Published online before print July 2, 2007, doi: 10.1161/CIRCULATIONAHA.107.689471
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(Circulation. 2007;116:535-544.)
© 2007 American Heart Association, Inc.
Molecular Cardiology |
Endothelial Nitric Oxide Synthase Plays an Obligatory Role in the Late Phase of Ischemic Preconditioning by Activating the Protein Kinase C
–p44/42 Mitogen-Activated Protein Kinase–pSer-Signal Transducers and Activators of Transcription1/3 Pathway
From the Institute of Molecular Cardiology, University of Louisville, Louisville, Ky.
Correspondence to Roberto Bolli, MD, Division of Cardiology, University of Louisville, Louisville, KY 40292 (e-mail rbolli{at}louisville.edu); or Yu-Ting Xuan, PhD, Division of Cardiology, University of Louisville, Louisville, KY 40292 (e-mail ytxuan01@louisville.edu).
Received January 9, 2007; accepted May 29, 2007.
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Abstract |
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Background— The role of endothelial nitric oxide synthase (eNOS) in ischemic preconditioning (PC) and cardioprotection is poorly understood. We addressed this issue using a genetic, rather than pharmacological, approach.
Methods and Results— In the nonpreconditioned state, eNOS–/– mice exhibited infarct sizes similar to those of wild-type mice. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (ischemic PC) induced late PC in wild-type mice; genetic deletion of eNOS abrogated the cardioprotection induced by late PC. In wild-type mice, ischemic PC induced membranous translocation of protein kinase C (PKC)
and an increase in pSer-MEK-1/2 and pTyr-p44/42 mitogen-activated protein kinase, nuclear pSer-signal transducers and activators of transcription (STAT)1 and pSer-STAT3, and nuclear STAT1/3 DNA binding activity, followed by upregulation of cyclooxygenase-2 protein and activity 24 hours later. All of these changes were abrogated in eNOS–/– mice. The NO donor diethylenetriamine/NO recapitulated the effects of ischemic PC.
Conclusions— In contrast to previous reports, we found that basal eNOS activity does not modulate infarct size in the nonpreconditioned state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts as the trigger of this process by activating the PKC-MEK-1/2-p44/42 mitogen-activated protein kinase pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac cyclooxygenase-2.
Key Words: ischemia • myocardial infarction • occlusion • stress
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Introduction |
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The protective effects of ischemic preconditioning (PC) occur in 2 distinct phases: an early phase that develops rapidly after the stimulus but dissipates within 2 to 3 hours and a late phase that becomes apparent 12 to 24 hours later and persists for
72 hours.1–12 Although nitric oxide (NO) is known to be involved in both phases,3,4,7,8,13,14 the role of endothelial NO synthase (eNOS; the major constitutive source of NO in the heart) in the late phase of ischemic PC remains poorly understood. Specifically, 2 fundamental issues are unresolved.
Clinical Perspective p 544
The first issue is whether eNOS is responsible for triggering this delayed adaptation of the heart to stress. The proposal put forth in previous investigations8,13,15 that eNOS-derived NO triggers the late phase of ischemic PC is based on the finding that pretreatment with L-nitro-arginine, a nonselective NOS inhibitor, blocks late PC8,15 and conversely that exposure to exogenous NO in the absence of ischemia induces a delayed protective effect.13 These studies, however, were performed using pharmacological inhibitors of NOS rather than genetic ablation of the protein. Furthermore, the use of L-nitro-arginine does not enable one to identify the source of the increased NO formation that triggers late PC. Constitutively expressed NOS includes both eNOS and neuronal NOS; in principle, either or both of these isoforms could contribute to the burst of NO generation that triggers the development of late PC.
The second unresolved issue pertains to the mechanism by which eNOS triggers late PC. We have previously demonstrated that the PC ischemia induces activation of Janus tyrosine kinase-1 and -2, followed by tyrosine phosphorylation and activation of signal transducers and activators of transcription (STAT)1 and 3, which results in transactivation of STAT-responsive genes such as cyclooxygenase-2 (COX-2).16,17 Full transcriptional activation of STATs, however, requires not only tyrosine phosphorylation (Tyr-701 in STAT1 and Tyr-705 in STAT3) but also serine phosphorylation (Ser-727 in both STAT1 and STAT3).18–21 We have recently found that Ser-727 phosphorylation of STAT1/3 is modulated by the protein kinase C (PKC)-Raf-1-MEK-1/2-p44/42 mitogen-activated protein kinase (MAPK) signaling cascade and is critical for the upregulation of COX-2 during late PC.17 The exact position of eNOS in this signaling scheme remains unclear. Because the promoter of the mouse COX-2 gene contains the interferon-
activation site consensus sequence for the binding of STATs16,22,23 and because ischemic PC is associated with increased constitutively expressed NOS activity and NO generation,14 as well as activation of PKC6,9, STAT1, and STAT3,16,17 we hypothesized that rapid activation of eNOS by the initial PC ischemia is the early signal that generates NO, which in turn activates a downstream PKC-Raf-1-MEK-1/2-p44/42 MAPK-pSer-STAT1/3 pathway, leading to induction of COX-2 protein and cardioprotection. However, it is unknown whether eNOS is necessary for serine phosphorylation of STATs and, if so, whether eNOS is proximal to the aforementioned pathway.
The objective of the present study was to determine the role of eNOS in ischemic PC using a genetic, rather than a pharmacological, approach. The following specific questions were addressed in a well-established murine model of myocardial infarction: Is eNOS necessary for the late phase of ischemic PC? If so, does eNOS trigger late PC through the activation of the PKC-Raf-1-MEK-1/2-p44/42 MAPK pathway, leading to Ser-727 phosphorylation of STAT1/3 during the initial PC ischemia and to subsequent upregulation of COX-2? Finally, can exogenous NO (NO donors) mimic eNOS-derived NO; ie, can it activate the PKC-Raf-1-MEK-1/2-p44/42 MAPK-pSer-STAT1/3 pathway and induce COX-2?
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Methods |
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We used eNOS–/– mice with deletion of the calmodulin binding domain.24 Wild-type (WT) control mice were C57BL/6 (The Jackson Laboratory, Bar Harbor, Me). Spontaneously hypertensive mice (high blood pressure mice [BPH]) were obtained from The Jackson Laboratory. All mice were maintained in sterile microisolator cages under pathogen-free conditions. The genotype of the eNOS–/– strain was verified by polymerase chain reaction, as previously described, using DNA prepared from tail samples taken at the end of the experiments.17 A total of 155 mice were used for the present study.
The murine model of late PC has previously been described in detail.16,25 Briefly, a nontraumatic balloon occluder was implanted around the middle left anterior descending coronary artery in pentobarbital-anesthetized mice. To prevent hypotension, blood from a donor mouse was given during surgery. Rectal temperature was maintained close to 37°C throughout the experiment.
Ischemic PC was elicited by a sequence of six 4-minute coronary occlusion/4-minute reperfusion (O/R) cycles. Control mice underwent 1 hour of open-chest state (sham control), and sutures were placed as in the PC groups. Mice used for studies of myocardial infarction underwent a 30-minute coronary occlusion, followed by 24 hours of reperfusion. At the conclusion of the study, the occluded/reperfused vascular bed and the infarct were identified by postmortem perfusion of the heart with phthalo blue dye and triphenyltetrazolium chloride, respectively. Infarct size was calculated as a percentage of the region at risk.16,25
The investigation consisted of 3 successive phases (A, B, and C). The objective of phase A was to determine whether eNOS is necessary for the development of the cardioprotective effects of late PC. Mice were assigned to 6 groups (Figure 1
A). On day 1, groups 1 (WT acute myocardial infarction [AMI]) and 3 (eNOS–/– AMI) underwent 1 hour of open-chest state without ischemia, whereas groups 2 (WT late PC [LPC]) and 4 (eNOS–/– LPC) underwent a sequence of six 4-minute coronary O/R cycles. On day 2, all mice underwent a 30-minute coronary occlusion. Groups 5 (BPH AMI) and 6 (BPH LPC) underwent a 30-minute occlusion on day 2 without (group 5) or with (group 6) six 4-minute coronary O/R cycles on day 1. The purpose of studying these spontaneously hypertensive mice26 was to determine whether high blood pressure, in itself, could affect infarct size.
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The objective of phase B was to determine whether eNOS is required for the activation of PKC and the MEK1/2-p44/42 MAPK pathway, the serine phosphorylation of STAT1/3, and the upregulation of COX-2 during late PC. Mice were assigned to 12 groups (Figure 1
B). Groups 8 (WT PC–5 minutes), 10 (eNOS–/– PC–5 minutes), 12 (WT PC–30 minutes), 14 (eNOS–/– PC–30 minutes), 16 (WT PC–24 hours), and 18 (eNOS–/– PC–24 hours) underwent six 4-minute coronary O/R cycles, whereas groups 7 (WT control–5 minutes), 9 (eNOS–/– control–5 minutes), 11 (WT control–30 minutes), 13 (eNOS–/– control–30 minutes), 15 (WT control–24 hours), and 17 (eNOS–/– control–24 hours) underwent 1 hour of open-chest state without occlusion. Mice were euthanized at 5 minutes (groups 7 through 10), 30 minutes (groups 11 through 14), or 24 hours (groups 15 through 18) after the open-chest state or the last reperfusion. Myocardial samples were removed rapidly from the ischemic-reperfused region or the left ventricle and frozen in liquid nitrogen until used.
The objective of phase C was to elucidate (by using the NO donor diethylenetriamine [DETA]/NO) whether exogenous NO is sufficient to activate PKC, MEK-1/2, and p44/42 MAPKs and to increase serine phosphorylation of STAT1/3 and expression of COX-2 protein. Mice were assigned to 6 groups (Figure 1C). Groups 20, 22, and 24 received the NO donor DETA/NO (5 mg/kg IV every 20 minutes 4 times), a dose that has previously been shown to elicit a powerful late PC effect in mice,27 whereas control groups 19, 21, and 23 received the same volume of PBS. Mice were euthanized 5 minutes (groups 19 and 20), 30 minutes (group 21 and 22), or 24 hours (groups 23 and 24) after the end of the infusion of DETA/NO or PBS. Myocardial samples were removed rapidly from the left ventricle and frozen in liquid nitrogen until used.
Cytosolic, membranous, and nuclear fractions were prepared as previously described.16,17 Western immunoblotting analysis was performed using standard techniques16,17; the antibodies are specified in Table I of the online Data Supplement. Equal loading was confirmed by staining with Ponceau-S.16,17 The DNA binding activity of STAT1/3 was measured with electrophoretic mobility shift assays.16,17 A synthetic double-stranded probe with the sequence 5'-GATCAGCTTCATTTCCCGTAAATCCCTA-3' (Gibco, Carlsbad, Calif) was end-labeled using [-32P]ATP (3000 Ci/mmol, Amersham, Uppsala, Sweden) and T4 polynucleotide kinase. This oligonucleotide has the consensus sequence for interferon- activation site elements (italics).16,17 Prostaglandins (PGs) were extracted using ODS-silica reverse-phase columns.11 The myocardial content of PGE2, PGF2
, and 6-keto-PGF1 was determined by with enzyme immunoassay kits (PGE2 and PGF2 enzyme immunoassay kits from Cayman Chemical, Ann Arbor, Mich; 6-keto-PGF1a kit from Amersham Life Science, Buckinghamshire, UK) and expressed as picograms per milligram of protein.11
Data are reported as mean±SEM, and sample sizes are stated in the figures. Statistical comparisons were performed with 1-way ANOVA, followed by unpaired Student t test with Bonferroni correction as appropriate. In all Western analyses, the content of the specific protein of interest was expressed as a percentage of the corresponding protein in the anterior left ventricular wall of control mice.16,17
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Phase A
As expected, BPH mice exhibited greater left ventricular weight; in addition, heart rate was
15% faster in BPH mice preconditioned 24 hours earlier (Tables 1 and 2). Except for these differences, there were no other significant differences among groups with respect to heart weight, weight of the region at risk, rectal temperature, or heart rate (Tables 1 and 2).
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Infarct size in WT sham control mice (WT AMI, group 1) averaged 62.7±2.4% of the region at risk (Figure 2). When WT mice were preconditioned with six 4-minute coronary O/R cycles on day 1 (WT LPC, group 2), the infarct size induced by a 30-minute coronary occlusion 24 hours later (day 2) was reduced to 33.5±3.5% of the risk region, indicating the development of a robust late PC effect. However, when eNOS–/– mice were preconditioned with the six 4-minute coronary O/R cycles on day 1 (group 4), the infarct size on day 2 was not decreased compared with group 1 (WT AMI) (Figure 2), indicating that eNOS plays an obligatory role in mediating the delayed cardioprotective effects of late PC. Nonpreconditioned eNOS–/– mice (group 3; Figure 2) exhibited infarct sizes similar to those of the corresponding WT mice (group 1), indicating that the eNOS mutation did not affect the extent of ischemia/reperfusion injury under basal (unstressed) conditions.
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eNOS–/– mice have higher blood pressure than WT mice,24 a difference that was confirmed by our measurements (104±4.9 mm Hg in eNOS–/– mice [n=4] versus 81±1.1 mm Hg in WT mice [n=9]). Because hypertension in itself may modulate ischemia/reperfusion injury, we examined infarct size in BPH mice, a spontaneously hypertensive strain that has values of blood pressure similar to those of eNOS–/– mice.26 In BPH mice (group 5), the infarct size induced by the 30-minute occlusion was 55.6±1.9% of the region at risk (Figure 2), which was not significantly different from that in WT mice (group 1). Preconditioning BPH mice 24 hours before the 30-minute occlusion (group 6) reduced infarct size to 26.4±6.5% of the region at risk (Figure 2), indicating that late PC was fully manifest and implying that the failure of eNOS–/– mice to exhibit a late PC phenotype (group 4) was not due to hypertension. Measurements of blood pressure confirmed that the levels in BPH mice (99 mm Hg; n=2) were similar to those in eNOS–/– mice (see above).
Phase B
Having established that eNOS is obligatorily required for the development of late PC, we sought to elucidate the mechanism responsible. As illustrated in Figure 3A through 3F, disruption of the eNOS gene blocked the membranous translocation of PKC 5 minutes later and the increase in pSer-MEK-1/2 and pTyr-p44/42 MAPKs observed in WT mice 30 minutes after ischemic PC. The total levels of MEK-1/2 and p44/42 MAPKs were not changed by the six 4-minute coronary O/R cycles or by deletion of eNOS (Figure 3A through 3F). Figure 4A and 4B shows that in WT mice the six 4-minute coronary O/R cycles markedly increased the nuclear content of pSer-STAT1 and pSer-STAT3. All of these changes were inhibited by deletion of eNOS (Figure 4A and 4B). Furthermore, in WT mice, the STAT1/3 DNA binding activity increased strikingly at 30 minutes after the ischemic PC stimulus (Figure 4C and 4D); this increase was suppressed by >80% in eNOS–/– mice. Targeted disruption of the eNOS gene in itself did not alter the binding activity of STAT1/3 compared with WT control mice (Figure 4C and 4D). Collectively, these data indicate that eNOS plays a necessary role in triggering the PKC-MEK-1/2-p44/42 MAPK signaling cascade and the subsequent activation of STAT1/3.
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Next, we examined the role of eNOS in COX-2 upregulation. In WT mice, ischemic PC induced a marked increase in COX-2 protein (Figure 5A and 5B) 24 hours later. This was associated with increased myocardial levels of PGE2 (Figure 6A), PGF2 (Figure 6B), and 6-keto-PGF1 (Figure 6C), which are indicative of increased COX-2 activity (because the ischemic PC protocol does not upregulate COX-1 protein28). Both the increased COX-2 protein expression (Figure 5A and 5B) and the increased COX-2 activity (Figure 6A and 6C) were abrogated in eNOS–/– mice.
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Phase C
To determine whether eNOS-derived NO is not only necessary but also sufficient to trigger the PKC-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade and the serine and tyrosine phosphorylation of STAT1/3, we examined the effects of administering exogenous NO with the NO donor DETA/NO. Administration of DETA/NO rapidly increased the PKC content in the membranous fraction, with a corresponding decrease of this isoform in the cytosolic fraction (Figure 7A and 7B) 5 minutes later, indicating activation of this enzyme. DETA/NO also induced a robust increase in pSer-MEK-1/2 (Figure 7C and 7D) and pTyr-p44/42 MAPK (Figure 7E and 7F) 30 minutes later but had no effect on the total levels of these kinases (Figures 7C-7F). In addition, DETA/NO induced a striking increase in the phosphorylated forms of STAT1 and STAT3 (Figure 8A and 8B) and in the DNA binding activity of STAT1/3 (Figure 8C and 8D). Finally, administration of DETA/NO increased the expression of COX-2 protein 24 hours later (Figure 8E and 8F) but had no effect on COX-1 expression.
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Discussion |
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Although extensive evidence indicates a pivotal role of NO as a cardioprotectant (reviewed elsewhere15), the role of eNOS in myocardial ischemia/reperfusion injury and specifically in late PC remains unclear. Studies of late PC have not resolved either the isoform involved or the mechanism whereby NOS initiates late PC.14 The present study was conducted to address these unanswered questions.
The salient findings can be summarized as follows. First, eNOS is obligatorily required for the development of the cardioprotective effects of late PC. Second, eNOS activity triggers late PC by activating the PKC-Raf-1-MEK-1/2-p44/42 MAPK pathway, which leads to serine phosphorylation of STAT1 and STAT3 and subsequent upregulation of COX-2. Third, administration of exogenous NO is sufficient to reproduce the effects of eNOS, ie, to activate PKC and the downstream MEK-1/2-p44/42 MAPK-pSer-STAT1/3 pathway with the consequent increase in COX-2 protein expression.
To the best of our knowledge, this is the first study to demonstrate that eNOS plays an obligatory role in the delayed cardioprotection afforded by ischemic PC and that it does so by activating the PKC-MEK-1/2-p44/42 MAPK-pSer-STAT1/3 pathway, leading to upregulation of STAT-dependent genes, including COX-2. This is also the first study to demonstrate that exogenous NO (NO donors) can replicate the effects of eNOS activation, ie, that NO donors recruit the aforementioned pathway, resulting in increased COX-2 protein expression in the heart. In contrast, we found no evidence that constitutive expression of eNOS modulates myocardial ischemia/reperfusion injury in the nonpreconditioned state in our mouse model. Collectively, the results indicate that eNOS is an important defense mechanisms during stress but not under basal conditions and plays a necessary role in late PC. These data have broad implications for our understanding of how eNOS participates in the response of the heart to stress in general.
Role of eNOS in Late PC
Previous studies of the role of eNOS in late PC are limited by the use of a pharmacological approach; moreover, the specific NOS isoform responsible for generating the NO that triggers late PC (eNOS versus neuronal NOS) has not been resolved, and the mechanism whereby NOS triggers late PC remains unknown. Our finding that genetic ablation of eNOS completely blocked the delayed infarct-sparing effects of ischemic PC (Figure 2) demonstrates that eNOS plays an obligatory role in this process. However, this finding does not elucidate whether eNOS serves as a trigger of this adaptation (acting on day 1) or as a mediator of the protection (acting on day 2). To distinguish between these 2 possibilities, we investigated whether deletion of eNOS interferes with the activation of an early signaling cascade (the PKC-MEK-1/2-p44/42 MAPK-pSer-STAT1/3 pathway) that we have previously shown to be responsible for the initiation of the late PC adaptation on day 1.17 We reasoned that if eNOS acts as a trigger on day 1, this pathway will not be recruited in eNOS–/– mice; conversely, if eNOS acts as a mediator on day 2, deletion of this protein should have no effect on the recruitment of the early signaling pathway on day 1. Our finding that targeted disruption of eNOS completely blocked or markedly reduced the increase in PKC membranous translocation (Figure 3A and 3B), pSer-MEK-1/2 (Figure 3C and 3D), pTyr-p44/42 MAPKs (Figure 3E and 3F), pSer-STAT1/3 (Figure 4A and 4B), and STAT1/3 DNA binding activity (Figure 4C and 4B) 30 minutes after ischemic PC, as well as the subsequent upregulation of COX-2 protein (Figure 5A and 5B) and activity (Figure 6A through 6C), demonstrates that this pathway is downstream of eNOS and is dependent on eNOS activity; ie, eNOS is an obligatory trigger of the adaptive response of late PC.
Effect of Exogenous NO on the PKC-p44/42 MAPK-pSer-STAT1/3 Pathway and on COX-2
The results of phases A and B indicate that eNOS-dependent NO generation is necessary to trigger the late phase of ischemic PC. To determine whether increased NO availability in itself is sufficient to recruit the same signaling mechanisms that are activated by eNOS during ischemic PC, we studied the effects of the NO donor DETA/NO given at the same dose previously shown to induce a delayed cardioprotective effect (NO-induced late PC)27 in mice. Our finding that DETA/NO, in the absence of ischemia, induced translocation of PKC to the particulate fraction (Figure 7A and 7B) and increased pSer-MEK-1/2 (Figures 7C and 7D), pTyr-p44/42 MAPKs (Figures 7E and 7F), pSer-STAT1/3 (Figures 8A and 8B), and STAT1/3 DNA binding activity (Figures 8C and 8D) demonstrates that exogenous NO can emulate the actions of endogenous NO with respect to the mobilization of the PKC-p44/42 MAPK-pSer-STAT1/3 signaling cascade. These results corroborate the concept that eNOS is involved in late PC at a very early stage, as a trigger of this signaling pathway. In addition, the observation that administration of DETA/NO resulted in induction of COX-2 (Figures 8E and 8F) is the first evidence that NO donors can upregulate COX-2 levels in the heart.
Conclusions
We have demonstrated that, in our murine model, eNOS plays a fundamentally different role in the nonpreconditioned versus preconditioned state. We have found that basal eNOS activity does not modulate infarct size in the nonpreconditioned (naïve) state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts by triggering the activation of the PKC-MEK-1/2-p44/42 MAPK pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as COX-2. This is the first evidence that eNOS controls the serine phosphorylation of STAT1 and STAT3 and modulates the expression of COX-2 in the heart, revealing a novel action of this NOS isoform in cardiac biology. In addition, we have found that the effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac COX-2. Collectively, the present findings expand our understanding of the molecular mechanisms whereby eNOS and NO contribute to cardioprotection and to the response of the heart to stress in general.
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Acknowledgments |
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Sources of Funding
The present study was supported in part by National Institutes of Health grants R01 HL-65660, HL-55757, HL-68088, HL-70897, HL-76794, and P01 HL-78825.
Disclosures
None.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia. Circulation. 1986; 74: 1124–1136.
[Abstract/Free Full Text] - Marber MS, Latchman DS, Walker JM, Yellon DM. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation. 1993; 88: 1264–1272.
[Abstract/Free Full Text] - Bolli R. The late phase of preconditioning. Circ Res. 2000; 87: 972–983.
[Abstract/Free Full Text] - Yellon DM, Downey JM. Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev. 2003; 83: 1113–1151.
[Abstract/Free Full Text] - Guo Y, Jones WK, Xuan YT, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, Bolli B. The late phase of ischemic preconditioning is abrogated by targeted disruption of the iNOS gene. Proc Natl Acad Sci U S A. 1999; 96: 11507–11512.
[Abstract/Free Full Text] - Ping P, Zhang J, Qiu Y, Tang X-L, Manchikalapudi S, Cao X, Bolli R. Ischemic preconditioning induces selective translocation of protein kinase C isoforms and
in the heart of conscious rabbits. Circ Res. 1997; 81: 404–414.[Abstract/Free Full Text] - 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. Circ Res. 1997; 81: 1094–1107.
[Abstract/Free Full Text] - Bolli R, Bhatti ZA, Tang XL, Qiu Y, Zhang Q, Guo Y, Jadoon AK. Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxide. Circ Res. 1997; 81: 42–52.
[Abstract/Free Full Text] - Qiu Y, Ping P, Tang XL, Manchikalapudi S, Rizvi A, Zhang J, Takano H, Wu WJ, Teschner S, Bolli R. Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits. J Clin Invest. 1998; 101: 2182–2198.[Medline]
[Order article via Infotrieve]
- Xuan YT, Tang XL, Banerjee S, Takano H, Li RC, Han H, Qiu 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.[Abstract/Free Full Text] - Shinmura K, Tang XL, Wang Y, Xuan YT, Liu SQ, Takano H, Bhatnagar A, Bolli R. Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits. Proc Natl Acad Sci U S A. 2000; 97: 10197–10202.
[Abstract/Free Full Text] - Guo Y, Bao W, Wu WJ, Shinmura K, Tang XL, Bolli R. Evidence for an essential role of cyclooxygenase-2 as a mediator of the late phase of ischemic preconditioning in mice. Basic Res Cardiol. 2000; 95: 479–484.[CrossRef][Medline]
[Order article via Infotrieve]
- Takano H, Tang XL, Qiu Y, Guo Y, French BA, Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res. 1998; 83: 73–84.
[Abstract/Free Full Text] - Xuan YT, Tang XL, Qiu Y, Banerjee S, Takano H, Han H, Bolli R. Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits. Am J Physiol. 2000; 279: H2360–H2371.
- Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning. J Mol Cell Cardiol. 2001; 33: 1897–1918.[CrossRef][Medline]
[Order article via Infotrieve]
- Xuan YT, Guo Y, Han H, Zhu Y, Bolli R. An essential role of the JAK-STAT pathway in ischemic preconditioning. Proc Natl Acad Sci U S A. 2001; 98: 9050–9055.
[Abstract/Free Full Text] - Xuan YT, Guo Y, Zhu Y, Wang OL, Rokosh G, Messing RO, Bolli R. Role of the protein kinase C--Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning. Circulation. 2005; 112: 1971–1978.
[Abstract/Free Full Text] - Akira S. Roles of STAT3 defined by tissue-specific gene targeting. Oncogene. 2000; 19: 2607–2611.[CrossRef][Medline]
[Order article via Infotrieve]
- Levy DE, Darnell JE Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002; 3: 651–662.[CrossRef][Medline]
[Order article via Infotrieve]
- Wen Z, Zhong Z, Darnell JE Jr. Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell. 1995; 82: 241–250.[CrossRef][Medline]
[Order article via Infotrieve]
- Shen Y, Schlessinger K, Zhu X, Meffre E, Quimby F, Levy DE, Darnell JE Jr. Essential role of STAT3 in postnatal survival and growth revealed by mice lacking STAT3 serine 727 phosphorylation. Mol Cell Biol. 2004; 24: 407–419.
[Abstract/Free Full Text] - Horvath CM, Wen Z, Darnell JE. A Stat protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain. Genes Dev. 1995; 9: 984–994.
[Abstract/Free Full Text] - Decker T, Kovarik P, Meinke A. GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J Interferon Cytokine Res. 1997; 17: 121–134.[Medline]
[Order article via Infotrieve]
- Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE, Sherman PA, Sessa WC, Smithies O. Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1996; 93: 13176–13181.
[Abstract/Free Full Text] - Guo Y, Wu WJ, Qiu Y, Tang XL, Yang Z, Bolli R. Demonstration of an early and a late phase of ischemic preconditioning in mice. Am J Physiol. 1998; 275: H1375–H1387.[Medline]
[Order article via Infotrieve]
- Schlager G, Sides J. Characterization of hypertensive and hypotensive inbred strains of mice. Lab Anim Sci. 1997; 47: 288–292.[Medline]
[Order article via Infotrieve]
- Guo Y, Stein AB, Wu WJ, Zhu X, Tan W, Li Q, Bolli R. Late preconditioning induced by NO donors, adenosine A1 receptor agonists, and {delta}1-opioid receptor agonists is mediated by iNOS. Am J Physiol. 2005; 289: H2251–H2257.
- Xuan YT, Guo Y, Zhu Y, Han H, Langenbach R, Dawn B, Bolli R. Mechanism of cyclooxygenase-2 upregulation in late preconditioning. J Mol Cell Cardiol. 2003; 35: 525–537.[CrossRef][Medline]
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CLINICAL PERSPECTIVE
Preconditioning (PC) is the most powerful and reproducible cardioprotective intervention identified to date, and mounting evidence suggests that it is effective in protecting human myocardium. Thus, PC represents an attractive strategy for inducing cardioprotection. PC consists of an early phase and a late phase (which occur 1 to 3 hours and 1 to 3 days after the PC stimulus, respectively). Although nitric oxide (NO) is known to be a key player in late PC, the role of endothelial NO synthase (eNOS) (the major constitutive source of NO in the heart) in late PC remains unclear. Here, we demonstrate that eNOS plays an obligatory role in triggering the development of the cardioprotective effects of late PC by activating a downstream protein kinase C-MEK-1/2-p44/42 mitogen-activated protein kinase signaling cascade that leads to recruitment of the signal transducers and activators of transcription (STAT)1 and STAT3 and the consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are recapitulated by exogenous NO (NO donor administration), implying that nitrates can upregulate cardiac cyclooxygenase-2. Collectively, these results indicate that eNOS is an important defense mechanisms during stress and plays a necessary role in late PC. These data expand our understanding of the molecular mechanisms whereby eNOS and NO participate in the response of the heart to stress in general.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA. 107.689471/DC1.
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