This Article Abstract Full Text (PDF) All Versions of this Article: 89/10/915    most recent hh2201.099452v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, T. C. Articles by Kukreja, R. C. Search for Related Content PubMed PubMed Citation Articles by Zhao, T. C. Articles by Kukreja, R. C. Related Collections Genetically altered mice Ischemic biology - basic studies Physiological and pathological control of gene expression

(Circulation Research. 2001;89:915.)
© 2001 American Heart Association, Inc.

Integrative Physiology

p38 Triggers Late Preconditioning Elicited by Anisomycin in Heart

Involvement of NF-B and iNOS

Ting C. Zhao, Mohiuddin M. Taher, Kristoffer C. Valerie, Rakesh C. Kukreja

From the Division of Cardiology, Departments of Medicine (T.C.Z., R.C.K.), Surgery (M.M.T.), and Radiation Oncology (K.C.V.), Medical College of Virginia, Virginia Commonwealth University, Richmond, Va.

Correspondence to Rakesh C. Kukreja, PhD, Professor of Medicine, Division of Cardiology, Box 281, Medical College of Virginia, Virginia Commonwealth University, 1101 E Marshall St, Richmond, VA 23298. E-mail rakesh{at}hsc.vcu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—— We investigated the role of stress-activated p38 MAP kinase (p38/SAPK-2) signaling in delayed preconditioning of the heart. Adult male out-bred ICR mice were treated with p38 activator, anisomycin (0.1 mg/kg IP), or vehicle (5% DMSO). Twenty-four hours later, hearts were perfused in Langendorff mode and subjected to 30 minutes of ischemia and 30 minutes of reperfusion. Improvement in postischemic recovery of end-diastolic pressure and reduction in infarct size was observed, which was abolished by SB203580, a specific p38 inhibitor, and pyrrolidinediethyldithiocarbamate (PDTC), the NF-B inhibitor, but not by PD 98059, a specific inhibitor for MEK1 or 2. Transient increase in p38 phosphorylation was observed 15 minutes after anisomycin treatment which subsided by 30 minutes. Electrophoretic mobility shift assay demonstrated rapid activation of NF-B DNA binding with anisomycin, peaking at 30 minutes. Western blot confirmed the accumulation of p50 and p65 in nuclear extracts after anisomycin treatment. Anisomycin-induced NF-B DNA binding activity was inhibited by SB203580 and PDTC. Expression of inducible nitric oxide synthase (iNOS) mRNA, protein, and nitric oxide (NO) synthesis were enhanced in anisomycin-treated mice. SB203580 and PDTC blocked the increased expression of iNOS and increase in synthesis of NO. Selective iNOS inhibitor S-methylisothiourea abolished the protective effect of anisomycin. Furthermore, postischemic cardioprotective effect of anisomycin was absent in mice with targeted ablation of iNOS gene but not in the wild-type B6.129 mice. For the first time, these results suggest that direct pharmacological activation of p38 triggers delayed preconditioning by signaling mechanism involving NF-B activation and synthesis of NO from iNOS.

Key Words: anisomycin • p38 mitogen-activated protein kinase • nuclear factor-B • nitric oxide • ischemia

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Brief ischemia before a second sustained ischemia decreases myocardial infarction.¹ This cardioprotective phenomenon, known as ischemic preconditioning (IPC), has been classified into two temporally distinct phases: an early or acute phase that wanes within 2 to 4 hours, and a late phase of IPC that becomes manifest 24 to 72 hours later.^2,3 Recent studies suggest an important role of MAP kinase family, particularly p38, in the early phase of IPC,⁴ but the results have been controversial.⁵ It has been shown that p38 and MAPKAP kinase-2 are strongly activated by ischemia in the perfused rat heart.⁶ Direct activation of p38 by anisomycin mimicked IPC in the hearts,^7,8 myocytes,⁹ and hepatocytes against hypoxic insults.¹⁰ However, the role of p38 in the late preconditioning has not been investigated thoroughly. Dana et al¹¹ showed that late preconditioning induced by transient activation of adenosine A₁ receptor with 2-chloro-N⁶-cyclopentyladenosine (CCPA) was accompanied by significant rise in p38 activity. We further demonstrated that CCPA-induced delayed protection was abolished by p38 inhibitor SB203580 in the mouse heart,¹² suggesting an essential role of p38 in protection. Also, recently we showed that heat stress–induced delayed protection was mediated by MAP kinases.¹³ However, we should consider the fact that IPC or possibly heat shock may trigger a number of additional signaling pathways, including activation of G-protein–coupled receptors, protein kinase C, and MAP kinases such as p44/42, p46/54 (JNK 1/2), and MAPKAP kinase-2.^6,14–17 These signaling molecules can potentially induce the synthesis of effector protein(s) of cardioprotection either individually or in concert. Therefore, it is difficult to elucidate the direct role of a specific MAP kinase such as p38 in cardioprotection using an IPC model. Accordingly, a desirable approach would be to use an agent/drug that selectively targets the signaling molecule of interest. Furthermore, the downstream signaling mechanism(s) by which p38 phosphorylation transduces the stress signal at the level of transcription or posttranslation leading to the development of late preconditioning is not fully understood. Recent studies suggest that IPC triggered activation and translocation of nuclear transcription factor B (NF-B) in the heart,¹⁸ which has been shown to be essential in the development of delayed preconditioning in vivo.¹⁹ In addition, iNOS has been implicated unequivocally in the late phase of cardioprotection induced by pharmacological agents^20,21–23or IPC.^24,25 Accordingly, we hypothesized that direct activation of p38 would trigger delayed preconditioning via downstream activation of NF-B and synthesis of iNOS. In the present study, we used anisomycin, an antibiotic isolated from Streptomyces griseolus that inhibits protein synthesis in eukaryotic ribosomes.²⁶ It inhibits translation by binding to 60S ribosomal subunits and blocking peptide bond formation. More recently, anisomycin has been shown to activate the stress-activated p38 MAP kinase cascades and SAPK/JNK, which makes it a useful tool to study the biological role of phosphorylation pathways in mammalian cells.^27–29 The goals of the present study were as follows: (1) to show if anisomysin induces delayed cardioprotective effect in mouse heart following ischemia/reperfusion; (2) to demonstrate if the late preconditioning effect is mediated by p38 phosphorylation and activation of transcription factor NF-B; (3) to determine the role of p38 activation in the expression of iNOS, synthesis of nitric oxide (NO) and to provide their cause and effect in delayed cardioprotection. Our results suggest that targeted phosphorylation of p38 with anisomycin leads to the delayed cardioprotective effect, which is mediated by NF-B activation and synthesis of iNOS. Also, the anisomycin-induced delayed cardioprotection is absent in mice with targeted ablation of iNOS gene.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Adult male out-bred mice (ICR Strain, 30 to 40 g) were supplied by Harlan (Indianapolis, Ind). Adult males with targeted disruption of iNOS gene (-/-) B6.129 and B6.129 wild-type mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) The care and use of animals were conducted in accordance with the guidelines of the Committee on Animals of Virginia Commonwealth University and the National Institutes of Health (DHHS publication No. 80-23, revised).

Chemicals and Supplies
Anisomycin was obtained from RBI; SB203580 and PD98059 were obtained from Calbiochem; gel electrophoresis supplies were obtained from BioRad Laboratories; pyrrolidinediethyldithiocarbamate (PDTC) and all other chemicals were obtained from Sigma Chemical. The antibodies for Western blotting were obtained from the following companies: phospho-specific p38 MAPK (Thr 180/Tyr 182), New England Biolabs; and p38, iNOS, p50, p65, and rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, Calif).

Langendorff’s Isolated Heart Perfusion
The methodology of Langendorff’s isolated perfused mouse heart preparation and measurement of contractile function have been described previously in detail.^20,23,30 Transverse slices of the heart were stained in 10% TTC for 30 minutes followed by measurement of the infracted areas using computer morphometry as described previously.^20,21,23 The infarct size was measured after 30 minutes of reperfusion, which is sufficient for accurate detection of infarct size as described by Schaper et al.³¹

Experimental protocol
Cardiac Function and Infarct Studies
Mice were randomized into 12 groups and received the following drugs intraperitoneal 24 hours before sacrifice. (1) Vehicle (n=11): 5% DMSO; (2) anisomycin (n=7): treatment with anisomycin (0.1 mg/kg); (3) SB203580+anisomycin (n=6): SB203580 (1.0 mg/kg) was injected 30 minutes before anisomycin; (4) SB203580 (n=5): treatment with SB203580 alone; (5) PDTC+anisomycin (n=6): PDTC (150 mg/kg) was injected 30 minutes before anisomycin; (6) PDTC (n=5): treatment with PDTC alone; (7) PD98059+ anisomycin (n=9): PD 98059 (1.0 mg/kg) was injected 30 minutes before anisomycin; (8) PD 98059 (n=6): pretreatment with PD 98059 alone; (9) S-methylisothiourea (SMT)+anisomycin (n=7): iNOS inhibitor SMT (3 mg/kg) was injected 30 minutes before anisomycin; (10) SMT (n=6): treatment with SMT alone; (11) anisomycin+iNOS-KO (n=6): treatment of iNOS gene knockout mice with anisomycin; (12) anisomycin+B6.129 (n=7): treatment of the wild-type B6.129 mice with anisomycin.

p38 Phosphorylation
A subset of animals were treated with anisomycin, and 10, 15, and 30 minutes later, the hearts were removed for measurement of p38 phosphrylation.

NF-B Binding Activity
To determine the time course of NF-B binding activity, another set of mice were treated with either DMSO or anisomycin, and 15, 30, 60, and 120 minutes later, left ventricular (LV) samples were harvested and stored frozen at -70°C until analyzed for NF-B by electrophoretic mobility shift assay (EMSA). Another subset of mice was treated with drugs as described in Group 1 to 6. The tissue samples were collected 30 minutes later for measurement of NF-B binding activity. These mice were not subjected to ischemia/reperfusion protocol.

Expression of iNOS and NO Synthesis
Mice were treated with anisomycin, and 6, 10, 16, and 24 hours later, LV tissue was harvested and frozen in liquid nitrogen for measurement of iNOS mRNA by RT-PCR. Another subset of mice (n=3 to 4) was treated as described in Group 1 to 6, and LV tissue was collected 24 hours later to measure iNOS protein and NO.

Western Blot Analysis
Frozen tissue samples were ground in liquid nitrogen and lysed in the Tris buffer (pH 7.9) containing 140 mmol/L NaCl, 1.5 mmol/L MgCl₂, 1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L DTT, 0.5% NP-40, 0.5 mmol/L sodium orthovanadate, and protease inhibitors (0.5 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, and 1 µg/mL leupeptin). The samples were homogenized using a Polytron (Brinkman) and microcentrifuged at 14 000 rpm for 10 minutes. The protein content of the supernatant was determined using the DC-protein assay (BioRad). Western blots were performed as per the manufacturer’s instructions (Cell Signaling Technology). The protein was separated by SDS-PAGE using 12% SDS for p38, 10% SDS for p50, and p65 NF-B proteins or 8% SDS for iNOS protein. The gels were incubated with the respective primary antibody, washed, and visualized by incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibody BioRad (Hercules, Calif) and ECL Chemiluminescence Detection Reagent from Amersham Pharmacia Biotech.

Electrophoretic Mobility Shift Assay
Preparation of nuclear extract and EMSA were performed according to the method of Taher et al.³² A double-stranded 22-mer oligonucleotide with the sequence 5'-AGTTGAGGGGACTTTAGGC-3' (Promega Corp) was end-labeled using [-³²p] ATP (ICN) and T4 polynucleotide kinase according to the manufacturer’s instructions. To ensure the specificity of NF-B, gel competition assay was performed by incubating nuclear extracts with 50- and 100-fold molar excess of unlabeled (cold) doubled-stranded NF-B before addition of [³²p]-labeled NF-B oligonucleotide.

RT-PCR of iNOS
Total cellular RNA was isolated from LV tissue with Trizol reagent (Gibco BRL, Life Technologies). RNA (5 µg) was reverse transcribed to generate cDNA using standard methodology. The reverse-transcribed cDNA (10 µL) was amplified in a final volume of 100 µL by PCR under standard conditions (1.5 mmol/L MgCl₂, 200 µmol/L dNTP, and 4.0 U Taq polymerase) with 50 µmol/L of primers for iNOS based on the sequence for murine iNOS cDNA. The primers were synthesized with the following sequences: iNOS, up: 5'-TGTCGCAGCTCCCTATCTTG-3'; down: 5'-CATTG-GCCAGCTGCTTTTGC-3'. The experimental conditions for PCR were 30 cycles for 60 seconds at 94°C, 55°C, and 72°C. After amplification, 10 µL of PCR product per lane was resolved on 1.5% agarose gel containing ethidium bromide in 1x TBA buffer. Bands were confirmed under UV fluorescence and purified by using a Qiagen purification kit. The nucleotide sequences for iNOS was determined by the same up primer used for PCR reaction and a DNA sequencer. The reproducibility of iNOS RT-PCR was confirmed by at least 2 samples in each group.

Measurement of NO
NO was measured with a Sievers nitric oxide analyzer (model 280), as described previously.²⁰ Briefly, hearts were collected, powdered under liquid nitrogen, and homogenized at 4°C with a Polytron PT 20 (4 bursts of 5 seconds each) in 5 volumes of PBS (pH 7.4). They were then centrifuged at 14 000 rpm for 10 minutes and the pellets discarded. Samples were deproteinized with 200° proof ethyl alcohol at 0°C in a 1:2 v/v mix and incubated 30 minutes at 0°C followed by centrifugation at 14 000 rpm for 5 minutes. The pellets were discarded and the supernatant was used to measure inactivated NO in the form of nitrate, nitrite, and S-nitrosocompounds by reducing these to NO with vanadium (III)–HCl. NO was then measured based on a gas-phase chemiluminescent reaction between NO and ozone with a NOA 280 (Sievers Instruments).

Statistical Analysis
The results are expressed as the mean±SEM. Difference among the groups were analyzed by one-way analysis of variance (ANOVA). Statistical differences were considered significant with a value of P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infarct Size
Myocardial infarct size was 30.9±2.4% in the vehicle-treated hearts, which was reduced to 10.9±3.4% after treatment with anisomycin (P<0.05, Figure 1). SB303580, the inhibitor of p38, abolished the protective effect of anisomycin as indicated by increase in the infarct size to 30.0±3.1% (P<0.05). The infarct size in SB203580-treated mice was 28.3±2.6%, which was not different from the vehicle group (P>0.05). However, PD 98059, the inhibitor of ERK, failed to block the infarct limiting effect of anisomycin, ie, 13.8±2.9% versus 10.9±3.4% with anisomycin (P>0.05). Also, PD 98059 had no effect on infarct size in the control hearts that were not pretreated with anisomycin (infarct size: 29.0±1.4% versus 30.9±2.4% in the vehicle group). PDTC, a potent inhibitor of NF-B, blocked the infarct limiting effect of anisomycin while having no effect in the control hearts, ie, 25.9±2.7% versus 30.9±2.4% in the vehicle control group. SMT, a selective iNOS inhibitor, when administrated 30 minutes before ischemia and reperfusion blocked the protective effect of anisomycin as indicated by increase in infarct size to 23.8±3.6%. The infarct size in SMT-treated control mice was 26.0±4.0% that was also not different compared with the SMT+anisomycin or vehicle treated control group. Treatment of iNOS gene knockout mice with anisomycin failed to reduce infarct size (25.2±4.4% versus 30.9±2.4% in the vehicle group, P>0.05). However, treatment of B6.129 wild-type strain of mice with anisomycin still exhibited a reduction in the infarct size (8.7±2.4% versus 30.9±2.4% in the vehicle group, Figure 1).

View larger version (52K): [in this window] [in a new window]   Figure 1. Effect of anisomycin on myocardial infarct size after ischemia/reperfusion. The description of groups and drug dosages are provided under experimental protocol "Cardiac Function and Infarct Studies" in Materials and Methods. Values represent mean±SE. Aniso indicates anisomycin; SB, SB203580; SMT, S-methylisothiourea; PDTC, pyrrolidinediethyldithiocarbamate; iNOS-KO, inducible nitric oxide synthase knockout mice; and B6.129, wild-type littermates of iNOS.

Ventricular Function
Myocardial functional parameters LVSP, LVEDP, heart rate, and coronary flow were not significantly different between groups during preischemia (Table). Postischemic LVEDP improved in the anisomysin-treated group (4.4±1.4 mm Hg) as compared with the vehicle group (25.0±3.6 mm Hg, P<0.05, Figure 2A). Anisomycin-induced improvement in LVEDP was abolished by SB203580 (30.1±3.1 mm Hg), PDTC (21.3±4.5 mm Hg, P<0.05), and SMT (17.4± 2.9 mm Hg, P<0.05), but not by PD98059 (3.7±1.5 mm Hg, P>0.05). LVEDP was elevated in iNOS knockout mice (21.8±4.7 mm Hg) as compared with B6.129 wild-type strain mice (3.0±2.1 mm Hg, P<0.05). The recovery of postischemic LVDP was 57.2±7.1 mm Hg in the vehicle group, which increased to 75.2±3.4 mm Hg in the anisomycin group (P>0.05, Figure 2B). LVDP was 61.8±5.8, 66.0±8.7, and 55.7±11.5 mm Hg in the SB203580, PDTC, and SMT groups, respectively (P>0.05 versus anisomycin treated group). Anisomycin induced improvement of LVDP was not blocked by PD98059 (84.0±5.4 mm Hg). Postischemic recovery of LVDP in the iNOS gene knockout mice was lower as compared with B6.129 wild-type mice (50.1±9.7 versus 70.0±3.2 mm Hg) after anisomycin treatment. An identical trend in the changes in the recoveries of rate pressure product (Figure 2C) was observed. Postischemic recoveries of heart rate and coronary flow were similar in all the groups (not shown).

View this table: [in this window] [in a new window]   Table 1. Preischemic Baseline Hemodynamic Data

View larger version (70K): [in this window] [in a new window]   Figure 2. Postischemic recovery of left ventricular end-diastolic pressure (A), developed pressure (B), and rate pressure product (C) 24 hours after anisomycin treatment. Refer to Figure 1 legend for group abbreviations. The description of groups and drug dosages are provided under experimental protocol "Cardiac Function and Infarct Studies" in Materials and Methods.

p38 Phosphrylation
Phosphrylated p38 was present at low levels in the vehicle-treated mice. A mild increase in phosphrylated p38 was detected 10 minutes after treatment with anisomycin, which reached maximum level at 15 minutes and declined by 30 minutes (Figure 3A). Quantitative analysis showed that anisomycin elicited a 4-fold increase in p38 phosphorylation at 15 minutes after treatment. Total p38 expression remained unaltered (Figure 3B).

View larger version (40K): [in this window] [in a new window]   Figure 3. Time course of p38 phosphorylation by anisomycin: after treatment with anisomycin, myocardial samples were obtained at 0, 10, 15, and 30 minutes later as described under experimental protocol "p38 Phosphorylation." A, Western blots of tissue extracts with an antibody against phospho p38. The blots were stripped and probed again with an antibody against total p38. B, Densitometric analysis of phosphorylated p38. Results were normalized by arbitrarily setting the densitometry of control group. Results are mean±SE, n=6, *P<0.05 vs control. No significant change in total p38 was observed.

NF-B Binding Activity
NF-B binding activity was low or undetectable in vehicle-treated mice. Administration of anisomycin resulted in a rapid increase in NF-B binding activity at 15 minutes, which peaked at 30 minutes (Figure 4A). Quantitative analysis showed that NF-B binding activity increased 4.3-fold at 30 minutes (P<0.05 versus control) and returned to values not significantly different from control group after 2 hours of treatment (Figure 4B). Western blot showed accumulation of NF-B p50 and p65 protein in nuclear extract after anisomycin treatment (Figure 4C). The anisomycin-induced increase in NF-B binding activity was attenuated by pretreatment with SB203580 and PDTC (Figure 5A). SB203580 and PDTC had no effect on NF-B binding activity (Figure 5A and 5B). Incubation of nuclear extracts with 50-fold molar excess of cold oligonucleotide inhibited formation of the NF-B/DNA complex after anisomycin treatment confirming the specificity of binding (Figure 5C).

View larger version (33K): [in this window] [in a new window]   Figure 4. Time course of NF-B DNA binding and p50/p65 accumulation after anisomycin treatment. Nuclear extracts were prepared as described in detail under Materials and Methods. The extracts were incubated with the 32P-labeled NF-B oligonucleotide probe to determine the binding activity using EMSA. A, Specific binding of NF-B complex is indicated by an arrow. B, Densitometric analysis of NF-B DNA binding activity. C, Western blot of p50 and p65. Results were normalized by arbitrarily setting the densitometry of control group and expressed as mean±SE (n=3). *P<0.05 vs Control.

View larger version (51K): [in this window] [in a new window]   Figure 5. Effect of SB203580 and PDTC on NF-B DNA binding activity. Mice were treated with SB203580 and PDTC 30 minutes before administration of anisomycin as described under experimental protocol "Cardiac Function and Infarct Studies" in Materials and Methods. EMSA was performed to measure NF-B DNA binding activity in nuclear extracts as described under Materials and Methods. A, EMSAs performed after various treatments. B, Densitometric analysis of NF-B DNA binding activity (*P<0.05 vs anisomycin, n=3). C, Competition assay of NF-B DNA binding activity. The binding activity assay was performed in the absence or presence of increasing 50- and 100-fold molar excess of unlabeled probe.

Expression of iNOS and Synthesis of NO
Enhanced RT-PCR product of the predicted size (492 bp) was identified beginning at 6 hours, which remained elevated up to 24 hours after anisomycin administration (Figure 6). The RT-PCR product showed 100% homology with murine macrophage iNOS sequence. iNOS protein was observed in vehicle group, which was significantly elevated by 24 hours after anisomycin treatment (Figure 7A). Densitometric analysis showed a 2.1-fold increase in iNOS protein at 24 hours after anisomycin treatment as compared with control (P<0.05), which decreased with SB203580 and PDTC. SB203580 and PDTC had little effect on iNOS protein (Figure 7B). Furthermore, pretreatment with anisomycin caused increase in NO levels 24 hours later compared with vehicle group (8.0±1.0 versus 4.9±0.9 nmol/mg · protein, P<0.05, Figure 7C), which was significantly decreased by SB203580 (2.80±0.07 nmol/mg · protein) and PDTC (2.4±0.3 nmol/mg · protein).

View larger version (20K): [in this window] [in a new window]   Figure 6. RT-PCR showing time course of iNOS transcription by anisomycin treatment. PCR products were amplified from cDNA of iNOS using murine iNOS-specific primers (see Materials and Methods for details).

View larger version (33K): [in this window] [in a new window]   Figure 7. Western blot analysis showing iNOS expression and NO synthesis with anisomycin. Mice were treated with SB203580 and PDTC before administration of anisomycin as described under experimental protocol "Cardiac Function and Infarct Studies" in Materials and Methods. Twenty-four hours later, the myocardial tissue was processed and analyzed for iNOS by Western blots using an antibody against iNOS. NO was measured using Siever’s NO analyzer. A, Western blot; B, Densitometric analysis of iNOS protein; and C, NO content. (*P<0.05 vs anisomycin, n=3 to 4/group).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results show that selective activation of p38 with anisomycin triggered delayed cardioprotection in the heart, as indicated by reduction in infarct size and significant improvement of postischemic LVEDP after global ischemia and reperfusion. In addition, these results provide direct evidence for the causative role of p38 in triggering downstream signaling involving activation of NF-B, expression of iNOS, and increased synthesis of NO leading to delayed cardioprotection.

The role of p38 signaling in acute IPC has been extensively investigated, although conflicting results have been obtained.⁵ Maulik et al⁶ suggested the involvement of tyrosine kinase-phospholipase D as a potential signaling pathway in IPC. Weinbrener et al⁴ showed an increase of p38 phosphorylation in the preconditioned heart that was blocked by adenosine receptor antagonist. Sakamoto et al³³ also demonstrated partial blockade of the cardioprotective effect of CCPA with SB203580 in the isolated perfused rat heart. A transient dual phosphorylation and activation of p38 occurred during preconditioning,³⁴ which was attenuated during sustained ischemia/reperfusion and was associated with improved functional recovery. In contrast, SB203580 decreased myocardial apoptosis,³⁵ improved postischemic cardiac function,³⁶ and decreased LDH release in myocytes after ischemia.⁹ The role of p38 in late preconditioning has been described after pharmacological activation of adenosine A₁ receptor. Increased expression of phospho-specific p38 was observed 24 hours after stimulation with A₁ receptor in the rabbit and mouse hearts, which was blocked by genistein¹¹ and SB203580.¹² These studies suggested that p38 pathway as a potential distal effector of late preconditioning. In the present studies, administration of SB203580 before anisomycin abolished the protection suggesting that p38 acts as a trigger of delayed protection. It is not clear which specific isoform(s) of p38, ie, , ß, , or are phosphorylated with anisomycin in the present studies. However, it is well known that SB 203580 is a potent inhibitor of p38 and ß, but has little effect on and .³⁷ Thus, based on our data on blockade of anisomycin-induced delayed cardioprotection with SB 203580, it appears that p38 and ß isoforms may be involved. Further studies are needed to resolve the isoform-selective effects of anisomycin. The role of SAPK/JNK, however, remains speculative, because so far no specific antagonist for this kinase has been developed. Previous studies have shown that anisomycin causes a low level dual-phosphorylation of p44/42 MAP kinase in HepG2 cells.³⁸ However, pretreatment of mice with PD98059, a specific inhibitor for MEK1 or 2, the upstream activator of p42/44 MAP kinase,³⁹ failed to abolish anisomycin-mediated delayed cardioprotection. In contrast, both p44/42 MAP kinase and p38 have been suggested to be integral components of delayed pharmacological preconditioning induced by activation of opioid receptor and were proposed to be acting via parallel pathways.⁴⁰

In the present studies, we observed a rapid transient increase in NF-B DNA binding activity within 30 minutes after treatment with anisomycin, which was attenuated by 2 hours. SB203580 and PDTC, the specific inhibitor of NF-B,⁴¹ not only blocked the DNA binding but also abolished the delayed cardioprotection. It has been suggested that p38 signals through the transcription factor NF-B.^18,42 NF-B exists in the cytoplasm of many cells complexed to an inhibitor, IB. Treatment of cells with TNF-, IL-1, and LPS leads to phosphorylation and proteolytic degradation of IB and release of NF-B, which then translocates to the nucleus and binds its cognate DNA sequence on responsive genes. NF-B activation has also reported to be essential for the development of delayed PC in vivo.¹⁹ Our results show that peak p38 phosphorylation preceded the maximum NF-B activation after anisomycin treatment suggesting the possibility that NF-B is the downstream target of p38 in the signaling cascade. The mechanism by which stimulation of p38 increases NF-B DNA binding is not clear. It is likely that p38 increased NF-B binding activity by phosphorylation of its inhibitor IB or indirectly via intermediate kinases.^18,43

Our results also demonstrated that anisomycin caused rapid transcription of iNOS mRNA, which peaked by 6 to 24 hours later. Additionally, anisomycin caused increased transcription of iNOS gene and NO synthesis 24 hours later, which was attenuated by SB203580, suggesting the role of p38 activation in regulating iNOS expression. Similarly, anisomycin was shown to increase mRNA levels, iNOS activity, and protein in rat aortic smooth muscle cells.⁴⁴ Although anisomycin is a protein synthesis inhibitor, it can efficiently activate transcription of certain genes, including either the iNOS or the c-fos.⁴⁵ Anisomycin-induced iNOS protein synthesis was dependent on activation of NF-B because pretreatment with PDTC attenuated the increase in iNOS protein and NO levels.

Finally, we observed that inhibition of iNOS or targeted ablation of this gene in mice resulted in abrogation of anisomycin-induced delayed cardioprotection, suggesting an essential role of NO in p38-triggered protection. It is likely that NO is a mediator in this protective effect because SMT was administered 24 hours after anisomycin treatment. Previous studies have shown that NO is the possible trigger as well as the mediator for PC⁴⁶ because administration of a NO-synthase inhibitor L-nitroarginine and aminoguanidine abolished the delayed PC against myocardial stunning and infarction.^24,25 We have shown that NO plays a prominent role both in mediating this delayed cardioprotective response induced by adenosine A₁/A₃ receptor activation,^23,47 mitoK_ATP channel opener, diazoxide,⁴⁸ and monophosphoryl lipid A or its synthetic analogue, RC522.^20,21 These studies also showed an absence of delayed protection in the iNOS knockout homozygous mutant mice. Therefore, it appears that there is significant redundancy in the signaling pathways induced either by direct pharmacological treatment or stress (IPC) in the heart.

In conclusion, we have provided evidence that targeted activation of p38 by anisomycin triggers downstream signaling pathway leading to the rapid transient activation of NF-B, transcription and synthesis of iNOS, as well as NO leading to the development of long-lasting cardioprotective effect. These effects of p38 activation were observed in the absence of confounding effects of activation of membrane receptors and ensuing signaling cascade. The selective inhibition of either the trigger, ie, p38 or the mediators (NF-B or iNOS) resulted in the abrogation of delayed cardioprotective effect. In addition, the absence of delayed cardioprotective response in the iNOS gene-knockout mice further provides genetic evidence of the essential role of iNOS in this signaling cascade. We propose that novel drugs/therapies could be developed based on targeted transient activation of p38 that may lead to late preconditioning effect in the ischemic heart.

	Acknowledgments

 
This work was supported in part by HL-51045 and HL-59469 (R.C.K.) and a Grant-in-Aid (0160420 U) from the American Heart Association, Mid-Atlantic Consortium (T.C.Z.).

Received August 6, 2001; revision received September 20, 2001; accepted September 20, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124–1136.
2. Van Winkle DM, Thornton J, Downey JM. The natural history of preconditioning: cardioprotection depends on duration of transient ischemia and time to subsequent ischemia. Coron Artery Dis. 1991; 2: 613–619.
3. Baxter GF, Goma FM, Yellon DM. Characterisation of the infarct-limiting effect of delayed preconditioning: timecourse and dose-dependency studies in rabbit myocardium. Basic Res Cardiol. 1997; 92: 159–167.
4. Weinbrenner C, Liu GS, Cohen MV, Downey JM. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J Mol Cell Cardiol. 1997; 23: 2383–2391.
5. Ping P, Murphy E. Role of p38 mitogen-activated protein kinases in preconditioning: a detrimental factor or a protective kinase? Circ Res. 2000; 86: 921–922.
6. Maulik N, Watanabe M, Zu YL, Huang CK, Cordis GA, Schley JA, Das DK. Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts. FEBS Letters. 1996; 396: 233–237.
7. Sato M, Cordis GA, Maulik N, Das DK. SAPKs regulation of ischemic preconditioning. Am J Physiol. 2000; 279: H901–H907.
8. Baines CP, Liu GS, Birincioglu M, Critz SD, Cohen MV, Downey JM. Ischemic preconditioning depends on interaction between mitochondrial K_ATP channels and actin cytoskeleton. Am J Physiol. 1999; 276: H1361–H1368.
9. Mackay K, Mochly-Rosen D. An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia. J Biol Chem. 1999; 274: 6272–6279.
10. Carini R, Grazia De Cesaris M, Splendore R, Domenicotti C, Paola Nitti M, Adelaide Pronzato M, Albano E. Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes. Hepatology. 2001; 33: 131–139.
11. Dana A, Skarli M, Papakrivopoulou J, Yellon DM. Adenosine A₁ receptor induced delayed preconditioning in rabbits: induction of p38 mitogen-activated protein kinase activation and Hsp27 phosphorylation via a tyrosine kinase- and protein kinase C-dependent mechanism. Circ Res. 2000; 86: 921–932.
12. Zhao TC, Hines DS, Kukreja RC. Adenosine-induced late preconditioning in mouse hearts: role of p38 MAP kinase and mitochondrial K_ATP channels. Am J Physiol. 2001; 280: 278–285.
13. Tekin D, Xi L, Zhao TC, Tajero-Taldo MI, Atluri S, Kukreja RC. Mitogen-activated protein kinases mediate heat shock-induced delayed protection in mouse heart. Am J Physiol. 2001; 281: H523–H532.
14. Okubo S, Xi L, Bernardo NL, Yoshida K-I, Kukreja RC. Myocardial precondiitoning: basic concepts and mechanisms. Mol Cell Biochem. 1999; 196: 3–12.
15. Ping P, Zhang J, Huang S, Cao X, Tang XL, Li RC, Zheng YT, Qiu Y, Clerk A. PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits. Am J Physiol. 2001; 277: H1771–H1785.
16. Ping P, Zhang J, Cao X, Li RC, Kong D, Tang XL, Qiu Y, Manchikalapudi S, Auchampach JA, Black RG, Bolli R. PKC-dependent activation of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious rabbits. Am J Physiol. 1999; 276: H1468–H1481.
17. Okubo S, Bernardo NL, Elliott GT, Hess ML, Kukreja RC. Tyrosine kinase signaling in action potential shortening and expression of HSP72 in late preconditioning. Am J Physiol. 2000; 279: H2269–H2276.
18. Maulik N, Sato G, Price BD, Das DK. An essential role of NFB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett. 1998; 429: 365–369.
19. Xuan YT, Tang XL, Banerjee S, Takano H, Li RC, Han H, Qui Y, Li JJ, Bolli R. Nuclear factor-B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res. 1999; 84: 1095–1109.
20. Xi L, Jarrett NC, Hess ML, Kukreja RC. Essential role of inducible nitric oxide synthase in monophosphoryl lipid A-induced late cardioprotection: evidence from pharmacological inhibition and gene knockout mice. Circulation. 1999; 99: 2157–2163.
21. Xi L, Salloum F, Tekin D, Jarrett NC, Kukreja RC. Glycolipid RC-552 induces delayed preconditioning-like effect via iNOS-dependent pathway in mice. Am J Physiol. 2000; 277: H2418–H2424.
22. Xi L, Kukreja RC. Pivotal role of nitric oxide in delayed pharmacological preconditioning against myocardial infarction. Toxicology. 2000; 155: 37–44.
23. Zhao T, Xi L, Chelliah J, Levasseur JE, Kukreja RC. Inducible nitric oxide synthase mediates delayed myocardial protection induced by activation of adenosine A₁ receptors: evidence from gene-knockout mice. Circulation. 2000; 102: 902–907.
24. Bolli R, Manchikalapudi S, Tang XL, Takano H, Qiu Y, Guo Y, Zhang Q, Jadoon AK. The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase: evidence that nitric oxide acts both as a trigger and as a mediator of the late phase of ischemic preconditioning. Circ Res. 1997; 81: 1094–1107.
25. Takano H, Manchikalapudi S, Tang XL, Qiu Y, Rizvi A, Jadoon AK, Zhang Q, Bolli R. Nitric oxide synthase is the mediator of late preconditioning against myocardial infarction in conscious rabbits. Circulation. 1998; 98: 441–449.
26. Barbacid M, Vazquez D. [³H]Anisomycin binding to eukaryotic ribosomes. J Mol Biol. 1974; 84: 603–623.
27. Mahadevan LC. Signaling and superinduction. Nature. 1991; 349: 747–748.
28. Cano E, Hazzalin CA, Mahadevan LC. Identification of anisomycin-activated kinases and p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos-cjun induction. Mol Cell Biol. 1994; 14: 7352–7362.
29. Nahas N, Molski TF, Fernandez GA, Sha’afi RI. Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists. Biochem J. 1996; 318: 247–253.
30. Xi L, Hess ML, Kukreja RC. Ischemic preconditioning in isolated perfused mouse heart: reduction in infarct size without improvement of postischemic ventricular function. Mol Cell Biochem. 1998; 186: 69–77.
31. Schaper W, Binz K, Sass S, Winkler B. Influence of collateral blood flow and of variations in MVO₂ on tissue-ATP content in ischemic and infarcted myocardium. J Mol Cell Cardiol. 1987; 19: 19–37.
32. Taher MM, Baumgardner T, Dent P, Valerie KC. Genetic evidence that stress-activated p38 MAP kinase is necessary but not sufficient for UV activation of HIV gene expression. Biochemistry. 2001; 38: 13055–13062.
33. Sakamoto K, Urushidani T, Nagao T. Translocation of HSP27 to sarcomere induced by ischemic preconditioning in isolated rat hearts. Biochem Biophys Res Comm. 2000; 269: 137–142.
34. Marais E, Genade S, Huisamen B, Strijdom JG, Moolman JA, Lochner A. Activation of p38 MAPK induced by a multi-cycle ischemic preconditioning protocol is associated with attenuated p38 MAPK activity during sustained ischaemia and reperfusion. J Mol Cell Cardiol. 2001; 33: 769–778.
35. Barancik M, Hatun P, Strohm C, Kilian S, Schaper W. Inhibition of the cardiac p38-MAPK pathway by SB203580 delays ischemic cell death. J Cardiovasc Pharmacol. 2000; 35: 474–483.
36. Ma XL, Kumar S, Gao F, Louden CS, Lopez BL, Christopher TA, Wang C, Lee JC, Feuerstein GZ, Yue TL. Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation. 1999; 99: 1685–1691.
37. Lee JC, Kassis S, Kumar S, Badger A, Adams JL. p38 Mitogen-activated protein kinase inhibitors: mechanisms and therapeutic potential. Pharmacol Ther. 1999; 82: 389–397.
38. Dhawan P, Bell A, Kumar A, Golden C, Mehta KD. Critical role of p42/44 MAPK activation in anisomycin and hepatocyte growth factor-induced LDL receptor expression: activation of Raf-1/MEK-1/p42/44 MAPK cascade alone is sufficient to induce LDL receptor expression. J Lipid Res. 1999; 40: 1911–1919.
39. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995; 92: 7686–7689.
40. Fryer RM, Hsu AK, Gross GJ. ERK and p38 MAP kinase activation are components of opioid-induced delayed cardioprotection. Basic Res Cardiol. 2001; 96: 136–142.
41. Schreck R, Meier B, Mannel DN, Droge W, Baeuerle PA. Dithiocarbamates as potent inhibitors of nuclear factor B activation in intact cells. J Exp Med. 1992; 175: 1181–1194.
42. Liang F, Gardner DG. Mechanical strain activates BNP gene transcription through a p38/NF-B–dependent mechanism. J Clin Inv. 1999; 104: 1603–1612.
43. Ghosh S, Baltimore D. Activation in vitro of NF- B by phosphorylation of its inhibitor IkB. Nature. 1990; 344: 678–682.
44. Marczin N, Go CY, Papapetropoulos A, Catravas JD. Induction of nitric oxide synthase by protein synthesis inhibition in aortic smooth muscle cells. Br J Pharmacol. 1998; 123: 1000–1008.
45. Oguchi S, Weisz A, Esumi H. Enhancement of inducible-type NO synthase gene transcription by protein synthesis inhibitors: activation of an intracellular signal transduction pathway by low concentrations of cycloheximide. FEBS Lett. 1994; 338: 326–330.
46. Bolli R, Dawn B, Tang X-L, Qiu Y, Ping P, Xuan YT, Jones WK, Takano H, Guo Y. The nitric oxide hypothesis of late preconditioning. Basic Res Cardiol. 1998; 93: 325–328.
47. Zhao TC, Kukreja RC. Adenosine A₃ receptor induces late preconditioning by activation of nuclear factorB and nitric oxide synthase in mouse heart. J Mol Cell Cardiol. 2001; 33: A137.Abstract.
48. Ockaili R, Emani VR, Okubo S, Brown M, Krottapalli K, Kukreja RC. Opening of mitochondrial K_ATP channel induces early and delayed cardioprotective effect: role of nitric oxide. Am J Physiol. 1999; 277: H2425–H2434.

This article has been cited by other articles:

				W. Chai, Y. Wu, G. Li, W. Cao, Z. Yang, and Z. Liu Activation of p38 mitogen-activated protein kinase abolishes insulin-mediated myocardial protection against ischemia-reperfusion injury Am J Physiol Endocrinol Metab, January 1, 2008; 294(1): E183 - E189. [Abstract] [Full Text] [PDF]

				P. L. Hermonat, D. Li, B. Yang, and J. L. Mehta Mechanism of action and delivery possibilities for TGF{beta}1 in the treatment of myocardial ischemia Cardiovasc Res, May 1, 2007; 74(2): 235 - 243. [Abstract] [Full Text] [PDF]

				W. Chai and Z. Liu p38 Mitogen-Activated Protein Kinase Mediates Palmitate-Induced Apoptosis But Not Inhibitor of Nuclear Factor-{kappa}B Degradation in Human Coronary Artery Endothelial Cells Endocrinology, April 1, 2007; 148(4): 1622 - 1628. [Abstract] [Full Text] [PDF]

				R. M. Bell, J. E. Clark, D. J. Hearse, and M. J. Shattock Reperfusion kinase phosphorylation is essential but not sufficient in the mediation of pharmacological preconditioning: Characterisation in the bi-phasic profile of early and late protection Cardiovasc Res, January 1, 2007; 73(1): 153 - 163. [Abstract] [Full Text] [PDF]

				T. Zhao, P. Parikh, S. Bhashyam, H. Bolukoglu, I. Poornima, Y.-T. Shen, and R. P. Shannon Direct Effects of Glucagon-Like Peptide-1 on Myocardial Contractility and Glucose Uptake in Normal and Postischemic Isolated Rat Hearts J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1106 - 1113. [Abstract] [Full Text] [PDF]

				M. Massip-Salcedo, A. Casillas-Ramirez, R. Franco-Gou, R. Bartrons, I. Ben Mosbah, A. Serafin, J. Rosello-Catafau, and C. Peralta Heat Shock Proteins and Mitogen-activated Protein Kinases in Steatotic Livers Undergoing Ischemia-Reperfusion: Some Answers Am. J. Pathol., May 1, 2006; 168(5): 1474 - 1485. [Abstract] [Full Text] [PDF]

				Y.-Q. Zhu and X.-D. Tan TFF3 modulates NF-{kappa}B and a novel negative regulatory molecule of NF-{kappa}B in intestinal epithelial cells via a mechanism distinct from TNF-{alpha} Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1085 - C1093. [Abstract] [Full Text] [PDF]

				R. Ockaili, R. Natarajan, F. Salloum, B. J. Fisher, D. Jones, A. A. Fowler III, and R. C. Kukreja HIF-1 activation attenuates postischemic myocardial injury: role for heme oxygenase-1 in modulating microvascular chemokine generation Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H542 - H548. [Abstract] [Full Text] [PDF]

				M. Zhang, Y.-J. Xu, H. K. Saini, B. Turan, P. P. Liu, and N. S. Dhalla Pentoxifylline attenuates cardiac dysfunction and reduces TNF-{alpha} level in ischemic-reperfused heart Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H832 - H839. [Abstract] [Full Text] [PDF]

				L. Xi, M. Taher, C. Yin, F. Salloum, and R. C. Kukreja Cobalt chloride induces delayed cardiac preconditioning in mice through selective activation of HIF-1{alpha} and AP-1 and iNOS signaling Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2369 - H2375. [Abstract] [Full Text] [PDF]

				E. O. McFalls, M. Hou, R. J. Bache, A. Best, D. Marx, J. Sikora, and H. B. Ward Activation of p38 MAPK and increased glucose transport in chronic hibernating swine myocardium Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1328 - H1334. [Abstract] [Full Text] [PDF]

				A. Das, R. Ockaili, F. Salloum, and R. C. Kukreja Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1455 - H1460. [Abstract] [Full Text] [PDF]

				J. Zhang, P. Ping, T. M. Vondriska, X.-L. Tang, G.-W. Wang, E. M. Cardwell, and R. Bolli Cardioprotection involves activation of NF-{kappa}B via PKC-dependent tyrosine and serine phosphorylation of I{kappa}B-{alpha} Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1753 - H1758. [Abstract] [Full Text] [PDF]

				T. C. Zhao and R. C. Kukreja Protein kinase C-{delta} mediates adenosine A3 receptor-induced delayed cardioprotection in mouse Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H434 - H441. [Abstract] [Full Text] [PDF]

				H. H. Patel, R. M. Fryer, E. R. Gross, R. A. Bundey, A. K. Hsu, M. Isbell, L. O.V. Eusebi, R. V. Jensen, S. R. Gullans, P. A. Insel, et al. 12-Lipoxygenase in Opioid-Induced Delayed Cardioprotection: Gene Array, Mass Spectrometric, and Pharmacological Analyses Circ. Res., April 4, 2003; 92(6): 676 - 682. [Abstract] [Full Text] [PDF]

				F. Salloum, C. Yin, L. Xi, and R. C. Kukreja Sildenafil Induces Delayed Preconditioning Through Inducible Nitric Oxide Synthase-Dependent Pathway in Mouse Heart Circ. Res., April 4, 2003; 92(6): 595 - 597. [Abstract] [Full Text] [PDF]