An Inhibitor of p38 Mitogen-activated Protein Kinase Protects Neonatal Cardiac Myocytes from Ischemia
1999; Elsevier BV; Volume: 274; Issue: 10 Linguagem: Inglês
10.1074/jbc.274.10.6272
ISSN1083-351X
AutoresKatrina Mackay, Daria Mochly‐Rosen,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoCellular ischemia results in activation of a number of kinases, including p38 mitogen-activated protein kinase (MAPK); however, it is not yet clear whether p38 MAPK activation plays a role in cellular damage or is part of a protective response against ischemia. We have developed a model to study ischemia in cultured neonatal rat cardiac myocytes. In this model, two distinct phases of p38 MAPK activation were observed during ischemia. The first phase began within 10 min and lasted less than 1 h, and the second began after 2 h and lasted throughout the ischemic period. Similar to previous studies using in vivo models, the nonspecific activator of p38 MAPK and c-Jun NH2-terminal kinase, anisomycin, protected cardiac myocytes from ischemic injury, decreasing the release of cytosolic lactate dehydrogenase by approximately 25%. We demonstrated, however, that a selective inhibitor of p38 MAPK, SB 203580, also protected cardiac myocytes against extended ischemia in a dose-dependent manner. The protective effect was seen even when the inhibitor was present during only the second, sustained phase of p38 MAPK activation. We found that ischemia induced apoptosis in neonatal rat cardiac myocytes and that SB 203580 reduced activation of caspase-3, a key event in apoptosis. These results suggest that p38 MAPK induces apoptosis during ischemia in cardiac myocytes and that selective inhibition of p38 MAPK could be developed as a potential therapy for ischemic heart disease. Cellular ischemia results in activation of a number of kinases, including p38 mitogen-activated protein kinase (MAPK); however, it is not yet clear whether p38 MAPK activation plays a role in cellular damage or is part of a protective response against ischemia. We have developed a model to study ischemia in cultured neonatal rat cardiac myocytes. In this model, two distinct phases of p38 MAPK activation were observed during ischemia. The first phase began within 10 min and lasted less than 1 h, and the second began after 2 h and lasted throughout the ischemic period. Similar to previous studies using in vivo models, the nonspecific activator of p38 MAPK and c-Jun NH2-terminal kinase, anisomycin, protected cardiac myocytes from ischemic injury, decreasing the release of cytosolic lactate dehydrogenase by approximately 25%. We demonstrated, however, that a selective inhibitor of p38 MAPK, SB 203580, also protected cardiac myocytes against extended ischemia in a dose-dependent manner. The protective effect was seen even when the inhibitor was present during only the second, sustained phase of p38 MAPK activation. We found that ischemia induced apoptosis in neonatal rat cardiac myocytes and that SB 203580 reduced activation of caspase-3, a key event in apoptosis. These results suggest that p38 MAPK induces apoptosis during ischemia in cardiac myocytes and that selective inhibition of p38 MAPK could be developed as a potential therapy for ischemic heart disease. The heart is subjected to episodes of ischemia followed by reperfusion in a number of situations, including angina, myocardial infarction, and cardiac surgery, and these stresses can result in cell injury and death. Part of the cellular response to ischemia/reperfusion is activation of several members of the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; LDH, lactate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; ATF2, activating transcription factor-2; calcein AM, calcein acetoxymethyl ester; PI, propidium iodide. family. In many different cell types, p38 MAPK and c-Jun NH2-terminal kinase (JNK) family members are activated predominantly by cellular stresses or inflammatory signals, e.g. hyperosmolarity, chemical or heat stress, endotoxin, and cytokines (1Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2413) Google Scholar, 2Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar, 3Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2041) Google Scholar, 4Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar), whereas the extracellular signal-regulated kinases (ERKs) are activated by mitogenic stimuli (5Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (997) Google Scholar). In the isolated perfused rat heart, p38 MAPK is activated by global ischemia, and activation is maintained during reperfusion (6Bogoyevitch M.A. Gillespie-Brown J. Ketterman A.J. Fuller S.J. Ben-Levy R. Ashworth A. Marshall C.J. Sugden P.H. Circ. Res. 1996; 79: 162-173Crossref PubMed Scopus (490) Google Scholar, 7Yin T. Sandhu G. Wolfgang C.D. Burrier A. Webb R.L. Rigel D.F. Hai T. Whelan J. J. Biol. Chem. 1997; 272: 19943-19950Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). In the same model, neither ERKs nor JNKs are activated by ischemia, whereas reperfusion after ischemia activates JNK (6Bogoyevitch M.A. Gillespie-Brown J. Ketterman A.J. Fuller S.J. Ben-Levy R. Ashworth A. Marshall C.J. Sugden P.H. Circ. Res. 1996; 79: 162-173Crossref PubMed Scopus (490) Google Scholar, 7Yin T. Sandhu G. Wolfgang C.D. Burrier A. Webb R.L. Rigel D.F. Hai T. Whelan J. J. Biol. 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Chem. 1997; 272: 16657-16662Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Ischemia and reperfusion also activate members of the MAPK family in kidney and liver differentially (7Yin T. Sandhu G. Wolfgang C.D. Burrier A. Webb R.L. Rigel D.F. Hai T. Whelan J. J. Biol. Chem. 1997; 272: 19943-19950Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 10Bradham C.A. Stachlewitz R.F. Gao W. Qian T. Jayadev S. Jenkins G. Hannun Y. Lemasters J.J. Thurman R.G. Brenner D.A. Hepatology. 1997; 25: 1128-1135Crossref PubMed Scopus (188) Google Scholar, 11Pombo C.M. Bonventre J.V. Avruch J. Woodgett J.R. Kyriakis J.M. Force T. J. Biol. Chem. 1994; 269: 26546-26551Abstract Full Text PDF PubMed Google Scholar). However it is not clear from these studies whether activation of these kinases is part of the protective response of the cell or if these signals mediate the cellular damage and death caused by ischemia or ischemia/reperfusion. Evidence suggests that myocardial ischemic cell death occurs by both apoptosis and necrosis (12Kajstura J. Cheng W. Reiss K. Clark W.A. Sonnenblick E.H. Krajewski S. Reed J.C. Olivetti G. Anversa P. Lab. Invest. 1996; 74: 86-107PubMed Google Scholar, 13Anversa P. Kajstura J. Circ. Res. 1998; 82: 1231-1233Crossref PubMed Scopus (79) Google Scholar). From the timing of p38 MAPK activation during ischemia and initiation of apoptosis, Yinet al. (7Yin T. Sandhu G. Wolfgang C.D. Burrier A. Webb R.L. Rigel D.F. Hai T. Whelan J. J. Biol. Chem. 1997; 272: 19943-19950Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) speculate that activation of p38 MAPK initiates the signal for apoptotic cell death. Indeed, p38 MAPK activation has been implicated in mediating apoptosis in several cell types (14Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5036) Google Scholar, 15Kawasaki H. Morooka T. Shimohama S. Kimura J. Hirano T. Gotoh Y. Nishida E. J. Biol. Chem. 1997; 272: 18518-18521Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 16Kummer J.L. Rao P.K. Heidenreich K.A. J. Biol. Chem. 1997; 272: 20490-20494Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). Recent studies in neonatal rat cardiac myocytes support a role for the α isoform of p38 MAPK in mediating apoptosis; overexpression of activated MAPK kinase 3b, which phosphorylates and activates p38 MAPK, induces apoptosis that is increased by coexpression of p38α and is decreased by expression of a dominant negative form of this isoform (17Wang Y. Huang S. Sah V.P. Ross Jr., J. Brown J.H. Han J. Chien K.R. J. Biol. Chem. 1998; 273: 2161-2168Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar). In contrast, a separate study demonstrates that activation of p38 MAPK can prevent apoptosis in neonatal rat cardiac myocytes (18Zechner D. Craig R. Hanford D.S. McDonough P.M. Sabbadini R.A. Glembotski C.C. J. Biol. Chem. 1998; 273: 8232-8239Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Furthermore, others have proposed that p38 MAPK activation mediates a phenomenon termed preconditioning, which confers cardiac protection from ischemia. Preconditioning is a highly effective method of protecting the heart from ischemic damage by subjecting it to sublethal periods of ischemia before the prolonged ischemia (6Bogoyevitch M.A. Gillespie-Brown J. Ketterman A.J. Fuller S.J. Ben-Levy R. Ashworth A. Marshall C.J. Sugden P.H. Circ. Res. 1996; 79: 162-173Crossref PubMed Scopus (490) Google Scholar, 8Knight R.J. Buxton D.B. Biochem. Biophys. Res. Commun. 1996; 218: 83-88Crossref PubMed Scopus (150) Google Scholar, 19Murry C. Jennings R.B. Reimer K.A. Circulation. 1986; 74: 1124-1136Crossref PubMed Scopus (7040) Google Scholar,20Downey J.M. Cohen M.V. Sideman S. Beyar R. Analytical and Quantitative Cardiology. Plenum Press, New York1997: 39-55Google Scholar). A protective function for p38 MAPK is supported by a recent study in which the role of p38 MAPK in preconditioning was examined in isolated rabbit cardiac myocytes (21Weinbrenner C. Liu G.-S. Cohen M.V. Downey J.M. J. Mol. Cell. Cardiol. 1997; 29: 2383-2391Abstract Full Text PDF PubMed Scopus (236) Google Scholar). Pretreatment with anisomycin, an activator of p38 MAPK, protects isolated rabbit cardiac myocytes against ischemia-induced cell fragility, leading to the suggestion that p38 MAPK protects the heart against ischemia. The addition of SB 203580, a selective inhibitor of p38 MAPK (22Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 23Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar), during a preconditioning treatment abolishes the protective effect of preconditioning, supporting the initial observation (21Weinbrenner C. Liu G.-S. Cohen M.V. Downey J.M. J. Mol. Cell. Cardiol. 1997; 29: 2383-2391Abstract Full Text PDF PubMed Scopus (236) Google Scholar). Similar results, showing that SB 203580 inhibits the protection afforded by ischemic preconditioning against myocardial infarction, were obtained using isolated rat hearts (24.Maulik, N., Yoshida, T., Zu, Y.-L., and Engelman, R. M. (1998) inVascular and Myocardial Aspects of Ischemic Heart Disease, Nevada.Google Scholar). Most previous ischemia studies have investigated MAPK activation in whole heart, which contains a large proportion of non-myocyte cells, mainly fibroblasts and endothelial cells. In the present study, we used primary cultures of neonatal rat cardiac myocytes and confirmed that p38 MAPK is activated in a model of ischemia which uses a glucose-free hypoxic incubation. We report that activation of p38 MAPK occurred in two distinct phases and that inhibition of p38 MAPK during the second phase protected cardiac myocytes from ischemic injury. These results are consistent with the hypothesis that sustained p38 MAPK mediates ischemia-induced cell injury and death in neonatal rat cardiac myocytes. All antibodies were used according to manufacturers' protocols. Anisomycin (Sigma) was dissolved in dimethyl sulfoxide (Me2SO) at 5 mg/ml and used to give a final Me2SO concentration less than 0.01%. SB 203580 (Calbiochem) was dissolved in Me2SO at 10 mmand used to give final a Me2SO concentration less than 0.1%. Primary cultures of ventricular myocytes from 1-day-old Sprague-Dawley rats were performed by gentle, serial trypsinization, as described previously (25Simpson P. Savion S. Circ. Res. 1982; 50: 101-116Crossref PubMed Scopus (446) Google Scholar) with modifications (26Johnson J.A. Gray M.O. Karliner J.S. Chen C.-H. Mochly-Rosen D. Circ. Res. 1996; 79: 1086-1099Crossref PubMed Scopus (80) Google Scholar). A preplating step was included to reduce the number of contaminating non-myocytes. Myocytes were plated at 800 cells/mm2 in 35- or 60-mm dishes (Falcon). Myocytes represented 90–95% of total adhering cells. Division of non-myocytes was prevented by the addition of 0.1 mm bromodeoxyuridine to medium for the first 4 days of culture. Cells were maintained at 37 °C in a 1% CO2 incubator in M-199 medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Hyclone), 50 units/ml penicillin, and 80 μm vitamin B12for the first 4 days. Vitamin C (80 μm) was present from day 2. On day 4, myocytes were placed in defined (10 μg/ml insulin, 10 μg/ml transferrin, 80 μm vitamin C, 50 units/ml penicillin, and 80 μm vitamin B12) M-199 medium. Myocytes exhibited a spontaneous contraction rate of approximately 250–300 beats/min, and cultures with a slower contraction rate were not used. The higher rate of contractionversus that seen by Simpson and Savion (25Simpson P. Savion S. Circ. Res. 1982; 50: 101-116Crossref PubMed Scopus (446) Google Scholar) is partly the result of the increased density of cultures used here. Experiments were performed on days 5 and 6 of culture. Ischemia was induced in a humidified 37 °C incubator within an air-tight Plexiglas glove box (Anaerobic Systems) maintained with 0.2–0.5% O2, 1% CO2, and the balance N2. Medium (defined minimal essential medium and Hank's balanced salt solution without glucose) was equilibrated to low O2 within the glove box for at least 90 min before commencing experiments. Inside the glove box, cells were washed twice with warm, preequilibrated medium before the addition of incubation medium (1.5 ml/35-mm dish). O2was measured using an electronic gas analyzer OXOR®II or Fyrite® (both from Bacharach). After ischemic or normoxic treatments, incubation medium was stored at 4 °C, and the same volume of cold buffer (10 mm Tris-HCl, pH 7.4, 1 mm EDTA) was added to the cells. The cells were scraped and lysed by trituration. Lysates were centrifuged at 4 °C at 16,000 × g for 15 min, and the supernatant was stored at 4 °C. LDH activity was measured from both medium (released LDH) and cell lysate (retained LDH) using a spectrophotometric assay (Sigma). Results were expressed as released LDH activity as a percent of total (released plus retained) LDH activity. After treatment, cells were placed on ice, and the incubation medium was aspirated and discarded. Cells were washed once with cold phosphate-buffered saline. Laemmli loading buffer at 2 × concentration (2% SDS, 20% glycerol, 0.04 mg/ml bromphenol blue, 0.12m Tris-HCl, pH 6.8, 0.28 m β-mercaptoethanol) was added (150 μl/35-mm dish). Cells were scraped and lysed by trituration. Samples were frozen in dry ice/ethanol then transferred immediately to −80 °C. Prior to electrophoresis, samples were heated to 95 °C for 5 min. Electrophoresis was performed with approximately 20 μg of protein/sample on 10% low ratio bisacrylamide (100:1, acrylamide:bisacrylamide). After Western blotting, filters were probed sequentially with dual phospho-p38 MAPK (Thr180Tyr182), total p38 MAPK (both from New England Biolabs), or ERK2 antiserum, and immunoreactivity was detected by enhanced chemiluminescence. ERK2 antiserum (DC3; obtained from Dr. J. E. Ferrell, Stanford University) was raised against XenopusERK2 (27Chou S.-Y. Baichwal V. Ferrell Jr., J.E. Mol. Biol. Cell. 1992; 3: 1117-1130Crossref PubMed Scopus (59) Google Scholar) and recognizes both non-phosphorylated and phosphorylated forms of ERK2. Antiserum was used at a dilution of 1/500. Blots were stripped by incubation in 62.5 mm Tris-HCl, pH 6.8, 100 mm β-mercaptoethanol, 2% SDS for 30 min at 50 °C followed by two washes with phosphate-buffered saline and 0.05% Tween, then blocking. Activation of p38 MAPK requires phosphorylation on both Thr180 and Tyr182, which is specifically recognized by the antibody used, and therefore activation is expressed as the ratio of dual phospho-p38 MAPK to total p38 MAPK immunoreactivity, which allows correction for differences in protein loading. Because dual phospho-p38 immunoreactivity was usually undetectable in control cells, results were normalized to the ratio of the 10-min ischemia sample. Phosphorylation of JNK was assayed as above, except that 50 μg of cell lysate protein was electrophoresed, and filters were probed with anti-active JNK (Promega). The p38 MAP kinase assay kit from New England Biolabs was used with a few modifications. Sodium orthovanadate (2 mm), 1 mm phenylmethylsulfonyl fluoride, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 20 μg/ml soybean trypsin inhibitor were added fresh to lysis buffer, and the cells were lysed by trituration. p38 MAPK was immunoprecipitated, its catalytic activity determined using the in vitro kinase assay to phosphorylate recombinant activating transcription factor-2 (ATF2), and the reaction mixture separated by SDS-PAGE. Western blots were probed with the ATF2 antibody provided in the kit (specific for phospho-Thr71), and immunoreactivity was detected by enhanced chemiluminescence. Filters were stripped as above and reprobed with anti-p38 MAPK. The ratio of phospho-ATF2 to p38 MAPK immunoreactivity was determined for each sample, and then results were expressed as fold activation over control. Cardiac myocytes (one 100-mm dish/treatment) were treated, and then the incubation medium was aspirated and discarded. Cells were washed once with cold phosphate-buffered saline and then scraped into 800 μl of lysis buffer (10 mm Tris-HCl, pH 7.4, 1 mmEDTA, 1 mm EGTA, 1% Triton X-100, 2 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 20 μg/ml soybean trypsin inhibitor) and lysed by trituration. Samples were extracted on ice for 15 min, and then cell debris was removed by centrifugation at 15,000 × g for 10 min. A sample of supernatant was retained for electrophoresis. 20 μl of dual phospho-p38 MAPK (Thr180 Tyr182) antibody (New England Biolabs) was added to the remainder of the supernatant, and samples were rotated overnight at 4 °C. 60 μl of a 50% slurry of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) was added, and the samples were rotated for 2 h at 4 °C. The immunoprecipitates were washed four times with phosphate-buffered saline, electrophoresed on 10% low ratio bisacrylamide SDS-PAGE, then subjected to Western blot analysis with anti-p38 MAPK, anti-dual phospho-p38 MAPK (Thr180 Tyr182), or anti-p38α (Santa Cruz Biotechnology). Autoradiographs were scanned using an ArcusII flatbed scanner (AGFA) with FotoLookPS 2.07.2, and the band density was analyzed by NIH ImageTM. Preparation of nuclear proteins from cardiac myocytes was performed exactly as described by Clerk and Sugden (28Clerk A. Sugden P.H. Biochem. J. 1997; 325: 801-810Crossref PubMed Scopus (56) Google Scholar). Approximately 50 μg of nuclear protein was electrophoresed on 8% SDS-PAGE, and Western blot analysis was performed. ATF2 was detected using phosphorylation state-independent antibody from Santa Cruz Biotechnology. Cell death from ischemic or normoxic incubations was assessed using two dyes that distinguish between live and dead cells. Calcein acetoxymethyl ester (calcein AM; Molecular Probes) and propidium iodide (PI) were added to the incubation medium at final concentrations of 2 μm and 1 μg/ml, respectively, and dishes incubated at 37 °C for 15 min (ischemic samples were maintained under ischemic conditions during this incubation). Cells were viewed using a Zeiss microscope and a 40 × objective and were scored as live (green cytosolic fluorescence from calcein AM) or dead (red nuclear fluorescence from PI). After ischemic or normoxic incubation, cells were scraped into incubation medium to allow retention of any floating cells and were harvested by centrifugation. DNA was prepared by standard techniques (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Identical amounts of DNA (2 μg) were electrophoresed through 1.8% agarose and DNA visualized on a UV transilluminator. Samples were treated exactly as for analysis of p38 MAPK immunoreactivity except that samples were submitted to 12% SDS-PAGE, and filters were probed with anti-CPP32 (H-277) from Santa Cruz Biotechnology. Data were compared using Student'st test for observations between two samples with unequal variance, with one-tailed distribution. A p value of less than 0.05 was considered significant. Activation of MAPKs in primary neonatal rat cardiac myocytes in response to simulated ischemia was investigated. The model used combines two properties of ischemia: decreased energy source, because incubations are performed in the absence of glucose, and hypoxia, with oxygen levels between 0.2 and 0.5%. p38 MAPK activation was estimated by Western blot analysis using an antibody that specifically recognizes the dual phosphorylated (on residues Thr180 and Tyr182), active form of the enzyme. Antibody recognizing p38 MAPK regardless of its phosphorylation state was used to normalize for differences in protein loading. This antibody is specific for the α isoform of p38 MAPK, and the level detected remained constant throughout ischemia. Dual phosphorylation of p38 MAPK was observed within 10 min of ischemia, remained maximal until 30 min, then decreased but remained above basal until 180 min of ischemia (Fig.1, A and B). At later time points, dual phosphorylation increased again with a peak at 240 min and remained high for 420 min (Fig. 1, A andB). We and others (30Webster K.A. Discher D.J. Bishopric N.H. J. Mol. Cell. Cardiol. 1995; 27: 453-458Abstract Full Text PDF PubMed Scopus (69) Google Scholar) have observed that when cardiac myocytes are fed with fresh medium, they cease contracting for a period of time. In our study, cells stopped contracting for approximately 20 min, then spontaneous contraction recovered gradually to a normal rate within 60–90 min. Because this corresponds with the timing of the first phase of p38 MAPK activation, and a change of medium is required to induce ischemic conditions, we examined p38 phosphorylation levels after changing incubation medium and maintaining cells under normoxic conditions. Transient phosphorylation of p38 MAPK was observed after simply feeding fresh medium (Fig. 1, C and D). Cardiac myocytes can be maintained healthily in culture for up to 8 days with multiple changes of medium. Although this does not show that the initial ischemic p38 MAPK activation and that induced by changing medium are equivalent, these results do demonstrate that transient activation of p38 MAPK can occur without long term harmful effects to cardiac myocytes. To confirm that dual phosphorylation of p38 MAPK during ischemia truly reflected activation, p38 MAPK was immunoprecipitated and used in anin vitro kinase assay with recombinant ATF2 as a substrate. We observed more than a 6-fold increase in the phosphorylation of ATF2 over basal after 20 min of ischemia (Fig. 1 E) and more than a 4-fold increase after 25 or 30 min of ischemia (n = 1, data not shown). A similar activation ratio was obtained by incubating cells with anisomycin, an activator of p38 MAPK (Fig.1 E). Thus, p38 MAPK is indeed activated during ischemia. To determine if ischemia-induced phosphorylation is unique to p38 MAPK, we examined phosphorylation of other MAPKs. Phosphorylation of ERK2 (p42 MAPK) was estimated using a gel electrophoresis mobility shift assay with an antibody that detects both inactive (non-phosphorylated) and active (dual phosphorylated) ERK2. The reduced mobility form of ERK2, indicating phosphorylation as seen with 4β-phorbol 12-myristate 13-acetate treatment, was not observed at any time during prolonged ischemia (Fig. 2 A). Similarly, probing with anti-active JNK, which detects the dual phosphorylated active forms of both JNK1 and JNK2, showed little or no activation of JNK in 10–240 min of ischemia (Fig. 2 B) and no activation in 300, 360, or 420 min of ischemia (n = 1, data not shown). Taken together, our results indicate that changing medium transiently activates p38 MAPK, whereas ischemia results in a transient, then sustained activation of p38 MAPK, but not ERK2 or JNK, in primary cultures of neonatal rat cardiac myocytes; these results are consistent with previous studies performed in whole heart (6Bogoyevitch M.A. Gillespie-Brown J. Ketterman A.J. Fuller S.J. Ben-Levy R. Ashworth A. Marshall C.J. Sugden P.H. Circ. Res. 1996; 79: 162-173Crossref PubMed Scopus (490) Google Scholar, 7Yin T. Sandhu G. Wolfgang C.D. Burrier A. Webb R.L. Rigel D.F. Hai T. Whelan J. J. Biol. Chem. 1997; 272: 19943-19950Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 8Knight R.J. Buxton D.B. Biochem. Biophys. Res. Commun. 1996; 218: 83-88Crossref PubMed Scopus (150) Google Scholar). Release of the cytosolic enzyme LDH, caused by cell membrane leakage, was used to assess cell damage resulting from ischemia. Under the conditions used in this study, 7–9 h of ischemic incubation resulted in the release of between 45 and 60% of cellular LDH, whereas 7–9 h of normoxic incubation resulted in release of less than 6% of total LDH. To determine if the activation of p38 MAPK protects myocytes from ischemic stress or mediates the damage from ischemia, we first used anisomycin. Anisomycin is a protein synthesis inhibitor that activates MAPK family members and has been shown to protect myocytes from ischemia (21Weinbrenner C. Liu G.-S. Cohen M.V. Downey J.M. J. Mol. Cell. Cardiol. 1997; 29: 2383-2391Abstract Full Text PDF PubMed Scopus (236) Google Scholar). This result has been used to implicate p38 MAPK in the protective mechanism (21Weinbrenner C. Liu G.-S. Cohen M.V. Downey J.M. J. Mol. Cell. Cardiol. 1997; 29: 2383-2391Abstract Full Text PDF PubMed Scopus (236) Google Scholar). To confirm that anisomycin was protective in our model, cells were pretreated with anisomycin and then subjected to ischemia, either in the presence or absence of additional anisomycin. Fig.3 demonstrates that anisomycin protected myocytes from ischemia-induced injury; LDH release was reduced significantly when anisomycin was present either during the pretreatment only or during both the pretreatment and the prolonged ischemia. Anisomycin is a nonspecific reagent, and therefore these data do not prove that p38 MAPK activation is the mechanism by which anisomycin protects myocytes. More direct evidence for this would require inhibition of the anisomycin-induced protection by a selective p38 MAPK inhibitor. Therefore, we next used an inhibitor of p38 MAPK, SB 203580, which has been shown to inhibit p38 MAPK selectively over other MAPK family members and several other kinases (23Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar, 31Young P.R. McLaughlin M.M. Kumar S. Kassis S. Doyle M.L. McNulty D. Gallagher T.F. Fisher S. McDonnell P.C. Carr S.A. Huddleston M.J. Seibel G. Porter T.G. Livi G.P. Adams J.L. Lee J.C. J. Biol. Chem. 1997; 272: 12116-12121Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). Surprisingly, we found that the presence of SB 203580 during ischemia resulted in a significant dose-dependent decrease in LDH release from myocytes (Fig. 4 A). These data indicate that inhibition of p38 MAPK during ischemia protects myocytes from ischemic damage, whereas previous results have suggested that activation of p38 MAPK protects myocytes. As mentioned previously, activation of p38 MAPK was shown to occur in two phases in these cells (Fig. 1, A and B). To determine if inhibition of p38 MAPK during only one, or both phases, was necessary for the protection seen with SB 203580, the p38 MAPK inhibitor was added at different times during ischemia, and the effect on injury was examined. Adding the inhibitor to cells 45 min after the start of ischemia (which is after the first peak of p38 MAPK phosphorylation, Fig. 1 B) gave a level of protection not significantly different from that seen when the inhibitor was present from the start of ischemia (Fig. 4 B). SB 203580 is a reversible inhibitor; isolated p38 MAPK can be washed free of inhibitor and be fully active (23Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Galla
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