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• W2056590359 abstract "Toll-like receptor-4 (TLR4) and its signaling molecule interleukin-1 receptor-associated kinase (IRAK-1) play an important role in host defense and tissue inflammation. Intriguingly, systemic administration of lipopolysaccharide (LPS), the agonist for TLR4, confers a cardio-protective effect against ischemic injury. However, the mechanisms leading to the cardiac protection remain largely unknown. The present study was designed to investigate the role of TLR4 activation by LPS in protecting cardiomyocytes (CM) against apoptosis in an in vitro model of ischemia and to explore the downstream mechanisms leading to the protective effect. Incubation with LPS led to activation of IRAK-1 and protected CMs against serum deprivation (SD)-induced apoptosis as demonstrated by DNA laddering, histone-DNA fragment enzyme-linked immunosorbent assay, and activation of caspase-3. Phosphatidylinositol 3-kinase/Akt, extracellular signal-regulated kinase 1/2, and IκB kinase β appear to contribute to the anti-apoptotic effect of LPS since the specific inhibitors, wortmannin, PD98059, and dominant negative IKKβ transgene expression reversed the LPS effect. To assess whether LPS improves CM function, we examined intracellular Ca2+ transients and cell shortening in single adult rat CMs. SD for 6 h dramatically inhibited Ca2+ transients and CM contractility. LPS at 500 ng/ml significantly improved the [Ca2+]i transients and enhanced contractility in control CMs as well as in CMs subjected to SD. Importantly, transient ischemia led to rapid activation of IRAK-1 in cultured CMs and in adult rat myocardium. Adenovirus-mediated transgene expression of IRAK-1 but not its kinase-deficient mutant IRAK-1(K239S) protected CMs against SD-induced apoptosis. Taken together, these data suggest an important role of TLR4 signaling via IRAK-1 in protecting against SD-induced apoptosis. Toll-like receptor-4 (TLR4) and its signaling molecule interleukin-1 receptor-associated kinase (IRAK-1) play an important role in host defense and tissue inflammation. Intriguingly, systemic administration of lipopolysaccharide (LPS), the agonist for TLR4, confers a cardio-protective effect against ischemic injury. However, the mechanisms leading to the cardiac protection remain largely unknown. The present study was designed to investigate the role of TLR4 activation by LPS in protecting cardiomyocytes (CM) against apoptosis in an in vitro model of ischemia and to explore the downstream mechanisms leading to the protective effect. Incubation with LPS led to activation of IRAK-1 and protected CMs against serum deprivation (SD)-induced apoptosis as demonstrated by DNA laddering, histone-DNA fragment enzyme-linked immunosorbent assay, and activation of caspase-3. Phosphatidylinositol 3-kinase/Akt, extracellular signal-regulated kinase 1/2, and IκB kinase β appear to contribute to the anti-apoptotic effect of LPS since the specific inhibitors, wortmannin, PD98059, and dominant negative IKKβ transgene expression reversed the LPS effect. To assess whether LPS improves CM function, we examined intracellular Ca2+ transients and cell shortening in single adult rat CMs. SD for 6 h dramatically inhibited Ca2+ transients and CM contractility. LPS at 500 ng/ml significantly improved the [Ca2+]i transients and enhanced contractility in control CMs as well as in CMs subjected to SD. Importantly, transient ischemia led to rapid activation of IRAK-1 in cultured CMs and in adult rat myocardium. Adenovirus-mediated transgene expression of IRAK-1 but not its kinase-deficient mutant IRAK-1(K239S) protected CMs against SD-induced apoptosis. Taken together, these data suggest an important role of TLR4 signaling via IRAK-1 in protecting against SD-induced apoptosis. Studies employing pharmacologic inhibitors, transgene expression, and genetically modified animals have demonstrated that cardiomyocyte (CM) 1The abbreviations used are: CM, cardiomyocyte; LPS, lipopolysaccharide; SD, serum deprivation; DR, death receptor; TLR, Toll-like receptor; IRAK, interleukin-1 receptor-associated kinase; TNF, tumor necrosis factor; TRAF-6, TNFα receptor-associated factor-6; TAK1, TGF-β-activated kinase 1; NF, nuclear factor; ERK, extracellular signal-regulated kinase; m.o.i., multiplicity of infection; Ad, adenoviral vector; IRI, ischemia-reperfusion injury; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; IKK, IκB kinase; P-, phosphorylated; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide; MBP, myelin basic protein; ELISA, enzyme-linked immunosorbent assay; wt, wild type; LAD, left anterior descending coronary artery. apoptosis plays an important role in ischemic myocardial injury (1Yaoita H. Ogawa K. Maehara K. Maruyama Y. Circulation. 1998; 97: 276-281Crossref PubMed Scopus (569) Google Scholar, 2Matsui T. Tao J. del Monte F. Lee K.H. Li L. Picard M. Force T.L. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 2001; 104: 330-335Crossref PubMed Scopus (599) Google Scholar, 3Fujio Y. Nguyen T. Wencker D. Kitsis R.N. Walsh K. Circulation. 2000; 101: 660-667Crossref PubMed Scopus (738) Google Scholar, 4Wencker D. Chandra M. Nguyen K. Miao W. Garantziotis S. Factor S.M. Shirani J. Armstrong R.C. Kitsis R.N. J. Clin. Investig. 2003; 111: 1497-1504Crossref PubMed Scopus (639) Google Scholar). CM apoptosis has been found in injured myocardium in patients who died of myocardial infarction (5Itoh G. Tamura J. Suzuki M. Suzuki Y. Ikeda H. Koike M. Nomura M. Jie T. Ito K. Am. J. Pathol. 1995; 146: 1325-1331PubMed Google Scholar, 6Olivetti G. Quaini F. Sala R. Lagrasta C. Corradi D. Bonacina E. Gambert S.R. Cigola E. Anversa P. J. Mol. Cell. Cardiol. 1996; 28: 2005-2016Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 7Saraste A. Pulkki K. Kallajoki M. Henriksen K. Parvinen M. Voipio-Pulkki L.M. Circulation. 1997; 95: 320-323Crossref PubMed Scopus (751) Google Scholar) and in animal models of infarction (8Kajstura J. Cheng W. Reiss K. Clark W.A. Sonnenblick E.H. Krajewski S. Reed J.C. Olivetti G. Anversa P. Lab. Investig. 1996; 74: 86-107PubMed Google Scholar, 9Sam F. Sawyer D.B. Chang D.L. Eberli F.R. Ngoy S. Jain M. Amin J. Apstein C.S. Colucci W.S. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: 422-428Crossref PubMed Google Scholar) and ischemia reperfusion (10Gottlieb R.A. Burleson K.O. Kloner R.A. Babior B.M. Engler R.L. J. Clin. Investig. 1994; 94: 1621-1628Crossref PubMed Scopus (1358) Google Scholar). Apoptosis is a tightly regulated and energy-dependent process that requires specialized cellular machinery. The central component of this machinery is a proteolytic system involving a family of proteases called caspases (11Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar). The biochemical and cellular hallmarks of apoptosis are characterized by nuclear and DNA fragmentation and condensation, membrane blebbing, and cellular shrinkage. LPS signal transduction involves multiple signaling proteins such as LPS binding protein, CD14, MD-2, and TLR4. TLR4 dimerizes and becomes activated upon association with LPS·MD2·CD14 complex. Activated TLR4 recruits downstream the serine-threonine kinases, IRAKs, through the adapter protein MyD88. All IRAKs are multidomain proteins consisting of a conserved N-terminal death domain and a central kinase domain (12Janssens S. Beyaert R. Mol. Cell. 2003; 11: 293-302Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). The death domain is a protein interaction motif implicated in binding of IRAKs to the upstream adapter protein MyD88. The recruited IRAK-1 becomes multiply phosphorylated, either by IRAK-1 itself or by IRAK-4. Phosphorylated IRAK-1 has reduced affinity for MyD88 and increased affinity for TRAF-6. A point mutation in the ATP binding pocket (K239S) creates a catalytically inactive IRAK-1, IRAK-1(K239S) (13Knop J. Martin M.U. FEBS Lett. 1999; 448: 81-85Crossref PubMed Scopus (90) Google Scholar). IRAK-1 and TRAF-6 complex then activate TAK1 through a process involving cytosol translocation of TAK1 and two TAK1-binding proteins from membrane to cytosol and ubiquitination of TRAF-6. Activated TAK1 then phosphorylates IKKα/β as well as mitogen-activated protein kinase (MAPK) kinases, leading to activation of NF-κB and c-Jun NH2-terminal kinase/p38 MAPKs, respectively (14Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1651) Google Scholar). LPS also activates ERK in macrophages (15Watters J.J. Sommer J.A. Pfeiffer Z.A. Prabhu U. Guerra A.N. Bertics P.J. J. Biol. Chem. 2002; 277: 9077-9087Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and endothelial cells (16Wong F. Hull C. Zhande R. Law J. Karsan A. Blood. 2004; 103: 4520-4526Crossref PubMed Scopus (28) Google Scholar). Although innate immunity signaling components such as TLR4 are present predominantly in immune cells (17Medzhitov R. Janeway Jr., C. N. Engl. J. Med. 2000; 343: 338-344Crossref PubMed Scopus (1755) Google Scholar), other tissues and organs such as the heart also possess functionally intact innate immune systems (18Cowan D.B. Poutias D.N. Del Nido P.J. McGowan Jr., F.X. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: 619-629Crossref PubMed Google Scholar, 19Frantz S. Kobzik L. Kim Y.D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Investig. 1999; 104: 271-280Crossref PubMed Scopus (569) Google Scholar). The heart expresses at least three receptors involving TLR signaling: CD14, TLR2, and TLR4 (18Cowan D.B. Poutias D.N. Del Nido P.J. McGowan Jr., F.X. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: 619-629Crossref PubMed Google Scholar, 19Frantz S. Kobzik L. Kim Y.D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Investig. 1999; 104: 271-280Crossref PubMed Scopus (569) Google Scholar, 20Frantz S. Kelly R.A. Bourcier T. J. Biol. Chem. 2001; 276: 5197-5203Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). A growing body of evidence suggests that the myocardial innate immune system may play an important role in mediating the inflammatory response and modulating cardiac function in response to LPS (21Knuefermann P. Nemoto S. Misra A. Nozaki N. Defreitas G. Goyert S.M. Carabello B.A. Mann D.L. Vallejo J.G. Circulation. 2002; 106: 2608-2615Crossref PubMed Scopus (117) Google Scholar, 22Baumgarten G. Knuefermann P. Nozaki N. Sivasubramanian N. Mann D.L. Vallejo J.G. J. Infect. Dis. 2001; 183: 1617-1624Crossref PubMed Scopus (189) Google Scholar). Intriguingly, evidence from several lines of investigation suggests that LPS may activate intracellular survival mechanisms and protect the myocardium against ischemia and reperfusion injury. For example, in animal models, administration of LPS or its non-pyrogenic derivative mono-phosphoryl lipid A reduces cardiac arrhythmia and myocardial infarction after ischemia and reperfusion injury (IRI) (23Brown J.M. Grosso M.A. Terada L.S. Whitman G.J. Banerjee A. White C.W. Harken A.H. Repine J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2516-2520Crossref PubMed Scopus (267) Google Scholar, 24Eising G.P. Mao L. Schmid-Schonbein G.W. Engler R.L. Ross J. Cardiovasc. Res. 1996; 31: 73-81Crossref PubMed Scopus (41) Google Scholar, 25Song W. Furman B.L. Parratt J.R. Br. J. Pharmacol. 1996; 118: 2157-2163Crossref PubMed Scopus (43) Google Scholar, 26Song W. Furman B.L. Parratt J.R. Eur. J. Pharmacol. 1998; 345: 285-287Crossref PubMed Scopus (16) Google Scholar, 27Yamashita N. Hoshida S. Otsu K. Taniguchi N. Kuzuya T. Hori M. Br. J. Pharmacol. 1999; 128: 412-418Crossref PubMed Scopus (15) Google Scholar). In a rabbit model systemic administration of LPS before ischemia led to a reduction in infarct size by 54% (28Belosjorow S. Schulz R. Dorge H. Schade F.U. Heusch G. Am. J. Physiol. 1999; 277: H2470-H2475PubMed Google Scholar). Using an ex vivo Langendorff model, Meng et al. (29Meng X. Ao L. Brown J.M. Meldrum D.R. Sheridan B.C. Cain B.S. Banerjee A. Harken A.H. Am. J. Physiol. 1997; 273: H1894-H1902PubMed Google Scholar) demonstrated that prior systemic treatment of rats with LPS led to an improved cardiac function during reperfusion phase after transient ischemic insult as compared with the control saline-treated rats. Although the cardio-protective effects of LPS against IRI are well documented, its effect on myocardial apoptosis is unclear. LPS has been shown to activate parallel pro-apoptotic and survival pathways in other cell types. Activation of TLR4 triggers expression of cell survival and inflammatory genes via NF-κB-dependent mechanisms, although in endothelial cells, NF-κB activity seems dispensable for the LPS-induced cytoprotective activity (16Wong F. Hull C. Zhande R. Law J. Karsan A. Blood. 2004; 103: 4520-4526Crossref PubMed Scopus (28) Google Scholar). LPS also activates phosphatidylinositol 3-kinase/Akt pathways in some cell types (30Venkataraman C. Shankar G. Sen G. Bondada S. Immunol. Lett. 1999; 69: 233-238Crossref PubMed Scopus (38) Google Scholar, 31Vivarelli M.S. McDonald D. Miller M. Cusson N. Kelliher M. Geha R.S. J. Exp. Med. 2004; 200: 399-404Crossref PubMed Scopus (62) Google Scholar). In endothelial cells LPS induces apoptotic signaling through a mechanism involving TRAF-6-mediated c-Jun NH2-terminal kinase activation (32Hull C. McLean G. Wong F. Duriez P.J. Karsan A. J. Immunol. 2002; 169: 2611-2618Crossref PubMed Scopus (99) Google Scholar). However, LPS-induced apoptosis is often cell type-specific (most in endothelial cells) and depends on the simultaneous administration of cycloheximide or proteasome inhibitor to block the synthesis of endogenous survival molecules (33Bannerman D.D. Tupper J.C. Erwert R.D. Winn R.K. Harlan J.M. J. Biol. Chem. 2002; 277: 8048-8053Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 34Bannerman D.D. Tupper J.C. Ricketts W.A. Bennett C.F. Winn R.K. Harlan J.M. J. Biol. Chem. 2001; 276: 14924-14932Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The survival signals in LPS-treated endothelial cells appear to include both an inducible and constitutively active components (34Bannerman D.D. Tupper J.C. Ricketts W.A. Bennett C.F. Winn R.K. Harlan J.M. J. Biol. Chem. 2001; 276: 14924-14932Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 35Hu X. Yee E. Harlan J.M. Wong F. Karsan A. Blood. 1998; 92: 2759-2765Crossref PubMed Google Scholar). In the present study we examined the anti-apoptotic effect of TLR4 activation by LPS in an in vitro model of ischemia, i.e. hypoxia/serum deprivation (SD). LPS pretreatment led to improved survival and function of CMs subjected to SD. Employing transgene expression and pharmacological inhibitors, we explored the roles of IRAK-1 and other three survival molecules, Akt, ERK1/2, and IKKβ in LPS cardio-protective effects. Finally, we demonstrated that IRAK-1, an important component of TLR4 innate signaling, is dynamically activated in CMs subjected to hypoxia/reoxygenation and in myocardium subjected to coronary artery ligation. These results suggest that the anti-apoptotic effect of LPS-TLR4 signaling may play an important role in protecting CMs and that the myocardial innate immune system involving IRAK-1 may represent an intrinsic mechanism of cardio-protection. Materials—LPS (Escherichia coli 0111:B4), collagenase, myelin basic protein, and β-actin antibody were from Sigma. Antibodies for cleaved caspase-3, Akt, P-Akt, and P-ERK were from Cell signaling (Beverly, MA), and antibodies for IRAK-1 were from Pro-Sci Co. (catalog number 1007) and Santa Cruz (F4, sc-5288), respectively. Wortmannin and PD98059 were from Calbiochem. Cell death detection ELISA kits were purchased from Roche Molecular Biochemicals. Caspase-3 activity assay kits were from R&D Systems. Neonatal Rat CM Preparation—CMs were prepared from 1–2-day-old rats using collagenase and pancreatin. Briefly, the hearts were isolated, dissected from major vessels, and cut into small pieces. The heart tissues were then incubated in ADS buffer (116 mm NaCl, 20 mm HEPES, 0.8 mm Na2HPO4, 5.6 mm glucose, 5.4 mm KCl, and 0.8 mm MgSO4) containing 0.04% collagenase and 0.06% pancreatin (Worthington) at 37 °C for 20 min in a shaker. Cell suspension was slowly removed, and the remaining myocardial tissues were further incubated with the enzyme buffer. Cells in suspension were collected, spun, and resuspended in Dulbecco's modified Eagle's medium containing 10% horse serum, 5% fetal bovine serum, and 4.5% d-glucose. Fibroblasts were removed by plating cells on 10-cm dishes for 30 min. Neonatal CMs were then plated in Petri dishes and incubated in CO2 incubator at 37 °C for 3–4 days before experiments were performed. Adult Rat CM Isolation—Isolated adult ventricular cardiomyocytes were prepared using an enzymatic perfusion method as described by Martin et al. (36Martin B.J. Valdivia H.H. Bunger R. Lasley R.D. Mentzer Jr., R.M. Am. J. Physiol. 1998; 274: H8-H17PubMed Google Scholar). Briefly, the heart was perfused with an enzyme solution containing 0.08% collagenase Type II (Worthington) and 0.04% hyaluronidase (Sigma) in oxygenated Ca2+-free Krebs-Henseleit solution. The heart was perfused for 25 min, and the resulting cell suspension was filtered and triturated with Krebs-Henseleit solution containing 0.5 mm Ca2+ for 5 min. The cells were spun down at 1000 rpm for 1 min and then resuspended in Krebs-Henseleit buffer containing 1.0 mm Ca2+. Adult CMs were incubated in RPMI 1640 with 10% horse serum and 5% fetal bovine serum. In Vitro Model of Apoptosis and LPS Treatment—Three days after plating, cultured beating neonatal CMs were washed 3 times with serum-free RPMI 1640 medium containing 0.05% bovine serum albumin and incubated in the same medium. In some experiments cell cultures were then placed in a hypoxic chamber filled with humidified flowing hypoxic gas (95% nitrogen and 5% CO2) at 37 °C for 4–6 h. Our previous studies have demonstrated that SD alone for 4–24 h induces significant caspase activation and DNA fragmentation. Hypoxia leads to only modest additional activation of caspase-3 and DNA laddering in the SD model (37Chao W. Shen Y. Li L. Rosenzweig A. J. Biol. Chem. 2002; 277: 31639-31645Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Chao W. Matsui T. Novikov M.S. Tao J. Li L. Liu H. Ahn Y. Rosenzweig A. J. Gene Med. 2003; 5: 277-286Crossref PubMed Scopus (59) Google Scholar). For LPS experiments, cell cultures were pre-treated with saline or LPS at the indicated concentrations for 60 min. Cells were then washed three times and then incubated with either regular serum-containing RPMI medium or serum-free medium depending on experimental design. Second doses of fresh LPS were added to cell cultures accordingly. In some experiments LPS was added at the time of SD without preincubation. Apoptosis/Cell Viability Assays—For DNA laddering, cells were lysed, and cellular DNA was extracted with phenol:chloroform. 1.5 μg of genomic DNA fragments were treated with RNase, 32P-labeled by Klenow DNA polymerase, and then separated by electrophoresis in 1.8% agarose gel (37Chao W. Shen Y. Li L. Rosenzweig A. J. Biol. Chem. 2002; 277: 31639-31645Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Chao W. Matsui T. Novikov M.S. Tao J. Li L. Liu H. Ahn Y. Rosenzweig A. J. Gene Med. 2003; 5: 277-286Crossref PubMed Scopus (59) Google Scholar). For cell death ELISA, apoptosis-induced histone-DNA fragments were quantitated using cell death detection ELISA (37Chao W. Shen Y. Li L. Rosenzweig A. J. Biol. Chem. 2002; 277: 31639-31645Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Chao W. Matsui T. Novikov M.S. Tao J. Li L. Liu H. Ahn Y. Rosenzweig A. J. Gene Med. 2003; 5: 277-286Crossref PubMed Scopus (59) Google Scholar). For caspase activity assays, caspase-3 activity was examined by using the caspase colorimetric assay kit from R&D Systems as previously described (37Chao W. Shen Y. Li L. Rosenzweig A. J. Biol. Chem. 2002; 277: 31639-31645Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). For the MTT assay, neonatal CMs cultured in 24-well plates (6 × 105/well) were incubated in either regular RPMI medium or serum-free medium as indicated for 6 h. At the end of incubation, 1.5 ml of fresh regular RPMI medium containing 0.05% MTT was added into each well and incubated at 37 °C for 1 h. The MTT-containing medium was then removed, 1.5 ml of Me2SO was added in each well, and absorbance was measured at 570 nm. Immunoprecipitation, Western Blotting, and IRAK-1 Kinase Assay— CMs (∼2 × 106 cells) were scraped from 60-mm dishes in RPMI 1640 medium, spun down, and washed once with 15 ml of cold phosphate-buffered saline. Cell pellets or freshly frozen rat myocardial tissue (see “In Vivo Model of Ischemia Reperfusion”) were dissolved in 1 ml of cold lysis buffer (50 mm HEPES, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 20 mm β-glycerophosphate, 1 mm NaVO3, 1 mm NaF, 5 mm p-nitrophenyl phosphate, 1 mm benzamidine, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 mm leupeptin, aprotinin, and pepstatin) and incubated for 10 min at 4 °C. Cell lysate was then centrifuged at 10,000 rpm for 10 min, and supernatant was removed. For immunoprecipitation, 2.5 μg of polyclonal anti-IRAK-1 antibody (Pro-Sci Co.) was added to cell lysates containing equal amount of proteins and incubated at 4 °C for 3 h or overnight on a rotator. To harvest IRAK-1-antibody complex, 50 μl of pre-washed 50% slurry protein G (Amersham Biosciences) was added, incubated for 2 h at 4 °C, spun down at 14,000 rpm in an Eppendorf centrifuge, and washed 4 times with cold lysis buffer. Half of the protein G beads were used for IRAK-1 Western blotting (F4 antibody from Santa Cruz) as described previously (39Li L. Cousart S. Hu J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). The remaining beads were washed twice and incubated in 110 μl of kinase assay buffer (20 mm HEPES, 20 mm MgCl2, 20 mm β-glycerophosphate, 20 mm p-nitrophenyl phosphate, 1 mm EDTA, 1 mm NaVO3, and 1 mm benzamidine). To start IRAK-1 kinase assay, 2 μg of myelin basic protein (MBP) (Sigma), 5 μm cold ATP, and 50 μCi of [γ-32P]ATP were added to 110 μl of immunoprecipitates and incubated at 37 °C for 30 min. At the end of the assay, immunoprecipitates were mixed with SDS sample buffer, heated, and separated in 4–20% gradient SDS-PAGE. 32P-Labeled MBP was visualized by autoradiography. Measurement of Ca2+ Transient and Cell Shortening—Adult CMs were loaded with 5 μm Fluo 4-AM (Molecular Probe) at room temperature for 20 min. Cells attached to the coverslip were continuously perfused with Tyrode solution (130 mm NaCl, 5.4 mm KCl, 4 mm NaH2PO4, 0.5 mm MgCl2, 1 mm CaCl2, 25 mm HEPES, 22 mm glucose, 0.01 μg/ml insulin, pH 7.4 with NaOH) and electrically stimulated (0.5 Hz, 35 V, 100 ms). Ca2+ transients were measured using a Nikon Diaphot confocal microscope (Bio-Rad MR-1). Fluo 4-AM was excited at 488 nm, with emitted fluorescence measured at 515 nm. All images were taken in the line-scan mode (2 ms/line, 0.1–0.3 μm/pixel, with the scan line oriented along the long axis of CM, avoiding the nuclei of the cell). Ca2+ transients were analyzed with an automated computer program. All experiments were performed at room temperature. In Vivo Model of Ischemia Reperfusion—Sprague-Dawley rats (200–300 g) were anesthetized, and midline sternotomy was performed (38Chao W. Matsui T. Novikov M.S. Tao J. Li L. Liu H. Ahn Y. Rosenzweig A. J. Gene Med. 2003; 5: 277-286Crossref PubMed Scopus (59) Google Scholar). LAD was ligated with silk suture (5.0) ∼4 mm from its origin with a slipknot. Ischemia was confirmed by myocardial bleaching. 5 min into ischemia, fluorescent microspheres (300 μl, 10-μm FluoSpheres, Molecular Probes) were injected into the left ventricle cavity. For IRI, after 30 min the LAD ligature was released, and reperfusion was visually confirmed. For straight infarct, a permanent tie was used instead of a slipknot. In the sham-operated animals, a slipknot tie was passed under the LAD but not tied. To harvest heart tissues, animals were anesthetized and exsanguinated. The heart was removed, and ventricular chambers were opened, washed three times with cold phosphate-buffered saline, and cryopreserved in liquid nitrogen for subsequent DNA/protein analysis and IRAK kinase assay. Recombinant Adenoviral Vector—All Ad constructs have been made using the pAdEasy system as described (40He T. Zhou S. da Costa L. Yu J. Kinzler K. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar) and have been characterized. These included Ad vectors carrying cDNAs for IRAK-1, kinase-deficient mutant IRAK-1(K239S), IKKβ-dn (41Meiler S.E. Hung R.R. Gerszten R.E. Gianetti J. Li L. Matsui T. Gimbrone Jr., M.A. Rosenzweig A. J. Mol. Cell. Cardiol. 2002; 34: 349-359Abstract Full Text PDF PubMed Scopus (14) Google Scholar), and β-galactosidase/green fluorescent protein (controls). cDNAs for IRAK-1 and IKKβ-dn were kindly provided by Dr. Z. Cao and Dr. D. Goeddel, respectively, at Tularik. High titer stocks were prepared, and expression was confirmed in neonatal and/or adult CMs in vitro and in rat myocardium in vivo. Wild-type Ad contamination was excluded by the absence of PCR-detectable E1 sequences. Statistics—Unless stated otherwise, all data are expressed as the mean ± S.D. of at least three independent experiments and were analyzed with a two-tailed, unpaired Student's t test. The null hypothesis was rejected for p < 0.05. LPS Induces IRAK-1 Activation and Protects CMs against Hypoxia/SD-induced CM Apoptosis—We have established an in vitro kinase assay for IRAK-1 both in isolated neonatal rat CMs and in adult rat myocardium to test innate immunity signaling. As shown in Fig. 1, LPS activated IRAK-1 kinase in cultured CMs with enhanced phosphorylation of MBP. The IRAK-1 kinase activity was specific for the LPS-TLR4-signaling pathway as TNFα failed to stimulate IRAK-1 activity. A similar concentration of TNFα leads to NF-κB activation as demonstrated by gel shift (data not shown). The LPS-stimulated phosphorylation of MBP was dependent on IRAK-1, as the immunoprecipitates with control rabbit IgG from the LPS-stimulated CMs did not show any increase in IRAK-1 activity as compared with the nonspecific background (Fig. 1A). LPS activates IRAK-1 rapidly, reaching the peak within 30 min (Fig. 1B). An in vitro model of hypoxia and re-oxygenation was used to mimic in vivo ischemia and involved a brief incubation of CMs in SD and hypoxic conditions. The in vitro model has been demonstrated to induce significant CM apoptosis (37Chao W. Shen Y. Li L. Rosenzweig A. J. Biol. Chem. 2002; 277: 31639-31645Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Chao W. Matsui T. Novikov M.S. Tao J. Li L. Liu H. Ahn Y. Rosenzweig A. J. Gene Med. 2003; 5: 277-286Crossref PubMed Scopus (59) Google Scholar). As shown in Fig. 2A, the combination of hypoxia and serum deprivation for 24 h led to an increase in DNA laddering, and treatment of CMs with LPS at the time of SD substantially inhibited DNA fragmentation. Quantitative analysis using histone-DNA fragment ELISA further demonstrated that hypoxia/SD induced up to an 8.2-fold increase in DNA fragmentation within 9 h in CMs and a 2.4-fold increase in human umbilical vein endothelial cells within 5 h. LPS significantly inhibited hypoxia/SD-induced apoptosis by 45% in cultured rat CMs but failed to inhibit apoptosis in human umbilical vein endothelial cells, suggesting the cyto-protective effect of LPS is cell type-specific (Fig. 2B). To further explore the anti-apoptotic effect of LPS, caspase-3 cleavage and activity were measured. LPS dramatically inhibited the pro-caspase-3 cleavage in a dose-dependent manner (Fig. 2C). Similar to what we documented previously (37Chao W. Shen Y. Li L. Rosenzweig A. J. Biol. Chem. 2002; 277: 31639-31645Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), hypoxia/SD induced a time-dependent cleavage and activation of pro-caspase-3, both of which were correlated well with each other. LPS treatment led to a decrease in caspase-3 activity for up to 6 h of SD (Fig. 2, D and E). Consistent with its anti-apoptotic effect, LPS also improved the viability of serum-deprived CMs as demonstrated by MTT assay (Fig. 2F). LPS Improves Function of Adult CMs Subjected to SD—To learn whether activation of TLR4 innate signaling by LPS improves CM function in an in vitro model of ischemia, cultured adult rat CMs were pre-treated with LPS (500 ng/ml) or saline for 1 h in regular RPMI, washed, and incubated in the presence or absence of serum as indicated. Additional doses of LPS or saline were added after the wash. Five hours later [Ca2+]i transients were recorded (Fig. 3-A). It is noteworthy that only beating CMs were chosen to record [Ca2+]i transients and cell contractility in all four groups of CMs. Three-dimensional graphics as changes of fluorescence ratio ΔF/F0 (F0 is the base-line Ca2+ signal before depolarization, ΔF = F – F0) is presented in Fig. 3B. The average amplitudes (ΔF/F0) of Ca2+ transients from control CMs (+serum, saline, n = 36) was 3.1 ± 0.45 (Fig. 3C), the time-to-peak Ca2+ was 138 ± 41 ms (Fig. 3D), the time constant (τ) during reuptake and relaxation was 299 ± 65 ms (Fig. 3E), and the cell shortening was 10.1 ± 1.5% (Fig. 3F). Surprisingly, CMs treated with 500 ng/ml LPS in normal serum-containing conditions (+serum, LPS, 6 h, n = 31) demonstrated improved Ca2+ homeostasis and contractility with a 19% increase in the average amplitudes of Ca2+ (Fig. 3C), a 23% decrease in the time-to-peak Ca2+ (Fig. 3D), and a 35% increase in the cell shortening (Fig. 3F) but no signifi" @default.
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• W2056590359 date "2005-06-01" @default.
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• W2056590359 title "Lipopolysaccharide Improves Cardiomyocyte Survival and Function after Serum Deprivation" @default.
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