2.5% fetal bovine serum, and 1% penicillin/streptomycin. The cells were then plated onto 35-mm cell culture dishes precoated with 20 μg/ml mouse laminin in phosphate-buffered saline with 1% penicillin/streptomycin for 1 h. Cardiomyocytes were maintained under resting conditions in the incubator for at least 16 h before experimentation. Ischemia/reperfusion was simulated by incubating cardiomyocytes 30 min in an “ischemia buffer” containing 118 mm NaCl, 24 mm NaHCO3, 1.0 mm NaH2PO4, 2.5 mm CaCl2, 1.2 mm MgCl2, 20 mm sodium lactate, 16 mm KCl, 10 mm 2-deoxyglucose (pH adjusted to 6.2), followed by reoxygenation for 4 h. Reoxygenation was accomplished by replacing the ischemic buffer with normal medium under normoxic conditions. All of the studies were completed within 72 h after isolation.Cardiomyocytes were treated with PDTC (100 μm in PBS) for 1 h prior to undergoing sI/R. IKK was targeted by SC-514 (10 μm in DMSO for 1 h). In addition, IKKβ and IκB-α were targeted using adenoviral dominant negative IKKβ or IκBα (dnIKKβ or dnIκBα, respectively), as described previously (19Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Ad.GFP served as a control. Transfection efficiency of cardiomyocytes with these adenoviral vectors is near 100%. 24 h after infection, cells underwent sI/R.Splenocyte Isolation-Male C57BL/6 mice weighing ∼25–30 g and aged 3–4 months were sacrificed by cervical dislocation. The spleens from three animals were pooled and homogenized in a Dounce homogenizer in RPMI1640 plus 10% fetal calf serum. The cell suspension was then carefully overlaid on His-topaque 1083 (Sigma), centrifuged at 800 × g for 20 min at 22 °C, and the splenic mononuclear cells were carefully collected from the media/Ficoll interface and resuspended in RPMI 1640 plus 10% fetal calf serum. Splenocytes (5 × 106/ml) were incubated with and without cardiomyocyte-derived culture supernatants following simulated I/R and LPS (1 μg/ml) for 24 h at 37 °C. After incubation, supernatants were sampled and analyzed for IFN-γ production by ELISA (n = 6).Adenoviral Transduction-Replication-deficient recombinant adenovirus encoding green fluorescent protein (Ad.GFP), dnIKKβ (Ad.dnIKKβ), and phosphorylation-deficient IκB-α (S32A/S36A; Ad.dnIκB-α) have all been described before (19Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 20Reddy V.S. Harskamp R.E. van Ginkel M.W. Calhoon J. Baisden C.E. Kim I.S. Valente A.J. Chandrasekar B. J. Cell. Physiol. 2008; 215: 697-707Crossref PubMed Scopus (55) Google Scholar, 21Patel D.N. King C.A. Bailey S.R. Holt J.W. Venkatachalam K. Agrawal A. Valente A.J. Chandrasekar B. J. Biol. Chem. 2007; 282: 27229-27238Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Cardiomyocytes were infected with adenoviruses at a multiplicity of infection (MOI) of 100 at ambient temperature as described previously (19Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 20Reddy V.S. Harskamp R.E. van Ginkel M.W. Calhoon J. Baisden C.E. Kim I.S. Valente A.J. Chandrasekar B. J. Cell. Physiol. 2008; 215: 697-707Crossref PubMed Scopus (55) Google Scholar, 21Patel D.N. King C.A. Bailey S.R. Holt J.W. Venkatachalam K. Agrawal A. Valente A.J. Chandrasekar B. J. Biol. Chem. 2007; 282: 27229-27238Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). After 1 h, the adenovirus-containing medium was replaced with medium containing 0.5% bovine serum albumin. 24 h later, cardiomyocytes underwent ischemia/reoxygenation as described above. Adenoviral transduction did not result in cardiomyocyte death (data not shown).Lipid Peroxidation-Ischemic zones were assessed for lipid peroxidation (22Chandrasekar B. Colston J.T. Geimer J. Cortez D. Freeman G.L. Free Radic. Biol. Med. 2000; 28: 1579-1588Crossref PubMed Scopus (25) Google Scholar). Left ventricular tissue from sham-operated animals at 2.5 h served as controls. Frozen tissues were homogenized in 250 μl of ice-cold 20 mm Tris-HCl (pH 7.4). After centrifugation, 200 μl of supernatant were analyzed for lipid peroxidation products (malondialdehyde (MDA)/4-hydroxyalkenals (4-HNE)) using a lipid peroxidation assay kit (Calbiochem).Detection of Intracellular ROS-Intracellular ROS levels were visualized using the cell-permeable, redox-sensitive fluorophore, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Molecular Probes, Inc., Eugene, OR). Prior to undergoing simulated ischemia or ischemia/reoxygenation for the indicated time periods, cardiomyocytes were incubated in medium containing 30 μm non-fluorescent DCFH-DA for 1 h to obtain stable intracellular levels of the probe. Similar concentrations were maintained during simulated ischemia or ischemia/reoxygenation. Upon entry into the cell, DCFH-DA is initially converted into non-fluorescent 2′,7′-dichlorofluorescein (DCFH) by cellular esterase and then oxidized to fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of ROS (DCFH-DA → DCFH → DCF). DCF fluorescence was visualized by fluorescent microscopy. The intensity of DCF in the cells indicates increased intracellular levels of ROS. Image acquisition and analysis were performed as previously reported (23Swift L.M. Sarvazyan N. Am. J. Physiol. 2000; 278: H982-H990Crossref PubMed Google Scholar). In brief, cells were observed through a Plan-Apo ×60 oil immersion objective mounted on an inverted Olympus microscope with an Olympus LSM GB200 confocal imaging system attached. Excitation of dyes was achieved using the 488-nm line of a 15-milliwatt argon ion laser. All images were collected at room temperature. Mean fluorescence intensity was calculated by averaging area intensities from a number of outlined cells. For each condition described, six images of different cells were collected, and experiments were repeated at least three times.MPO Activity-MPO activity in ischemic LV tissue was analyzed as described previously (22Chandrasekar B. Colston J.T. Geimer J. Cortez D. Freeman G.L. Free Radic. Biol. Med. 2000; 28: 1579-1588Crossref PubMed Scopus (25) Google Scholar). In brief, cardiac tissue samples were first homogenized in 20 mm potassium phosphate (1:10, w/v) and then centrifuged at 20,000 × g for 30 min at 4 °C. The pellets were then frozen (-20 °C) for 12 h. After thawing, the pellet was added to a solution consisting of 0.5% hexadecyltrimethylammonium bromide dissolved in 50 mm potassium phosphate buffer (pH 6.0) containing 30 units/ml aprotinin. Each sample was sonicated for 1 min at 4 °C and then centrifuged at 40,000 × g for 30 min at 4 °C. Aliquots of the supernatant were reacted with a solution of O-dianisidine dihydrochloride (0.167 mg/ml) and 0.0005% H2O2, and absorbance at 405 nm was measured over time. MPO activity is defined as the quantity of enzyme degrading 1 μmol of peroxide/min at 37 °C and is expressed as units/g of tissue (wet weight).Transcription Factor Activation-DNA Binding Activity-Nuclear proteins were extracted from the ischemic zones of the myocardium from I/R-treated mice using a nuclear extraction kit (catalog number AY2002; Panomics, Fremont, CA). Formation of NF-κB protein-DNA complexes in postischemic myocardium and isolated cardiomyocytes was analyzed by an electrophoretic mobility shift assay (EMSA), and its subunit composition was analyzed by a supershift assay. In addition, NF-κB activation and its subunit composition were also analyzed by ELISA (TransAM™ TF ELISA kits; catalog number 43296; Active Motif, Carlsbad, CA) (19Chandrasekar B. Mummidi S. Mahimainathan L. Patel D.N. Bailey S.R. Imam S.Z. Greene W.C. Valente A.J. J. Biol. Chem. 2006; 281: 15099-15109Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 20Reddy V.S. Harskamp R.E. van Ginkel M.W. Calhoon J. Baisden C.E. Kim I.S. Valente A.J. Chandrasekar B. J. Cell. Physiol. 2008; 215: 697-707Crossref PubMed Scopus (55) Google Scholar, 21Patel D.N. King C.A. Bailey S.R. Holt J.W. Venkatachalam K. Agrawal A. Valente A.J. Chandrasekar B. J. Biol. Chem. 2007; 282: 27229-27238Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). EMSA was performed using IL18 gene-specific (sense, 5′-CCCTGATAAAATGTAGATTCCCTATTATAC-3) and mutant (sense, 5′-CCCTGATAAAATACCTATTCCCTATTATAC-3) oligonucleotides, as described previously (20Reddy V.S. Harskamp R.E. van Ginkel M.W. Calhoon J. Baisden C.E. Kim I.S. Valente A.J. Chandrasekar B. J. Cell. Physiol. 2008; 215: 697-707Crossref PubMed Scopus (55) Google Scholar, 21Patel D.N. King C.A. Bailey S.R. Holt J.W. Venkatachalam K. Agrawal A. Valente A.J. Chandrasekar B. J. Biol. Chem. 2007; 282: 27229-27238Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Specificity of NF-κB DNA binding activity was determined by competition with unlabeled consensus or mutant NF-κB oligonucleotides or consensus Oct1 (5′-TGT CGA ATG CAA ATC ACT AGA A-3′) oligonucleotides. In addition, labeled mutant NF-κB oligonucleotide also served as a control. In supershift assays, the nuclear protein extracts were incubated with antibodies (1 μg) p50 (sc-7178 X) or p65 (sc-109 X; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 40 min prior to EMSA. Activation of NF-κB in cardiomyocytes was also confirmed by reporter assays using adenoviral transduction with NF-κB (Ad-NF-κB-Luc, 50 MOI; kindly provided by John F. Engelhardt), as described previously (21Patel D.N. King C.A. Bailey S.R. Holt J.W. Venkatachalam K. Agrawal A. Valente A.J. Chandrasekar B. J. Biol. Chem. 2007; 282: 27229-27238Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Ad-MCS-Luc (Vector Biolabs) served as a control. Ad-β-galactosidase (50 MOI; Vector Biolabs) served as an internal control. β-Galactosidase activity in cell extracts was determined using a luminescent β-galactosidase detection kit II (BD Biosciences) (24Cortez D.M. Feldman M.D. Mummidi S. Valente A.J. Steffensen B. Vincenti M. Barnes J.L. Chandrasekar B. Am. J. Physiol. 2007; 293: H3356-H3365Crossref PubMed Scopus (191) Google Scholar), and the results are expressed as the ratio of firefly luciferase to β-galactosidase activity measured in relative light units.mRNA Analysis-Expression of IL-18, IL-18Rα, IL-18Rβ, and IL-18BP mRNA in the ischemic left ventricular tissue was analyzed by reverse transcription followed by real time quantitative PCR (qPCR) using an ABI Geneamp 7700 Sequence Detection System (PerkinElmer Life Sciences) according to the manufacturer's instructions, with SYBR Green fluorescence for amplicon detection. DNA-free total cellular RNA was isolated using the RNAqueous®-4PCR kit (Ambion). RNA quality was assessed by capillary electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All RNA samples had RNA integrity numbers greater than 8.9 (sc
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