Late Phase Of Ischemic Preconditioning Research Articles

Prostaglandin E2 mediates the late phase of ischemic preconditioning in the heart via its receptor subtype EP4

Ischemic preconditioning (IPC) describes a phenomenon wherein brief ischemia of the heart induces a potent cardioprotective mechanism against succeeding ischemic insult. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostanoid biosynthesis, is upregulated in the ischemic heart and contributes to IPC. Prostaglandin E2 (PGE2) protects the heart from ischemia–reperfusion (I/R) injury via its receptor subtype EP4. We sought to clarify the role of the PGE2/EP4 system in the late phase of IPC. Mice were subjected to four IPC treatment cycles, consisting of 5 min of occlusion of the left anterior descending coronary artery (LAD). We found that COX-2 mRNA was significantly upregulated in wild-type hearts at 6 h after IPC treatment. Cardiac PGE2 levels at 24 h after IPC treatment were significantly increased in both wild-type mice and mice lacking EP4 (EP4–/–). At 24 h after IPC treatment, I/R injury was induced by 30 min of LAD occlusion followed by 2 h of reperfusion and the cardiac infarct size was determined. The infarct size was significantly reduced by IPC treatment in wild-type mice; a reduction was not observed in EP4–/– mice. AE1-329, an EP4 agonist, significantly reduced infarct size and significantly ameliorated deterioration of cardiac function in wild-type mice subjected to I/R without IPC treatment. Furthermore, AE1-329 significantly enhanced the I/R-induced activation of Akt, a pro-survival kinase. We demonstrated that the PGE2/EP4 system in the heart plays a critical role in the late phase of IPC, partly by augmenting Akt-mediated signaling. These findings clarify the mechanism of IPC and may contribute to the development of therapeutic strategies for ischemic heart disease.

The COX-2/PGI2 Receptor Axis Plays an Obligatory Role in Mediating the Cardioprotection Conferred by the Late Phase of Ischemic Preconditioning

BackgroundPharmacologic studies with cyclooxygenase-2 (COX-2) inhibitors suggest that the late phase of ischemic preconditioning (PC) is mediated by COX-2. However, nonspecific effects of COX-2 inhibitors cannot be ruled out, and the selectivity of these inhibitors for COX-2 vs. COX-1 is only relative. Furthermore, the specific prostaglandin (PG) receptors responsible for the salubrious actions of COX-2-derived prostanoids remain unclear.ObjectiveTo determine the role of COX-2 and prostacyclin receptor (IP) in late PC by gene deletion.MethodsCOX-2 knockout (KO) mice (COX-2−/−), prostacyclin receptor KO (IP−/−) mice, and respective wildtype (WT, COX-2+/+ and IP+/+) mice underwent sham surgery or PC with six 4-min coronary occlusion (O)/4-min R cycles 24 h before a 30-min O/24 h R.ResultsThere were no significant differences in infarct size (IS) between non-preconditioned (non-PC) COX-2+/+, COX-2−/−, IP+/+, and IP−/− mice, indicating that neither COX-2 nor IP modulates IS in the absence of PC. When COX-2−/− or IP−/− mice were preconditioned, IS was not reduced, indicating that the protection of late PC was completely abrogated by deletion of either the COX-2 or the IP gene. Administration of the IP selective antagonist, RO3244794 to C57BL6/J (B6) mice 30 min prior to the 30-min O had no effect on IS. When B6 mice were preconditioned 24 h prior to the 30-min O, IS was markedly reduced; however, the protection of late PC was completely abrogated by pretreatment of RO3244794.ConclusionsThis is the first study to demonstrate that targeted disruption of the COX-2 gene completely abrogates the infarct-sparing effect of late PC, and that the IP, downstream of the COX-2/prostanoid pathway, is a key mediator of the late PC. These results provide unequivocal molecular genetic evidence for an essential role of the COX-2/PGI2 receptor axis in the cardioprotection afforded by the late PC.

Identification of inducible nitric oxide synthase in peripheral blood cells as a mediator of myocardial ischemia/reperfusion injury

Although the late phase of ischemic preconditioning is known to be mediated by increased inducible nitric oxide synthase (iNOS) activity, controversy persists regarding the role of iNOS in ischemia/reperfusion (I/R) injury and, specifically, whether this protein is protective or detrimental. We hypothesized that iNOS is protective in myocytes but detrimental in inflammatory cells. To test this hypothesis, we created chimeric mice with iNOS-deficient peripheral blood cells by transplanting iNOS knockout (KO) bone marrow in wild-type (WT) mice after lethal irradiation. 2months later, the mice underwent a 30-min coronary occlusion followed by 24h of reperfusion. In WT naïve mice (iNOS(+/+) naïve; group I, n=17), infarct size was 56.9±2.8% of the risk region. In iNOS KO naïve mice with whole-body iNOS deletion (iNOS(-/-) naïve; group II, n=10), infarct size was comparable to group I (53.4±3.5%). When irradiated WT mice received marrow from WT mice (iNOS(+/+) chimera; group III, n=10), infarct size was slightly reduced versus group I (44.3±3.2%), indicating that irradiation and/or transplantation slightly decrease I/R injury. However, when WT mice received marrow from iNOS KO mice (iNOS(-/-) chimera; group IV, n=14), infarct size was profoundly reduced (22.8±2.1%, P<0.05 vs. group III), indicating that selective deletion of iNOS from peripheral blood cells (with no change in myocardial iNOS content) induces protection against myocardial infarction. Together with our previous work showing the cardioprotective actions of NO donors, iNOS gene therapy, and cardiac-specific overexpression of iNOS, these data support a complex, dual role of iNOS in myocardial infarction (i.e., protective in myocytes but deleterious in blood cells). To our knowledge, this is the first study to identify a critical role of iNOS in peripheral blood cells as a mediator of myocardial I/R injury. The results support heretofore unknown differential actions of iNOS depending on cell source and have important translational implications.

Carbon monoxide induces a late preconditioning-mimetic cardioprotective and antiapoptotic milieu in the myocardium

A growing body of evidence indicates that carbon monoxide (CO), once perceived merely as a poisonous gas, exerts antiapoptotic and cytoprotective effects. Using a water-soluble CO-releasing molecule (CORM) tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), we previously reported that CO induces a delayed protection against myocardial infarction similar to that observed in the late phase of ischemic preconditioning (PC). In the current study, we investigated the molecular mechanisms underlying this cardioprotective effect. The impact on apoptotic signaling pathways was first examined in the setting of ischemia/reperfusion injury. Mice were pretreated with CORM-3 or iCORM-3 (which does not release CO) and subjected to coronary occlusion/reperfusion 24h later. In mice that received CORM-3, there was a significant reduction in markers of apoptosis (cleaved lamin A, cleaved caspase-3, and cleaved PARP-1) after ischemia/reperfusion injury. To elucidate the mechanism of CORM-3-induced cardioprotection we further examined the activation of transcription factors and induction of cardioprotective and apoptosis modulating proteins. Infusion of CORM-3 rapidly activated the stress-responsive transcription factors nuclear factor kappaB (NF-κB), signal transducers and activators of transcription (STAT)1, STAT3, and NF-E2-related factor-2 (Nrf2). This was followed 24h later by upregulation of cardioprotective proteins (heme oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and extracellular superoxide dismutase [Ec-SOD]) and antiapoptotic proteins involving both the mitochondria-mediated (Mcl-1) and the death receptor-mediated (c-FLIPS and c-FLIPL) apoptosis pathways. We conclude that CO released by CORM-3 triggers a cardioprotective signaling cascade that recruits the transcription factors NF-κB, STAT1/3, and Nrf2 with a subsequent increase in cardioprotective and antiapoptotic molecules in the myocardium leading to the late PC-mimetic infarct-sparing effects. This article is part of a Special Issue entitled ‘Possible Editorial’.

Glucagon-Like Peptide-1 Increases Myocardial Glucose Uptake via p38α MAP Kinase–Mediated, Nitric Oxide–Dependent Mechanisms in Conscious Dogs With Dilated Cardiomyopathy

Background— We have shown that glucagon-like peptide-1 (GLP-1[7–36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1–induced myocardial glucose uptake are unknown. Methods and Results— Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7–36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20±7 nmol/min/g; DCM+GLP-1: 61±12 nmol/min/g; P =0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98±2 density units; DCM: 256±58 density units; P =0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1–induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38α MAP kinase activity (DCM+vehicle: 97±22 pmol ATP/mg/min; DCM+GLP-1: 170±36 pmol ATP/mg/min; P =0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151±13 density units; DCM+GLP-1: 306±12 density units; P =0.001), and GLUT-1 translocation (DCM+vehicle: 21±3% membrane bound; DCM+GLP-1: 39±3% membrane bound; P =0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38α MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro- l -arginine. Conclusions— GLP-1 stimulates myocardial glucose uptake through a non–Akt-1–dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

Ferilnic nirate produces delayed preconditioning against myocardial ischemia and reperfusion injury in rats

To determine whether ferilnic nirate (FLNT) can precondition the rat heart against myocardial ischemia/reperfusion (I/R) damage and its mechanism, two groups of experiments were conducted. In the first group of experiments, rats were divided among four treatment groups: sham group; solvent with I/R (I/R control group); FLNT pretreatment with I/R (I/R FLNT group); and late ischemic preconditioning group (LPC group). In the second group of experiments without I/R, rats were divided into two treatment groups: control group and FLNT group. The results indicated that myocardial infarct size and the levels of creatine kinase and lactate dehydrogenase in the sera of the I/R FLNT group were significantly lower and the level of nitric oxide molecule and Mn-containing superoxide dismutase were significantly elevated in the heart tissue compared with I/R control group. The protein expression ratio of Bcl-2/Bax in heart tissue was significantly elevated in the I/R FLNT group. These results demonstrate FLNT is able to precondition rat hearts against myocardial ischemia/reperfusion damage to a similar level as that achieved via the late phase of ischemic preconditioning. The mechanism may involve the up-regulation of nitric oxide and the strengthening of anti-oxidant and anti-apoptosis cellular functions.

Ischemic Pre-Conditioning Enhances the Mobilization and Recruitment of Bone Marrow Stem Cells to Protect Against Ischemia/Reperfusion Injury in the Late Phase

The aim of this study was to investigate whether the mobilization and recruitment of bone marrow stem cells (BMSCs) contribute to cardioprotection in the late phase after ischemic pre-conditioning (IPC). IPC is an innate phenomenon in which brief exposure to sublethal ischemia provides tissue protection from subsequent ischemia/reperfusion (I/R) injury. A delayed cardioprotection also occurs after IPC, but the precise mechanism is unclear. IPC was created with 4 cycles of 5-min occlusion and reperfusion of the abdominal aorta in mice. Heart I/R injury was induced by occluding the left anterior descending artery for 30 min immediately (early phase) or 24 h (late phase) after IPC. Serum vascular endothelial growth factor and stromal cell-derived factor-1alpha levels were increased significantly 1 and 3 h after IPC, but CD34+ and CD34+/flk-1+ stem cells in the peripheral blood were increased significantly 12 and 24 h after IPC (p < 0.05). Compared with the control treatment, both the early and late phases of IPC protected the heart against I/R injury. However, the recruitment of BMSCs was significantly greater in the heart when I/R injury was induced in late phase than in the early phase of IPC (p < 0.01). Interestingly, the blockade of the recruitment of BMSCs significantly attenuated the cardioprotective effect of IPC in the late phase (p < 0.01) but did not change in the early phase. Cardioprotection was observed in the early and late phases of IPC; however, the enhanced mobilization and recruitment of BMSCs played an important role in the late phase of IPC.

The Hsp70.3 isoform of Hsp70, but not Hsp70.1, contributes to NF‐kappaB‐dependent cardioprotection of late ischemic preconditioning

It has been shown by our lab and others that the transcription factor NF‐κB is necessary for the development of the late window of protection after ischemic preconditioning (IPC) in the heart. However, the specific mechanisms and downstream gene expression profiles that underlie the cardioprotection of NF‐κB in late IPC remain unknown. To investigate NF‐κB‐dependent gene expression in IPC, we conducted a directed microarray analysis to compare the gene expression patterns of wild‐type and NF‐κB dominant negative mice 3.5 hrs after an IPC stimulus. Among the potential cardioprotective candidates identified by gene array analysis as being regulated in an NF‐κB‐dependent manner after IPC were both isoforms of heat shock protein 70 (Hsp70.1 and Hsp70.3). An in vivo mouse model of IPC followed 24 hours later by a 30 minute ischemia and reperfusion was used to assess the impact of Hsp70 isoform expression on late IPC induced cardioprotection. As previously shown by others, an Hsp70.1/70.3 double knockout mouse model failed to exhibit a cardioprotective phenotype during the late window of IPC. However, the Hsp70.1 single isoform knockout exhibited late IPC cardioprotection similar to that of wild‐type mice. In conclusion, this work demonstrates that while both Hsp70.1 and Hsp70.3 expression are induced by NF‐κB after an IPC stimulus, only the Hsp70.3 isoform contributes to cardioprotection in the late phase of IPC. This work was supported by NIH grant R01 HL63034 (WKJ) and NSF award number 0333377.

Cardiac Myocyte–Specific Expression of Inducible Nitric Oxide Synthase Protects Against Ischemia/Reperfusion Injury by Preventing Mitochondrial Permeability Transition

Inducible nitric oxide synthase (iNOS) is an obligatory mediator of the late phase of ischemic preconditioning, but the mechanisms of its cardioprotective actions are unknown. In addition, it remains unclear whether sustained elevation of iNOS in myocytes provides chronic protection against ischemia/reperfusion injury. Constitutive overexpression of iNOS in transgenic mice (alpha-myosin heavy chain promoter) did not induce contractile dysfunction and did not affect mitochondrial respiration or biogenesis, but it profoundly decreased infarct size in mice subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion. In comparison with wild-type hearts, isolated iNOS-transgenic hearts subjected to ischemia for 30 minutes followed by 40 minutes of reperfusion displayed better contractile recovery, smaller infarct size, and less mitochondrial entrapment of 2-deoxy-[(3)H]-glucose. Reperfusion-induced loss of NAD(+) and mitochondrial release of cytochrome c were attenuated in iNOS-transgenic hearts, indicating reduced mitochondrial permeability transition. The NO donor NOC-22 prevented permeability transition in isolated mitochondria, and mitochondrial permeability transition-induced NAD(+) loss was decreased in wild-type but not iNOS-null mice treated with the NO donor diethylene triamine/NO 24 hours before ischemia and reperfusion ex vivo. iNOS-mediated cardioprotection was not abolished by atractyloside. Reperfusion-induced production of oxygen-derived free radicals (measured by electron paramagnetic resonance spectroscopy) was attenuated in iNOS-transgenic hearts and was increased in wild-type hearts treated with the mitochondrial permeability transition inhibitor cyclosporin A. Cardiomyocyte-restricted expression of iNOS provides sustained cardioprotection. This cardioprotection is associated with a decrease in reperfusion-induced oxygen radicals and inhibition of mitochondrial swelling and permeability transition.

The Early Phase of Preconditioning is Abrogated by COX‐2 Inhibition, but not by a Specific Prostaglandin Receptor Antagonist

COX‐2 is an important mediator of cardiovascular protection after ischemia and reperfusion and is essential for ischemic preconditioning (PC), mediated by the synthesis of PGE2 and/or PGI2. Recently, the importance of the PGI2 in the late phase of ischemic preconditioning has been established. The present study was designed to determine the contribution of PGI2 to the early phase of ischemic preconditioning. Male New Zealand white rabbits were administered the prostaglandin receptor (IP) antagonist RO3244794 (3mg/kg), nimesulide (5mg/kg), or vehicle thirty minutes prior to PC, 30 minutes of ischemia and 4 hours of reperfusion. PC reduced myocardial infarct size as a percent of area at risk (12.0 +/− 2.2 vs. control 42.3 +/− 2.9; P &lt; 0.001) and treatment with the COX‐2 inhibitor nimesulide abolished the protective effect (41.0 +/− 2.7). Surprisingly, treatment with the IP receptor antagonist had no effect on PC (17.0 +/− 3.1; P &lt; 0.001 vs. control). The data suggest that COX‐2 does not protect the myocardium from the early phase of preconditioning through the production of PGI2. These results differ from those seen in the late phase of PC, where RO3244794 abrogated the protective effects of PC, suggesting that the upregulation of COX‐2 differentially modulates protection in the early and late phases of ischemic preconditioning.

Abstract 213: The Late Phase of Ischemic Preconditioning Is Abrogated by a Specific Prostaglandin I2 Receptor Antagonist

Previous studies have shown that cyclooxygenase-2 (COX-2) mediates the late phase of ischemic preconditioning (PC) by producing various cardioprotective prostanoids including prostacyclin (PGI 2 ) and prostaglandin (PG) E 2 (PGE 2 ). Prostanoids are known to act via activation of specific receptors. However, the specific PG receptors responsible for the salubrious actions of COX-2 in PC and myocardial ischemia/reperfusion injury remain unclear. The PGI 2 receptor (IP) mediates the actions of PGI 2 and PGE 2 , the two major COX-2-derived prostanoids with cardioprotective properties. Accordingly, the goal of this study was to determine the role of the IP in late PC. C57BL/6 mice underwent a 30-min coronary occlusion (O) followed by 24 h of reperfusion (R). Administration of the IP receptor selective antagonist, RO 3244794 (group III, 10 mg/kg i.p.), 30 min prior to the 30-min O had no appreciable effect on infarct size compared with untreated and vehicle-treated groups (68.4 ± 1.2 % vs. 63.3 ± 3.1 % and 65.6 ± 3.9 % of the risk region, respectively). When mice were preconditioned with six cycles of 4-min O/4-min R 24 h prior to the 30-min O, infarct size was markedly reduced (group IV, 33.2 ± 2.9 %), indicating a late PC effect. In the vehicle-treated late PC group, infarct size (group V, 37.2 ± 4.9 %) was similar to the late PC group, indicating the vehicle had no effect. However, the protective effect of late PC was completely abrogated by administration of RO 3244794 30 min before the 30-min O (group VI, 63.8 ± 4.5 %). We conclude that the IP receptor is an obligatory mediator of the late phase of ischemic PC in vivo , suggesting that pharmacologic manipulations of this receptor may be therapeutically useful.

Abstract 3313: Smoking Abolishes Ischemic Preconditioning-Induced Augmentation of Endothelium-Dependent Vasodilation: Role of Endothelium-Derived Nitric Oxide and Circulating Endothelial Progenitor Cells

Background: Several studies have shown that both early and late effects of ischemic preconditioning (IPC) protect against myocardial injury following ischemic reperfusion. Recently, we have shown that repetition of IPC stimulus augments endothelium-dependent vasodilation in forearm circulation of healthy subjects through increases in nitric oxide (NO) production and number of endothelial progenitor cells (EPCs) under a local condition. The purpose of this study was to evaluate the late effects of IPC on endothelial function in smokers. Methods and Results: Late phase of IPC was induced by upper limb ischemia (cuff inflation of over 200 mmHg for 5 minutes) six times a day for one month. We evaluated forearm blood flow (FBF) responses to acetylcholine (ACh) and to sodium nitroprusside (SNP) before and after IPC stimulus in 15 male smokers (27±7 yr) and 15 male non-smokers (26±5 yr). FBF was measured using a strain-gauge plethysmography. The IPC stimulus significantly increased plasma concentration of vascular endothelial growth factor (VEGF) from 84.7±10.6 to 120.3±13.5 pg/mL (P&lt;0.05), circulating level of EPCs from 1258±198 to 1608±183 cells/mL (P&lt;0.05) and FBF responses to ACh from 17.3±4.9 to 24.8±6.9 mL/min/100 mL tissue (P&lt;0.05) in non-smokers, but these did not change in the smoker group. The FBF responses to SNP were similar before and after the IPC stimulus. Infusion of N G -monomethyl-L-arginine, a NO synthase inhibitor, completely eliminated the IPC stimulus-induced augmentation of FBF responses to ACh in non-smokers. Conclusions: These findings suggest that repetition of IPC stimulus may be a simple, safe, and feasible therapeutic technique for endothelial protection of peripheral vessels. However, smoking abolishes repetition of IPC stimulus-induced augmentation of endothelium-dependent vasodilation.

Abstract 1000: Dichotomous Roles Of TNF-α Receptors p55 And p75 In Acute Ischemia/reperfusion Injury And Late Ischemic Preconditioning

We have previously demonstrated that TNF-alpha signaling is critical for the development of protection afforded by the late phase of ischemic preconditioning (PC). In the current study, we investigated the roles of p55 (TNFR-I) and p75 (TNFR-II) in acute myocardial ischemia/reperfusion injury as well as late PC. Wild-type (WT, B6 and B6,129 strains), TNF-a−/−, p55−/−, p75−/−, and p55−/−/p75−/− double-knockout mice underwent a 30-min coronary occlusion followed by 4 h of reperfusion with or without six cycles of coronary occlusion/reperfusion (O/R) 24 h earlier. Six cycles of O/R reduced infarct size 24 h later in B6 as well as B6,129 WT mice, indicating a rob ust late PC effect (Figure ). This infarct-sparing effect of late PC was abolished in the absence of TNF-a, p55, p75, and both p55/p75, indicating that TNF-a signaling is critical for the development of late PC protection; and that signaling via both p55 and p75 is necessary for the development of protection. In nonpreconditioned TNF-a−/− and p75−/− mice, infarct size was similar to that observed in strain-matched WT mice (Figure ). However, infarct size in nonpreconditioned p55−/− mice was reduced compared with nonpreconditioned WT mice (46.8 ± 2.8% vs. 63.4 ± 3.2%, P &lt; 0.05, Figure ). These observations were confirmed via linear regression analysis of myocardial risk region and infarct size. We conclude that nonredundant TNF-a signaling via both p55 and p75 is crucial for the development of late PC protection. However, the reduction in infarct size in naïve p55−/− mice indicates a deleterious role of this receptor during acute myocardial ischemia/reperfusion injury.

The cardioprotection of the late phase of ischemic preconditioning is enhanced by postconditioning via a COX-2-mediated mechanism in conscious rats

The present study sought to determine whether the combination of late preconditioning (PC) with postconditioning enhances the reduction in infarct size. Chronically instrumented rats were assigned to a 45-min (subset 1) or 60-min (subset 2) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control) or were preconditioned 24 h before occlusion (PC), postconditioned at the onset of reperfusion following occlusion, or preconditioned and postconditioned without (PC + postconditioning) or with the COX-2 inhibitor celecoxib (3 mg/kg ip; PC + postconditioning + celecoxib) 10 min before postconditioning. Myocardial cyclooxygenase-2 (COX-2) protein expression and COX-2 activity (assessed as myocardial levels of PGE(2)) were measured 6 min after reperfusion in an additional five groups (control, PC, postconditioning, PC + postconditioning, and PC + postconditioning + celecoxib) subjected to a 45-min occlusion. PC alone reduced infarct size after a 45-min occlusion but not after a 60-min occlusion. Postconditioning alone did not reduce infarct size in either setting. However, the combination of late PC and postconditioning resulted in a robust infarct-sparing effect in both settings, suggesting additive cardioprotection. Celecoxib completely abrogated the infarct-sparing effect of the combined interventions in both settings. Late PC increased COX-2 protein expression and PGE(2) content. PGE(2) content (but not COX-2 protein) was further increased by the combination of both interventions, suggesting that postconditioning increases the activity of COX-2 induced by late PC. In conclusion, the combination of late PC and postconditioning produces additive protection, likely due to a postconditioning-induced enhancement of COX-2 activity.

Exogenous nitric oxide triggers classic ischemic preconditioning by preventing intracellular Ca 2+ overload in cardiomyocytes

The involvement of nitric oxide (NO) in the late phase of ischemic preconditioning is well established. However, the role of NO as a trigger or mediator of “classic preconditioning” remains to be determined. The present study was designed to investigate the effects of NO on calcium homeostasis in cultured newborn rat cardiomyocytes in normoxia and hypoxia. We found that treatment with the NO donor, sodium nitroprusside (SNP) induced a sustained elevation of intracellular calcium level ([Ca 2+] i) followed by a decrease to control levels. Elevation of extracellular calcium, which generally occurs during ischemia, caused an immediate increase in [Ca 2+] i and arrhythmia in cultures of newborn cardiomyocytes. Treatment with SNP decreased [Ca 2+] i to control levels and re-established synchronized beating of cardiomyocytes. A decrease in extracellular [Na +], which inhibits the Na +/Ca 2+ exchanger, did not prevent [Ca 2+] i reduction by SNP. In contrast, application of thapsigargin, an inhibitor of sarcoplasmic reticulum Ca 2+-ATPase (SERCA2a), increased [Ca 2+] i, and in its presence, SNP did not reduce [Ca 2+] i, indicating that Ca 2+ reduction is achieved via activation of SERCA2a. The results obtained suggest that activation of SERCA2a by SNP increases Ca 2+ uptake into the sarcoplasmic reticulum (SR) and prevents cytosolic Ca 2+ overload, which might explain the protective effect of SNP from hypoxic damage.

Endothelial Nitric Oxide Synthase Plays an Obligatory Role in the Late Phase of Ischemic Preconditioning by Activating the Protein Kinase Cε–p44/42 Mitogen-Activated Protein Kinase–pSer-Signal Transducers and Activators of Transcription1/3 Pathway

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. 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) epsilon 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. 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 epsilon-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.

Repetition of Ischemic Preconditioning Augments Endothelium-Dependent Vasodilation in Humans

Several studies have shown that both early and late effects of ischemic preconditioning (IPC) protect against myocardial injury after ischemic reperfusion. The purpose of this study was to evaluate the late effects of IPC on endothelial function in humans. Late phase of IPC was induced by upper limb ischemia (cuff inflation of over 200 mm Hg for 5 minutes) 6 times a day for 1 month. We evaluated forearm blood flow (FBF) responses to acetylcholine (ACh) and to sodium nitroprusside (SNP) before and after IPC stimulus in 30 young healthy men. FBF was measured using a strain-gauge plethysmograph. The IPC stimulus significantly increased plasma concentration of vascular endothelial growth factor (VEGF), circulating level of endothelial progenitor cells (EPCs), and FBF responses to ACh, but these did not change in the control group. The FBF responses to SNP were similar before and after the IPC stimulus. Infusion of N(G)-monomethyl-L-arginine, a nitric oxide synthase inhibitor, completely eliminated the IPC stimulus-induced augmentation of FBF responses to ACh. In the contralateral arms of subjects that received the IPC stimulus, FBF responses to ACh did not change, but levels of VEGF and circulating EPCs increased. These findings suggest that repetition of late IPC stimulus augments endothelium-dependent vasodilation in humans through increases in nitric oxide production and number of EPCs under a local condition. Repetition of IPC stimulus may be a simple, safe, and feasible therapeutic technique for endothelial protection of peripheral vessels.

The late phase of ischemic preconditioning induces a prosurvival genetic program that results in marked attenuation of apoptosis

Although the cardioprotection afforded by the late phase of ischemic preconditioning (PC) in ischemia/reperfusion (I/R) injury has been well studied, it is unknown whether this beneficial effect can be attributed to inhibition of apoptosis. We hypothesized that ischemic PC affords protection by suppressing apoptosis and examined the underlying mechanisms. Myocardial infarction was produced in mice (30-min coronary occlusion). In animals preconditioned 24 h earlier with six 4-min coronary occlusion/4-min reperfusion (O/R) cycles, there was a marked decrease in apoptosis as assessed by three different parameters: hairpin-1 assay, caspase-3 activity, and immunohistochemical analysis of active caspase-3 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1). This protective effect was accompanied by increased expression of multiple antiapoptotic proteins that regulate both the mitochondria-mediated (Bcl-x L and Mcl-1) and the death-receptor-mediated (c-FLIP L and c-FLIP S) pathway of apoptosis and by decreased expression of the proapoptotic protein Bad. This is the first demonstration that the late phase of ischemic PC attenuates cardiac apoptosis after ischemia/reperfusion injury and that this salubrious effect is associated with a complex genetic prosurvival program that results in modulation of several key proteins involved in both the mitochondrial and the death receptor pathways of apoptosis.

Targeted disruption of cox-2 gene abrogates the late phase of ischemic preconditioning

Pharmacologic studies with selective cyclooxygenase-2 (COX-2) inhibitors suggest that the late phase of ischemic preconditioning (PC) is mediated by inducible COX (COX-2). However, nonspecific effects of COX-2 inhibitors cannot be ruled out, and the selectivity of these inhibitors for COX-2 vs. COX-1 is only relative. Accordingly, we tested if targeted disruption of the COX-2 gene abrogates late PC in mice subjected to a 30-min coronary occlusion (O) and 24 h of reperfusion (R). In wild-type (WT) mice (group I), infarct size averaged 59.5±2.8% of the risk region (Figure). In mice homozygous for a null COX-2 allele (COX-2 knockout [KO], group II), infarct size was similar (62.0±2.2%), indicating that COX-2 does not modulate infarct size in the absence of PC. When WT mice were preconditioned 24 h earlier with six 4 min O/4-min R cycles (group V), infarct size was markedly reduced to 34.0±3.7%, indicating a late PC effect. In contrast, when COX-2 KO mice were preconditioned 24 h earlier with six 4-min O/4-min R cycles (group VI), infarct size was not reduced (59.8±3.0%). In conclusion, targeted disruption of the COX-2 gene completely abrogates the infarct-sparing effect of late PC, providing unequivocal molecular genetic evidence for an obligatory role of COX-2 in the cardioprotection afforded by the late phase of ischemic PC.

Redox Activation of Aldose Reductase in the Ischemic Heart

Aldose reductase (AR) reduces cytotoxic aldehydes and glutathione conjugates of aldehydes derived from lipid peroxidation. Its inhibition has been shown to increase oxidative injury and abolish the late phase of ischemic preconditioning. However, the mechanisms by which ischemia regulates AR activity remain unclear. Herein, we report that rat hearts subjected to ischemia, in situ or ex vivo, display a 2-4-fold increase in AR activity. The AR activity was not further enhanced by reperfusion. Activation increased Vmax of the enzyme without affecting the Km and decreased the sensitivity of the enzyme to inhibition by sorbinil. Enzyme activation could be prevented by pretreating the hearts with the radical scavenging thiol, N-(2-mercaptoproprionyl)glycine or the superoxide dismutase mimetic, Tiron, or by treating homogenates with dithiothreitol. In vitro, the recombinant enzyme was activated upon treatment with H2O2 and the activated, but not the native enzyme, formed a covalent adduct with the sulfenic acid-specific reagent dimedone. The enzyme activity in the ischemic, but not the nonischemic heart homogenates was inhibited by dimedone. Separation of proteins from hearts subjected to coronary occlusion by two-dimensional electrophoresis and subsequent matrix-assisted laser desorption ionization time-of-flight/mass spectrometry analysis revealed the formation of sulfenic acids at Cys-298 and Cys-303. These data indicate that reactive oxygen species formed in the ischemic heart activate AR by modifying its cysteine residues to sulfenic acids.