Preconditioning In Rat Heart Research Articles

Involvement of Estrogen Receptor and Mitochondrial KATP Channels in Cardioprotective Effect of Remote Aortic Preconditioning in Isolated Rat Heart

The present study was designed to investigate the role of estrogen receptor and KATP channel in cardioprotective effect of remote aortic preconditioning in rat heart. Remote preconditioning is a phenomenon in which brief episodes of ischemia and reperfusion to remote organs protect the target organ against sustained ischemia/ reperfusion induced injury. Protective effects of remote aortic preconditioning are well established in the heart, but their mechanisms still remain to be elucidated. Remote aortic preconditioning in rats was produced by introducing four episodes of ischemia and reperfusion, each comprising of 5 min occlusion and 5 min reperfusion, through abdominal aorta. Perfused rat heart was isolated and subjected to global ischemia for 30 min followed by reperfusion for 120 min. The degree of cardiac injury was assessed by analysing the level of lactate dehydrogenase, creatin kinase, reactive oxygen species and nitrite in coronary effluent. Myocardial infarct size was also estimated macroscopically using tetrazolium tri chloride staining. Ethylinestradiol (10 μg/kg, orally) as estrogen receptor agonist was noted to reproduce remote aortic preconditioning mediated cardioprotection in ovariectomized rat. However, administration of glibenclamide along with or prior to ethylinestradiol abolished cardioprotection. Moreover, glibenclamide a KATP channel blocker, abolish cardioprotective effect of remote aortic preconditioning with daidzein a caveolin inhibitor (0.2 mg/kg subcutaneous). Ethylinestradiol significantly increased the release of nitric oxide and restored the attenuated cardioprotective effect of remote aortic preconditioning. These data provide the evidence that estrogen receptor activation involved in cardioprotective effect of remote aortic preconditioning-mediated trough opening of KATP channels.

Cardioprotection by the transfer of coronary effluent from ischaemic preconditioned rat hearts: identification of cardioprotective humoral factors.

Ischaemic preconditioning (IPC) provides myocardial resistance to ischaemia/reperfusion (I/R) injuries. The protection afforded by IPC is not limited to the target tissue but extends to remote tissues, suggesting a mechanism mediated by humoral factors. The aim of the present study was to identify the humoral factors that are responsible for the cardioprotection induced by the coronary effluent transferred from IPC to naïve hearts. Isolated rat hearts were submitted to IPC (three cycles of 5min I/R) before 30-min global ischaemia and 60-min reperfusion. The coronary effluent (Efl_IPC) collected during IPC was fractionated by ultrafiltration in different molecular weight ranges (<3, 3-5, 5-10, 10-30, 30-50, and >50kDa) and evaluated for cardioprotective effects by perfusion before I/R in naïve hearts. Only the <3, 5-10 and <10kDa fractions of hydrophobic eluate reduced I/R injuries. The cardioprotective effect of the 5-10 fraction was blocked by KATP channel blockers and a PKC inhibitor. An Efl_IPC proteomic analysis revealed 14 cytoprotection-related proteins in 4-12kDa peptides. HSP10 perfusion protected the heart against I/R injuries. These data provide insights into the mechanisms of cardioprotection in humoral factors released by IPC. Cardioprotection is afforded by hydrophobic peptides in the 4-12kDa size range, which activate pathways that are dependent on PKC and KATP. Fourteen 4-12kDa peptides were identified, suggesting a potential therapeutic role for these molecules in ischaemic diseases. One of these, HSP10, identified by mass spectrometry, reduced I/R injuries and may be a potential candidate as a therapeutic target.

Effects of wortmannin on cardioprotection exerted by ischemic preconditioning in rat hearts subjected to ischemia-reperfusion.

Ischemic preconditioning (IPC) is one of the most powerful interventions to reduce ischemia-reperfusion injury. The aim of the present study was to investigate the involvement of the phosphatidylinositol-3-kinases (PI3Ks) family in cardioprotection exerted by IPC and the relationship between preservation of mitochondrial morphology and ATP synthesis capacity. In this regard, macroautophagy (autophagy) is considered a dynamic process involved in the replacement of aged or defective organelles under physiological conditions. IPC consisted of four 5-min cycles of ischemia-reperfusion followed by sustained ischemia. Wortmannin (W), a PI3K family inhibitor, was added to the perfusion medium to study the involvement of autophagy in the beneficial effects of IPC. In the present study, LC3-II/I expression was significantly increased in the IPC group when compared with the control group. The hearts subjected to IPC showed greater degradation of p62 than control groups, establishing the existence of an autophagic flow. Electron microscopy showed that IPC preserves the structural integrity of mitochondria after ischemia and at the end of reperfusion. Moreover, hearts subjected to IPC exhibited increased mitochondrial ATP synthesis. The beneficial effects of IPC were abolished by W in all trials of this study, abolishing the differences between the IPC and control groups. These results suggest that IPC could partly reduce injury by ischemia-reperfusion (I/R) by decreasing mitochondrial damage and promoting autophagy. Since W is a nonspecific inhibitor of the PI3Ks family, further research is required to confirm participation of PI3K in the response to IPC.

Studies of Ischemic Preconditioning Mechanisms in Langendorff Rat Heart Model the Impact of Phosphocreatin Kinase and ATP Sensitive Potasium Channels Pharmachological Openers and Blockers on Cardioprotection

Abstract Ischemic preconditioning (IPC) induced by administration of brief episodes of ischemia-reperfusion represents a protective mechanism of the heart against prolonged episodes of ischemia. Although the mechanism of ischemic preconditioning has been extensively studied, however, until now it is insufficient elucidated. Using a Langendorff rat heart model with 45 minutes ischemia followed by 120 minute reperfusion we aimed to evaluate the role of Phosphocreatin Kinase-C (PCK) in ischemic rat myocardium and demonstration of its involvment in the path of pharmachological preconditioning (PP) by using PKC activators 1,2-dioctanoyl-sn-glycerol (DOG) and inhibitors chelerythrine (CHE) and evaluation of the role of K ATP channels in pharmachological preconditioning (PP) mechanism by administration of a K ATP channel opener (Cromakalim) (CRK) or by blocking the opening of K ATP channels with glibenclamide(GLB). The activators of PCK(DOG) and of K ATP channel (CRK) limited the infarct size when perfused before lethal ischemia, mimicking the ischemic preconditioning in rat heart When activator of PCK DOG + GLB, a K ATP channel inhibitor were coperfused before the lethal ischemia, there was an increase in myocardial infarct size, expressed as a percentage of the area at risk, versus control, the cardioprotective effect of DOG being abolished by GLB. The same results were obtained when CHE the inhibitor of PCK was coperfused with CRK the activator of K ATP channel. The effect of CHE and GLB perfused at the beginning of IPC resulted in loss of protection accompanied by a significant increase in infarct size area. The finding that treatment with a DOG PKC activator and CRK activator of K ATP channel gives a similar degree of protection against infarction as that seen after ischemic preconditioning and that this protection can be blocked by CHE a PCK pharmachological inhibitor provides support for the hypothesis that PKC plays a pivotal role in ischemic preconditioning. Our data may have a significance in pharmachological preconditioning (PP) with decrease in infarct size as an end point.

Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.

Landiolol does not enhance the effect of ischemic preconditioning in isolated rat hearts

To determine the effect of landiolol on ischemic preconditioned rat hearts. Isolated perfused rat hearts were divided into 8 groups. In the control group, there was no treatment before the 30-min global ischemia. In the landiolol infused groups, landiolol (100, 300, and 500 microM) was infused without ischemic preconditioning (IPC). In other groups, hearts were pretreated with 2 episodes of 5-min global ischemia and reperfusion before the 30-min ischemia. During the preconditioning, landiolol (0, 100, 300, and 500 microM) was infused. Recoveries of coronary flow (CF) and myocardial oxygen consumption (MVO(2)) at the 120th min after global ischemia to 86 +/- 18 and 112 +/- 19% of the baseline in the IPC group was, respectively, significantly greater than that to 65 +/- 10 and 72 +/- 10% in the control group. Landiolol 300 microM also increased the CF and MVO(2) significantly (97 +/- 19 and 98 +/- 39%) compared to the control. IPC + landiolol 500 microM reduced the increase in LV end-diastolic pressure significantly compared to the control. IPC, landiolol (100, 300, and 500 microM), and IPC + landiolol (100, 300, and 500 microM) all decreased infarct sizes significantly to 23.5 +/- 15.2, 29.8 +/- 12.1, 30.2 +/- 13.3, 22.8 +/- 14.8, 21.6 +/- 7.8, 34.2 +/- 14.7 and 25.5 +/- 11.3% of the total left ventricular mass, respectively, compared to the control (53.3 +/- 12.5%), but there were no significant differences among these groups. IPC and landiolol have cardioprotective effects on ischemia-reperfusion injury in isolated rat hearts, but pretreatment with landiolol does not enhance the cardioprotective effect of IPC.

Mechanism of cardioprotective effect of erythropoietin-induced preconditioning in rat heart

Objective:The cardioprotective potential of human recombinant erythropoietin (alpha) (Epo) against ischemia-reperfusion-induced injury is well known. But, the underlying mechanisms are not well elucidated. The aim of this study was to characterize the mechanism involved in the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.Materials and Methods:The heart was mounted on a Langendorff apparatus. After 10 min of stabilization, four cycles of ischemic preconditioning (IPC) were given followed by 30 min of global ischemia and 120 min of reperfusion. Epo preconditioning was induced by four cycles of 5-min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution. Myocardial infarct size was estimated macroscopically using the triphenyltetrazolium chloride staining technique. The extent of myocardial injury was measured by release of lactate dehydrogenase and creatine kinase-MB in the coronary effluent.Results:The present study demonstrates that Epo preconditioning was almost as effective as IPC. Administration of Wortmannin (100 nM), a PI-3K inhibitor, or Chelerythrine (1 µM), a protein kinase-C (PKC) inhibitor, or AG490 (5 µM), a JAK-2 inhibitor, significantly attenuated the cardioprotective effects of Epo-induced preconditioning.Conclusion:Our result suggest that the cardioprotective potential of Epo-induced preconditioning in isolated rat heart was due to an interplay of the JAK-2, PI-3K and PKC pathways. Inhibition of any one of the three pathways was sufficient to block the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.

Enhanced expression of let-7f after ischemic preconditioning in rat heart is cardioprotective

Enhanced expression of let-7f after ischemic preconditioning in rat heart is cardioprotective

Dynamic changes in nitric oxide and mitochondrial oxidative stress with site-dependent differential tissue response during anoxic preconditioning in rat heart

In this study, dynamic changes in nitric oxide (NO) and mitochondrial superoxide (O(2)(*-)) were examined during anoxic preconditioning (AP) in rat heart model. AP and anoxia-reoxygenation (A/R) were performed on isolated hearts and single cardiomyocytes. The cellular insult in the form of infarct size and DNA damage were localized and correlated with NO synthases (endothelial and inducible) expression levels. The results showed that endocardium was the most affected region in AP groups, whereas the larger area of infarct was confined to mid- and epicardium in the A/R group. Interestingly, a high-level expression of immunofluorescent NO synthases was restricted to viable areas in the AP. In contrast to the gradual increase in O(2)(*-) level that occurred in the AP group, a sudden massive increase in its level was demonstrated at the onset of reoxygenation in the A/R group. The observed increase in NO production during reoxygenation in the AP group was attenuated by inducible NO synthase inhibitor. The study revealed, on a real-time basis, the role played by preconditioning for modulating NO and O(2)(*-) levels on behalf of cell survival. The results afford a better understanding of cardiac-adapting mechanism during AP and the role of inducible NO synthase in this important phenomenon.

Role of endogenous nitric oxide in classic preconditioning in rat hearts

Ischemic preconditioning (IPC) protects the heart against subsequent sustained ischemia reperfusion (RP). Despite many triggers and signaling pathways, which seem to be involved in IPC, the IPC-mechanisms remain a controversial issue. One of them is endogenous production of nitric oxide (NO). To assess the role of NO in IPC and its relation with glycogen and glycolysis, the effects of inhibiting NO synthase with L-NAME (50 μM) were examined in IPC rat hearts perfused with medium containing 10 mM glucose. Left ventricular developed pressure-rate product (RPP) and end diastolic pressure (EDP), lactate and glycogen contents, and cell viability were measured. Global ischemia (25 min) was followed by 30 min RP. IPC consisted in one cycle of 3 min ischemia–5 min RP. IPC reduced EDP and improved RP recovery of RPP. L-NAME had no effects on the non-IPC group but abolished these effects of IPC. IPC reduced ischemic decrease of glycogen and the acceleration of glycolysis, and improved cell viability. L-NAME did not affect these effects of IPC. The results suggest that NO is ineffective on the noxious effects of ischemia-RP in non-IPC hearts and on the effects of IPC on cell viability, glycogenolysis and glycolysis whereas it is only involved in functional protection.

Mechanism of reactive oxygen species generation after opening of mitochondrial KATP channels

opening of the ATP-sensitive K+ (KATP) channel mediates the cardioprotective effect induced by pathophysiological stressors such as ischemic preconditioning (IPC) ([15][1], [29][2]), heat shock ([19][3]), and pharmacological agents, including adenosine ([5][4]), ACh ([26][5]), opioids ([12][6]),

Antiapoptotic effect of calcitonin gene-related peptide on oxidative stress-induced injury in H9c2 cardiomyocytes via the RAMP1/CRLR complex

Calcitonin gene-related peptide (CGRP) plays an important role in the mediation of protective effects observed in situations such as ischemic preconditioning in rat hearts. In this study, we investigated in H9c2 rat cardiomyoblasts if the protective effect of CGRP could be linked to an inhibitory effect on the apoptotic pathway. We also determined the specificity of observed effects by treatment with adrenomedullin (ADM) in stress conditions generated by 100 μM hydrogen peroxide. Using MTT assays, we demonstrate that a pretreatment with CGRP decreases by half the loss of cell viability induced by H 2O 2. CGRP inhibits phosphatidylserine externalization, caspase 3 activation and DNA fragmentation due to oxidative stress. Using RT-PCR, we observed an increase in Bcl-2 mRNA expression induced by CGRP treatment. Dot blotting experiments showed that, in stress conditions, Bcl-2 protein level decreases while Bax is increased. CGRP administration prior to stress prevents these effects. The three-receptor activity modifying protein (RAMP) isotypes were detected by RT-PCR in H9c2 cells and in left ventricle rat tissue, RAMP1 and RAMP3 being the most abundant in both cases. RAMP1 expression was upregulated by CGRP while RAMP3 mRNA level was decreased. Cell viability assessed by MTT indicates that, contrary to CGRP, pretreatment of stressed cells with ADM, a RAMP2 agonist, fails to protect them while treatment with CGRP 8–37 (a RAMP1 and 2 inhibitor) abolished CGRP protective effect. Taken together, these data suggest that CGRP has antiapoptotic properties through the RAMP1/CRLR complex. CGRP could be used to prevent apoptosis in an ischemia–reperfusion context.

Gene transfer of 2 adrenergic receptor enhances cardioprotective effects of ischemic preconditioning in rat hearts after myocardial infarction

The purpose of the present study was to determine a role of beta(2) adrenergic receptor (beta(2)AR) in ischemic preconditioning (IPC) response. Post-myocardial infarction (MI) hearts were produced by ligating the left anterior descending coronary artery for 2 weeks. Post-MI hearts were transfected with the empty virus (Empty-vivo) or beta(2)AR cDNA (beta(2)AR-vivo) by intracoronary infusion of hemagglutinating virus of Japan-liposome. Empty-vivo or beta(2)AR-vivo hearts were subjected to Langendorff perfusion as Control or beta(2)AR hearts, respectively. IPC was undertaken in Control(IPC) and beta(2)AR(IPC+beta(2)AR). After global ischemia, seven hearts in each group were reperfused and normalized left ventricular peak developed pressures (LVPDP) and creatine phosphokinase (CPK) leakage were measured. beta(2)AR gene transfection was confirmed by measuring responsiveness to isoproterenol, real time RT-PCR and immunohistochemistory. IPC preserved LVPDP and reduced CPK leakage in IPC+beta(2)AR hearts as compared with IPC hearts. LVPDP was decreased in addition to increase in CPK leakage in beta(2)AR hearts as compared with Control. Expression of beta(2)AR and responsiveness to isoproterenol were increased in beta(2)AR-vivo as compared with Empty-vivo hearts. These results indicate that beta(2)AR are required to generate IPC effects, and that beta(2)AR gene transfection enhances IPC effects in post-MI hearts.

Activation of β2-adrenergic receptor plays a pivotal role in generating the protective effect of ischemic preconditioning in rat hearts

Background. Ischemic preconditioning (IPC) protects hearts against ischemia by reducing infarct size. However, IPC does not preserve cardiac function, such as left ventricular peak developed pressure (LVPDP). Moreover, IPC fails to protect the post-myocardial infarct (MI) heart. Design. Rat hearts were transfected with β2-adrenergic receptor (B2AR) cDNA by the hemagglutinating virus of Japan–liposome method. After the gene transfer, the hearts were perfused in a Langendorff mode and preconditioned with two cycles of 5 min of ischemia and reperfusion. After 20 min of global ischemia, the hearts were reperfused under aerobic conditions for 90 min. LVPDP was measured as an indicator of the cardiac function. Results. LVPDP of ischemic hearts was well preserved by the combination treatment of IPC and gene transfer of B2AR, but not IPC or gene transfer of B2AR alone. Moreover, the treatment was beneficial to even the post-MI heart. On the contrary, gene transfer of β-adrenergic receptor kinase 1 (BARK1) reduced the protective effect of IPC. We also found that the mRNA ratio of B2AR and BARK1 was well correlated with the preservation of the LVPDP. Conclusions. The combination treatment of IPC and gene transfer of B2AR protects cardiac function against ischemia and it shows the beneficial effect also in post-MI hearts.

Delayed ischemic preconditioning activates nuclear-encoded electron-transfer-chain gene expression in parallel with enhanced postanoxic mitochondrial respiratory recovery.

Delayed ischemic preconditioning promotes cardioprotection via genomic reprogramming. We hypothesize that molecular regulation of mitochondrial energetics is integral to this cardioprotective program. Preconditioning was induced by use of 3 episodes of 3-minute coronary artery occlusion separated by 5 minutes of reperfusion. Twenty-four hours later, infarct size was reduced by 58% after preconditioning compared with sham-operated controls (P<0.001). Cardiac mitochondria were isolated from sham and preconditioned rat hearts. Mitochondrial respiration and ATP production were similar between the groups; however, preconditioned mitochondria exhibit modest hyperpolarization of the inner mitochondrial membrane potential (> or =22% versus control, P<0.001). After 35-minute anoxia and reoxygenation, preconditioned mitochondria demonstrated a 191+/-12% improvement in ADP-sensitive respiration (P=0.002) with preservation of electron-transfer-chain (ETC) activity versus controls. This augmented mitochondrial recovery was eradicated when preconditioning was abolished by the antioxidant 2-mercaptopropionyl glycine (2-MPG). These biochemical modulations appear to be regulated at the genomic level in that the expression of genes encoding rate-controlling complexes in the ETC was significantly upregulated in preconditioned myocardium, with a concordant induction of steady-state protein levels of cytochrome oxidase, cytochrome c, and adenine nucleotide translocase-1. 2-MPG abolished preconditioning induction of these transcripts. Moreover, transcripts of nuclear regulatory peptides known to orchestrate mitochondrial biogenesis, nuclear respiratory factor-1 and peroxisome-proliferator-activated receptor gamma coactivator 1alpha, were significantly induced in preconditioned myocardium. Delayed preconditioned mitochondria display increased tolerance against anoxia-reoxygenation in association with modifications in mitochondrial bioenergetics, with concordant genomic induction of a mitochondrial energetic gene regulatory program. This program appears to be mediated by reactive oxygen species signaling.

Down-regulation of Connexin43 in Early Myocardial Ischemia and Protective Effect by Ischemic Preconditioning in Rat Hearts In Vivo

Down-regulation of Connexin43 in Early Myocardial Ischemia and Protective Effect by Ischemic Preconditioning in Rat Hearts In Vivo

Down-regulation of Connexin43 in Early Myocardial Ischemia and Protective Effect by Ischemic Preconditioning in Rat Hearts In Vivo

Connexin 43 (Cx43), a primary component of gap junctions, contributes to intercellular electrochemical communication. Cx43 undergoes dephosphorylation in early ischemia. We examined whether Cx43 is degraded in association with dephosphorylation during early myocardial ischemia and whether ischemic preconditioning (IP) affects the degradation after rat coronary artery occlusion. Male Sprague-Dawley rats underwent coronary artery occlusion for 1, 2, or 3 hours, or for 1 hour following treatment either with a calcineurin inhibitor (cyclosporine A), proteasome inhibitor (PSI), or lysosomal inhibitor (E64c), or following IP alone or after protein kinase C (PKC) inhibitor (chelerythrine) pretreatment. The IP was afforded by three cycles of 3 minute ischemia and 5 minute reperfusion. A large portion of the phosphorylated Cx43 (pCx43) in the membrane fraction was dephosphorylated, while a small portion was degraded at 1 hour of ischemia. The effects of the inhibitors were dephosphorylation and degradation by calcineurin and proteasome/lysosome, respectively. IP suppressed the decrease in pCx43 and increase in dCx43, while only the former was inhibited by the PKC inhibitor chelerythrine. The Cx43 mRNA level was reduced at 3 hours, but not at 1 hour of ischemia, irrespective of IP. We believe that Cx43 is dephosphorylated and degraded in early ischemia, whereas Cx43 transcription was suppressed at a later phase of ischemia.

Reactive hyperemia during early reperfusion as a determinant of improved functional recovery in ischemic preconditioned rat hearts

Objective Our study was undertaken to clarify the impact of the shear stress-induced reactive hyperemia (associated with reperfusion) in preconditioning-mediated protection. Methods In control rat hearts, a 40-minute preischemic perfusion (constant pressure: 70 mm Hg) period was followed by 25-minute global low-flow ischemia (constant flow: 0.3 mL/min) and 30-minute reperfusion (constant pressure). As preconditioning protocol, hearts underwent 2 cycles of 5-minute no-flow ischemia/5-minute reperfusion. Results Although coronary vasodilation in response to shear stress is severely impaired after global low-flow ischemia and reperfusion, it is fully preserved by ischemic preconditioning concomitantly with an improvement of left ventricular developed pressure. Restricting coronary peak flow to 100% of baseline at reperfusion reduced left ventricular recovery to the control level. N G-nitro- l-arginine methyl ester affects the restoration of reperfusion-reactive hyperemia and the improvement of contractile recovery afforded by ischemic preconditioning. However, if the time course of hyperemia was restored by forcibly reperfusing to 150% of baseline for 10 minutes and, therefore, by restricting final peak flow to 80% of baseline for 20 minutes, contractile function recovered to a high degree despite the presence of N G-nitro- l-arginine methyl ester. Conclusion We conclude that wall stretch and shear stress during reperfusion are necessary for the mediation phase of preconditioning.

Peroxynitrite triggers a delayed resistance of coronary endothelial cells against ischemia-reperfusion injury.

Experiments were designed to test whether nitric oxide (NO) and peroxynitrite trigger delayed coronary endothelial protection induced by preconditioning (PC) in rats. Prolonged ischemia reperfusion markedly reduced the response of isolated coronary arteries to acetylcholine, and this was prevented by PC performed 24 h earlier. The NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) administered during PC abolished its delayed endothelial protective effect, whereas the inducible NOS inhibitor N-(3(aminomethyl)benzyl)acetaminide had no effect. Delayed endothelial PC was also abolished by the peroxynitrite scavengers selenomethionine or uric acid given during PC. In parallel, the NO/peroxynitrite donor S-morpholinosydnonimine and authentic peroxynitrite, administered 24 h before prolonged ischemia-reperfusion mimicked endothelial PC, whereas the NO donor S-nitroso-N-acetylpencillamine had no effect. This suggests that peroxynitrite is an essential trigger of the delayed coronary endothelial protection induced by PC in rat hearts.

Preconditioning of rat hearts by adenosine A 1 or A 3 receptor activation

Our study in rat hearts examined whether activation of adenosine A 1 or A 3 receptors improved functional recovery and reduced apoptosis resulting from low-flow ischemia. Prior to 30 min low-flow ischemia (0.6 ml/min; 6% of baseline flow), Langendorff rat hearts were preconditioned with two 5-min cycles of (a) ischemia (PC; n=7), (b) infusion of 250 nM adenosine A 1 receptor agonist 2-chloro- N 6-cyclopentyladenosine (CCPA; n=6), or (c) infusion of 50 nM adenosine A 3 receptor agonist N 6-(3-iodobenzyl)-adenosine-5′- N-methyl-uronamide (IB-MECA; n=8). Recovery of function was improved in PC (71±3%), CCPA (68±6%) and IB-MECA (68±4%) groups compared to control hearts (46±5%; P<0.05). Cumulative release of total purines during ischemia–reperfusion was approx. 50% lower in PC, CCPA and IB-MECA groups compared to controls ( P<0.05) and was significantly correlated to the percentage functional recovery ( R 2=0.55; P<0.05). The number of cytosolic histone-associated-DNA fragments, a hallmark of apoptosis and measured by Enzyme Linked ImmunoSorbent Assay (ELISA), was small and not different between groups after 30 min reperfusion. However, CCPA (0.6±0.1 absorbance units) and MECA (0.7±0.1 units; P<0.05 vs. PC) decreased apoptosis after 150 min reperfusion compared to PC (1.4±0.3 units) and control (1.2±0.1 units) hearts. This study shows that adenosine triggers protection of function in preconditioned rat hearts via both the adenosine A 1 and A 3 receptor. In clinical practice, pharmacological stimulation of adenosine A 3 receptors may be advantageous over adenosine A 1 receptor activation due to a lack of contractile side-effects. In contrast to ischemic preconditioning, pharmacological stimulation of adenosine A 1 or A 3 receptors reduced apoptosis. Furthermore, total purine release may serve as a marker of the degree of functional protection.