Subsequent Sustained Ischemia Research Articles

Ischemic Preconditioning of Rat Livers from Non-Heart-Beating Donors Decreases Parenchymal Cell Killing and Increases Graft Survival after Transplantation

A critical shortage of donors exists for liver transplantation, which non-heart-beating cadaver donors could help ease. This study evaluated ischemic preconditioning to improve graft viability after non-heart-beating liver donation in rats. Ischemic preconditioning was performed by clamping the portal vein and hepatic artery for 10 min followed by unclamping for 5 min. Subsequently, the aorta was cross-clamped for up to 120 min. After 2 h of storage, livers were either transplanted or perfused with warm buffer containing trypan blue. Aortic clamping for 60 and 120 min prior to liver harvest markedly decreased 30-day graft survival from 100% without aortic clamping to 50% and 0%, respectively, which ischemic preconditioning restored to 100 and 50%. After 60 min of aortic clamping, loss of viability of parenchymal and nonparenchymal cells was 22.6 and 5.6%, respectively, which preconditioning decreased to 3.0 and 1.5%. Cold storage after aortic clamping further increased parenchymal and non-parenchymal cell killing to 40.4 and 10.1%, respectively, which ischemic preconditioning decreased to 12.4 and 1.8%. In conclusion, ischemic preconditioning markedly decreased cell killing after subsequent sustained warm ischemia. Most importantly, ischemic preconditioning restored 100% graft survival of livers harvested from non-heart-beating donors after 60 min of aortic clamping.

Intestinal Preconditioning Ameliorates Ischemia-Reperfusion Induced Acute Lung Injury in Rats: An Experimental Study

Phospholipases A(2) (PLA(2)) have been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) induced by intestinal ischemia-reperfusion (IIR). Intestinal ischemic preconditioning (IIP) has been shown to improve intestinal tolerance to subsequent sustained ischemia and limit the systemic inflammatory response. We tested the effect of IIP on the intestinal ischemia-reperfusion-induced ARDS, with particular focus on PLA(2). Rats were randomized into three groups: (1) sham surgery group (sGroup), 45 min sham intestinal ischemia-4 h reperfusion, (2) IIR group (IIRGroup), 45 min intestinal ischemia-4 h reperfusion, (3) IIP group (ipGroup), three cycles of intestinal ischemia for 4 min and reperfusion for 10 min followed by 45 min intestinal ischemia-4 h reperfusion. At the end of each experiment, blood gases were obtained and bronchoalveolar lavage (BAL) followed. Biochemical (total protein, PLA(2), PAF-AcH) and cytological parameters of the BAL fluid were quantified. Plasma MDA was measured as an indicator of systemic oxidative stress. Comparisons between groups were made using one-way ANOVA followed by post hoc comparison with a Tukey test or Mann-Whitney test when appropriate. Differences were considered significant if P < 0.05. Alveolar-arterial O(2) gradient values and wet to dry lung ratio were significantly (P < 0.05) increased in the IIRGroup and this increase was prevented in the ipGroup. Following the same pattern, BAL total protein, PLA(2), and PAF-AcH were significantly lower in the ipGroup. Ischemic preconditioning significantly abolished neutrophil count in BAL fluid. Plasma MDA was significantly lower in the ipGroup. Despite a significant tissue polymorphonuclear reduction, no significant lung or intestinal histologic damage score changes were revealed. Intestinal preconditioning protects IIR-induced lung injury, partly by modulating the arachidonic acid cascade.

Gap junctions in preconditioning against arrhythmias

See article by Papp et al . [10] (pages 396–405) in this issue . Ischemic preconditioning is a natural mechanism by which brief periods of ischemia–reperfusion initiate a complex cascade of events that culminate in the protection of the heart against a subsequent sustained ischemia, reducing infarct size, postischemic dysfunction, and the incidence of arrhythmias [1]. In essence, endogenous or exogenous triggers released during ischemic or pharmacological preconditioning activate mediators that act on end-effectors, preserving mitochondrial, cytoskeletal, and cell membrane integrity [2]. However, although much effort has been dedicated to the study of preconditioning mechanisms, these have not been clearly elucidated, and in particular the pathways involved in the protection against arrhythmias have not been as thoroughly investigated as those concerning infarct size. One of the proposed targets of preconditioning protection is gap junctions. These intercellular structures formed by two hemichannels of connexin-43 (Cx43) mediate the exchange of small molecules and ions between two adjacent cells and play an active role in the electrical synchronization of the heart. Phosphorylation of Cx43 regulates gap junction conductance and permeability. In the normoperfused heart, most Cx43 is phosphorylated, allowing cell-to-cell coupling and the propagation of depolarization in the myocardial syncytium. During the course of ischemia, however, … *Corresponding author. Tel.: +54 11 4378 1187; fax: +54 11 4381 0323. Email address: lascano{at}favaloro.edu.ar

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.

Ischemic Preconditioning does not alter NADH supported state 3 respiration or complex I activity.

Ischemic preconditioning (IPC) before sustained ischemia (ISC) decreases infarct size. We found that reversible chemical blockade of electron transport at complex I immediately before sustained ISC preserves mitochondrial respiration and decreases infarct size during ISC-reperfusion. We proposed that IPC would decrease complex I activity and attenuate state 3 respiration through complex I as a potential mechanism of cardioprotection. Langendorff-perfused rat hearts underwent IPC (3 cycles 5 min 37°C global ISC and 5 min reperfusion) or were perfused for 40 min without ISC as time controls. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated to measure respiration (natom O/min/mg protein) with complex I substrates (glutamate or pyruvate-malate). Maximally-expressed complex I activity (nmol NADH/min/mg) was measured as NADH:ubiquinone oxidoreductase in detergent-solubilized mitochondria. IPC did not decrease state 3 respiration. Complex I activity was also unaffected by IPC. IPC increased state 4 respiration in IFM (59 ± 7, n=6 vs 41 ± 5, n=5, p<0.05) indicating decreased coupling of respiration. Thus, the protection of IPC does not occur via a decrease in complex I activity or a decrease in respiration through complex I during subsequent sustained ISC. Alternatively, IPC decreases the coupling of respiration as a potential mechanism of cardioprotection.

Brief Episode of Myocardial Ischemia Before Prolonged Ischemia Attenuates Cardiac Sympathetic Nerve Injury

The aim of this study was to investigate the effects of brief ischemia before prolonged ischemia on cardiac sympathetic neural function. Brief ischemia inhibits the sympathetic neural release of norepinephrine (NE) during subsequent sustained ischemia. However, whether it can attenuate the neural function after sustained ischemia remains unknown. Sympathetic neural function was assessed using 123I-metaiodobenzylguanidine (MIBG) in patients who with (Group I) or without angina (Group II) within 3 days prior to acute myocardial infarction. In the rat experiment, cardiac interstitial NE (iNE) with or without pretreatment of 5-min coronary ligation was determined during a 30-min occlusion. Differences between MIBG and Thallium-201 for the total defect score were significantly greater in Group II than in Group I (6.1 +/- 4.0 vs 0.4 +/- 4.4). Levels of iNE were less in rats with a 5-min pretreatment (7.3 +/- 2.3 vs 18.6 +/- 5.9 x 10(3) pg/ml, p < 0.01) and MIBG uptake of ischemic region was greater (0.061 +/- 0.029 vs 0.031 +/- 0.011 %kg dose/g, p < 0.05) compared with rats without the pretreatment. A brief episode of ischemia attenuates the sympathetic neural injury caused by subsequent prolonged ischemia and this protective effect is associated with attenuation of NE release during the prolonged ischemia.

Preinfarction angina: No interference of coronary microembolization with acute ischemic preconditioning

Transient episodes of angina preceding acute myocardial infarction may both, protect the myocardium by ischemic preconditioning or damage it when associated with coronary microembolization. We now studied the potential loss of ischemic preconditioning with coronary microembolization. Anesthetized pigs (group 1; n = 8) were subjected to 90 min sustained low-flow ischemia. Group 2 ( n = 8) was subjected to coronary microembolization (i.e. microspheres; 42 μm ∅; 3000 per ml min –1 inflow) 35 min before sustained ischemia. In group 3, coronary microembolization was followed 10 min later by one cycle of ischemic preconditioning (10 min ischemia/15 min reperfusion) before subsequent sustained ischemia. Infarct size was determined after 2 h reperfusion by triphenyl tetrazolium chloride staining. Infarct size after sustained ischemia alone (group 1) was 19.4 ± 3.4% of the area at risk (mean ± S.E.M.). With coronary microembolization before sustained ischemia (group 2) infarct size was only slightly larger (23.6 ± 4.6%, ns). In group 3 with microembolization followed by ischemic preconditioning, infarct size was reduced to 12.7 ± 3.0% ( P < 0.05 vs. group 2). The relationships between infarct size and transmural blood flow in groups 1 and 3 were not different, giving the impression that ischemic preconditioning failed to protect microembolized myocardium. However, additional coronary microembolization shifted the relationship between infarct size and blood flow upwards to a larger infarct size at any given blood flow. Thus when comparing the relationship of group 3 to its true control (group 2), it was shifted downwards ( P < 0.05; analysis of covariance (ANCOVA)) indicating persistent protection of microembolized myocardium by ischemic preconditioning. Coronary microembolization induces additional infarction when superimposed on sustained ischemia but does not interfere with the endogenous protection by ischemic preconditioning.

Sublethal simulated ischemia promotes delayed resistance against ischemia via ATP-sensitive (K+) channels in murine myocytes: role of PKC and iNOS.

In this study, we examined whether sublethal simulated ischemia (SSI) induces delayed cellular protection in mouse cardiac myocytes, and whether the delayed cellular protection depends on the activation of protein kinase C-epsilon (PKC-epsilon), inducible nitric oxide synthase (iNOS), and ATP-sensitive K(+) (K(ATP)) channels against subsequent sustained simulated ischemia (SI). The following groups of mouse cardiac myocytes were studied: (a) SI: incubation with SI buffer for 1 h; (b) SSI: incubation with SSI buffer for 30 min; (c) SSI + PKC inhibitor, chelerythrine chloride (CCl): SSI and 1 micro M CCl; (d) SSI + iNOS inhibitor, S-methylthiourea (SMT): SSI and 100 nM SMT; (e) SSI + K(ATP) channel blocker, glibenclamide (Glb): SSI and 50 micro M Glb; (f) SSI + mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (5-HD): SSI and 50 micro M 5-HD. The release of lactate dehydrogenase into the medium and the amount remaining in the cells was measured, and A(1) adenosine receptor, PKC-epsilon, and iNOS were detected through western blot analysis. The delayed cellular protection acquired due to SSI showed a decreased release of lactate dehydrogenase (%) from 46.51 +/- 1.60 to 37.00 +/- 1.34 (p < 0.001) and was blocked by CCl (47.08 +/- 0.95), SMT (48.08 +/- 1.18), Glb (45.88 +/- 1.31), and 5-HD (47.20 +/- 1.56). Simultaneously, SSI-induced up-regulation of A(1) adenosine receptor, PKC-epsilon, iNOS, and opening of both membrane and mitochondrial K(ATP) channels also was observed compared with controls.

Ischemic preconditioning promotes a transient, but not sustained translocation of protein kinase C and sensitization of adenylyl cyclase.

Brief periods of ischemia precondition the heart and reduce the size of infarction caused by a subsequent sustained ischemia. The molecular memory of preconditioning, i.e., the molecule persistently activated by the preconditioning ischemia exhibiting protection during the infarct-inducing event, is subject to debate. Protein kinase C, adenylyl cyclase and beta-adrenergic receptors are candidates for this memory, supposedly their activation persists during several cycles of ischemia and reperfusion. The goal of this study was, therefore, to determine the activation of those signaling molecules after 1 - 3 cycles of myocardial ischemia and reperfusion. Rat hearts were perfused according to the method of Langendorff with 1 - 3 cycles of ischemia (5 min) and reperfusion (10 min). In the particulate fraction of these hearts, densities of b-adrenergic receptors, activities of adenylyl cyclase, and the activities and isozyme distributions of PKC-alpha, PKC-beta, and PKC- epsilon were determined. The ischemia-induced upregulation of beta-adrenergic receptors is not influenced by preconditioning. In contrast, the sensitization of adenylyl cyclase observed after 5 min of ischemia is lost after repetitive periods of ischemia and reperfusion. Translocation of protein kinase C to the particulate fraction could be shown after 1 and 2 periods of ischemia including all major cardiac isozymes, with a rapid relocation to the cytosol after every cycle of reperfusion. A third period of ischemia was unable to promote a repeated translocation of protein kinase C. The ischemia-induced regulation process of beta-adrenergic receptors is not influenced in preconditioning. Moreover, a sustained translocation of protein kinase C and a sustained sensitization of adenylyl cyclase are obviously no prerequisite for preconditioning after various cycles of ischemia and reperfusion. Thus, those signaling molecules do not seem to be operative for the preconditioning's memory. It is suggested that the initial, synergistic burst of sensitization of the adenylyl cyclase and of protein kinase C translocation induces myocardial protection very early in ischemia and reperfusion.

Ischemic and pharmacological preconditioning induces further delayed protection in transgenic mouse cardiac myocytes over-expressing adenosine A1 receptors (A1AR): role of A1AR, iNOS and K(ATP) channels.

In this study we examined the hypotheses that over-expression of the adenosine A(1) receptor (A(1)AR) in transgenic mouse cardiac myocytes (A(1)AR-tgm) induces cellular protection against subsequent sustained simulated ischemia (SI); that the cellular protection induced by over-expression of A(1)AR in A(1)AR-tgm is mediated through inducible nitric oxide synthase (iNOS) and K(ATP) channels. Sub-lethal simulated ischemia (SSI) and the A(1)AR agonists chloro- N(6)-cyclopentyl adenosine (CCPA) or (2 S)- N(6)-[2-endo-norbornyl]adenosine (S-ENBA) induce further, delayed cytoprotection, additional to the existing protection in A(1)AR-tgm. Cellular injury and cell viability was measured by the release of lactate dehydrogenase (LDH) or creatine kinase (CK) into the medium and the amount remaining in the cells. The cellular resistance acquired by cardiac myocytes due to the over-expression of A(1)AR was reflected by the reduced release of LDH (in units/liter) from 44.94+/-1.46 (wild-type mouse cardiac myocytes, wt) to 29.59+/-2.83 (A(1)AR-tgm, P<0.001). Conversely, LDH release from A(1)AR-tgm increased to 42.53+/-2.23 ( P<0.01) on exposure to 5-hydroxydecanoate (a mitochondrial K(ATP) channel blocker), to 45.93+/-2.90 ( P<0.01) on exposure to S-methylthiourea (an iNOS inhibitor) and to 56.04+/-3.00 ( P<0.01) on exposure to glibenclamide (a K(ATP) channel blocker). Treatment of A(1)AR-tgm is with SSI and the A(1)AR agonists chloro- N(6)-cyclopentyl adenosine (CCPA) or (2 S)- N(6)-[2-endo-norbornyl]adenosine (S-ENBA) decreased the release of LDH from 46.44+/-0.57 (A(1)AR-tgm) to 42.08+/-0.48 (A(1)AR-tgm plus SSI, P<0.05), 38.03+/-1.16 (A(1)AR-tgm plus CCPA, P<0.001) and 32.77+/-0.58 (A(1)AR-tgm plus S-ENBA, P<0.001). Our data suggest that the A(1)AR has a cytoprotective effect against subsequent sustained SI in A(1)AR-tgm and that the cellular protection induced by over-expression of A(1)AR in A(1)AR-tgm depends on iNOS and K(ATP) channels. Further, SSI and the A(1)AR agonists CCPA or S-ENBA induce further, delayed cytoprotection in A(1)AR-tgm.

Adenosine, adenosine receptors and myocardial protection: an updated overview.

Adenosine (Ado) accumulates in tissues under metabolic stress. On myocardial cells, the nucleoside interacts with various receptor subtypes (A(1), A(3), and probably A(2A) and A(2B)) that are coupled, via G proteins, to multiple effectors, including enzymes, channels, transporters and cytoskeletal components. Studies using Ado receptor agonists and antagonists, as well as animals overexpressing the A(1) receptor indicate that Ado exerts anti-ischemic action. Ado released during preconditioning (PC) by short periods of ischemia followed by reperfusion induces cardioprotection to a subsequent sustained ischemia. This protective action is mediated by A(1) and A(3) receptor subtypes and involves the activation and translocation of PKC to sarcolemmal and to mitochondrial membranes. PKC activation leads to an increased opening of ATP-sensitive K(+) (K(ATP)) channels. Recent studies implicate mitochondrial rather than sarcolemmal K(ATP) channels in the protective action of PC. Other effectors possibly contributing to cardioprotection by Ado or PC, and which seem particularly involved in the delayed (second window of) protection, include MAP kinases, heat shock proteins and iNOS. Because of its anti-ischemic effects, Ado has been tested as a protective agent in clinical interventions such as PTCA, CABG and tissue preservation, and was found in most cases to enhance the post-ischemic recovery of function. The mechanisms underlying the role of Ado and of mitochondrial function in PC are not completely clear, and uncertainties remain concerning the role played by newly identified potential effectors such as free radicals, the sarcoplasmic reticulum, etc. In addition, more studies are needed to clarify the signalling mechanisms by which A(3) receptor activation or overexpression may promote apoptosis and cellular injury, as reported by a few recent studies.

ATP-Sensitive potassium current alterations during ischemic preconditioning in guinea pig ventricular myocytes

Adenosine triphosphate sensitive K + channels (K ATP ) have been shown by numerous investigators to play an essential role in the mechanisms of cardioprotection.Thus far,however,the direct recordings of K ATP current during ischemic preconditioning (IPC) have never been reported.The whole cell patch clamp recording technique was used to directly investigate the K ATP current alterations during simulated ischemia (hypoxia,glucose free) and reperfusion in isolated guinea pig ventricular myocytes.Three groups were divided into:1) Baseline (BL):18 min normal solution perfusion; 2) IPC:3 cycles of IPC (3 min ischemia+3 min reperfusion);3) Glb:exposure to glibenclamide (1 μM/L) during 3 cycles of IPC.Then 15 min sustained ischemia and 5 min reperfusion were conducted in all three groups.The K ATP currents of IPC group were significantly increased after cycle 3 of IPC compared with BL group ( P 0.05).The increased K ATP currents also occurred 3～7 min after subsequent sustained ischemia,among which the most significant K ATP difference between two groups occurred at 5 min after sustained ischemia ( P 0.01).However,the opening extent of K ATP channel during late stage of subsequent sustained ischemia and reperfusion were similar between BL and IPC groups.These effects could be blocked by Glb.Conclusion:These results first directly demonstrated that K ATP is activated in IPC procedure,mainly through its opening during IPC period and early stage of subsequent sustained ischemia,but not during the late stage of sustained ischemia period and reperfusion.The mechanisms need to be further explored.

ATP-Sensitive potassium current alterations during ischemic preconditioning in guinea pig ventricular myocytes

Adenosine triphosphate sensitive K + channels (K ATP ) have been shown by numerous investigators to play an essential role in the mechanisms of cardioprotection.Thus far,however,the direct recordings of K ATP current during ischemic preconditioning (IPC) have never been reported.The whole cell patch clamp recording technique was used to directly investigate the K ATP current alterations during simulated ischemia (hypoxia,glucose free) and reperfusion in isolated guinea pig ventricular myocytes.Three groups were divided into:1) Baseline (BL):18 min normal solution perfusion; 2) IPC:3 cycles of IPC (3 min ischemia+3 min reperfusion);3) Glb:exposure to glibenclamide (1 μM/L) during 3 cycles of IPC.Then 15 min sustained ischemia and 5 min reperfusion were conducted in all three groups.The K ATP currents of IPC group were significantly increased after cycle 3 of IPC compared with BL group ( P 0.05).The increased K ATP currents also occurred 3～7 min after subsequent sustained ischemia,among which the most significant K ATP difference between two groups occurred at 5 min after sustained ischemia ( P 0.01).However,the opening extent of K ATP channel during late stage of subsequent sustained ischemia and reperfusion were similar between BL and IPC groups.These effects could be blocked by Glb.Conclusion:These results first directly demonstrated that K ATP is activated in IPC procedure,mainly through its opening during IPC period and early stage of subsequent sustained ischemia,but not during the late stage of sustained ischemia period and reperfusion.The mechanisms need to be further explored.

Preconditioning Enhanced Glucose Uptake Is Mediated by p38 MAP Kinase Not by Phosphatidylinositol 3-Kinase

Ischemia is reported to stimulate glucose uptake, but the signaling pathways involved are poorly understood. Modulation of glucose transport could be important for the cardioprotective effects of brief intermittent periods of ischemia and reperfusion, termed ischemic preconditioning. Previous work indicates that preconditioning reduces production of acid and lactate during subsequent sustained ischemia, consistent with decreased glucose utilization. However, there are also data that preconditioning enhances glucose uptake. The present study examines whether preconditioning alters glucose transport and whether this is mediated by either phosphatidylinositol 3-kinase (PI3K) or p38 MAP kinase. Langendorff-perfused rat hearts were preconditioned with 4 cycles of 5 min of ischemia and 5 min of reperfusion, with glucose as substrate. During the last reflow, glucose was replaced with 5 mM acetate and 5 mM 2-deoxyglucose (2DG), and hexose transport was measured from the rate of production of 2-deoxyglucose 6-phosphate (2DG6P), using (31)P nuclear magnetic resonance. Preconditioning stimulated 2DG uptake; after 15 min of perfusion with 2DG, 2DG6P levels were 165% of initial ATP in preconditioned hearts compared with 96% in control hearts (p < 0.05). Wortmannin, an inhibitor of PI3K, did not block the preconditioning induced stimulation of 2DG6P production, but perfusion with SB202190, an inhibitor of p38 MAP kinase, did attenuate 2DG6P accumulation (111% of initial ATP, p < 0. 05 compared with preconditioned hearts). SB202190 had no effect on 2DG6P accumulation in nonpreconditioned hearts. Preconditioning stimulation of translocation of GLUT4 to the plasma membrane was not inhibited by wortmannin. The data demonstrate that ischemic preconditioning increases hexose transport and that this is mediated by p38 MAP kinase and is PI3K-independent.

Ischemic preconditioning reduces Na(+) accumulation and cell killing in isolated rat hepatocytes exposed to hypoxia.

Short periods of ischemia followed up by reperfusion are known to protect the heart against injury caused by a subsequent sustained ischemia. This phenomenon, known as ischemic preconditioning, has also been recently shown to reduce ischemic liver damage, but the mechanisms involved are still unknown. By using isolated hepatocytes as an in vitro model of liver preconditioning, we have investigated the possible effect of preconditioning on intracellular pH and Na(+) homeostasis. Freshly isolated rat hepatocytes were preconditioned by 10 minutes of incubation under hypoxic conditions followed up by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Although preconditioning did not ameliorate adenosine triphosphate (ATP) depletion, preconditioned hepatocytes exhibited an increased resistance to cell killing during hypoxic incubation. Intracellular acidosis and Na(+) accumulation developing during hypoxia were appreciably reduced in preconditioned cells. The effects of preconditioning on intracellular pH, Na(+) homeostasis, and cytotoxicity were mimicked by stimulating protein kinase C (PKC) with 4beta-phorbol-12-myristate-13-acetate (PMA) or 1,2 dioctanoyl-glycerol (1,2 DOG). Conversely, inhibiting PKC with chelerythrine or blocking vacuolar proton ATPase (V-ATPase) with bafilomycin A(1) abolished the protection given by preconditioning or by PMA treatment on hypoxic acidosis, Na(+) overload, and hepatocyte killing. Similarly, the addition of Na(+) ionophore monensin also reverted the cytoprotection exerted by preconditioning. This indicated that ischemic preconditioning of isolated hepatocytes decreased cell killing during hypoxia by preventing intracellular Na(+) accumulation. We propose that, after preconditioning, the stimulation of PKC might activate proton extrusion through V-ATPase, thus, limiting intracellular acidosis and Na(+) overload promoted by Na(+)-dependent acid buffering systems.

Role of energy metabolism in the preconditioned heart--a possible contribution of mitochondria.

A brief period of ischemia and reperfusion has been shown to protect the myocardium against subsequent sustained ischemia and reperfusion injury, which is called "preconditioning". A great number of investigators have explored the mechanisms underlying this preconditioning-induced cardioprotection. This article dealt with possible mechanisms of energy metabolism and mitochondrial activity for preconditioning-induced cardioprotection. Particularly, the contribution of energy metabolites produced during a brief period of ischemia and reperfusion injury, as well as mitochondrial function that is modified by changes in mitochondrial ATPase activity, opening of mitochondrial ATP-dependent potassium channels and production of free radicals in mitochondria, to ischemic preconditioning is discussed.

Cardiac adaptation to ischemia-reperfusion injury.

Acute myocardial ischemia initiates a cascade of cellular events that lead to irreversible injury. We previously described the transient nature of heat-shock induced cardioprotection; treatment with a catalase inhibitor abolished the cytoprotective actions without affecting expression levels of HSP71. Repeated, transient ischemic episodes augment the ischemic tolerance of affected myocardium but the fundamental cytoprotective mechanism(s) for both "early" and "delayed" preconditioning remains unclear. Increased cellular induction of protooncogenes, heat shock genes, and downstream effector proteins might play critical roles in the cytoprotection afforded by delayed preconditioning. We measured c-fos, c-jun, c-myc, and hsp70 induction in preconditioned (2 x 5-min ischemia/10-min reperfusion) and control rabbit hearts that either underwent 30- or 120-min coronary occlusion and 60-min reperfusion, or did not undergo subsequent sustained ischemia; the latter hearts were allowed to recover for 0, 1, 3, 6, 24, 48, 72, or 96 hours. Both c-fos and c-jun in ischemic tissue were strongly induced by ischemia-reperfusion injury and preconditioning pretreatment. However, expression levels diminished significantly by 1-h reperfusion and remained depressed during the 96-h recovery period. Hsp70 (inducible) mRNA expression levels were highest primarily in ischemic myocardium after 6-h recovery post-preconditioning; Hsp70 levels in ischemic myocardium were slightly stronger after 48-h recovery but subsequently diminished to barely detectable levels by 96-h post-preconditioning. Induction of c-fos and c-jun preceded that of Hsp70. These findings support the concept that upregulation of immediate early genes and heat shock genes plays an important role in myocardial adaptation to acute ischemic stress.

Renal ischemia/reperfusion remotely improves myocardial energy metabolism during myocardial ischemia via adenosine receptors in rabbits: effects of “remote preconditioning”

ObjectivesThis study examined the changes in myocardial energy metabolism during myocardial ischemia after “remote preconditioning” and investigated the involvement of adenosine receptors in the mechanisms of this effect.BackgroundRecent studies have indicated that a brief period of ischemia and reperfusion (ischemic preconditioning, PC) in a remote organ reduces myocardial infarct size (IS) protecting against subsequent sustained myocardial ischemia. However, the mechanisms of “remote PC” remain unclear. We assessed myocardial energy metabolism during sustained myocardial ischemia and reperfusion after renal PC (RPC), in comparison with that after myocardial PC (MPC) in open-chest rabbits. It has been established that adenosine receptors are involved in the mechanisms of MPC.MethodsRabbits that had been anesthetized with halothane were divided into six groups. The control (CNT) group underwent 40-min coronary occlusion followed by 120 min reperfusion. Before the procedure, the MPC group underwent an additional protocol of 5 min coronary artery occlusion and 20 min reperfusion, and the RPC group received a 10 min episode of renal artery occlusion and 20 min reperfusion. In additional experimental groups, 8 sulfophenyltheophylline (SPT, 10 mg/kg), an adenosine receptor inhibitor, was intravenously injected before the 40 min myocardial ischemia (SPT, MPC + SPT and RPC + SPT groups, respectively). Myocardial levels of phosphocreatine (PCr), ATP and intracellular pH (pHi) were measured by 31P-NMR spectroscopy.ResultsRPC and MPC delayed the decreases in ATP levels, preserved pHi during 40-min myocardial ischemia and resulted in better recovery of ATP and PCr during 120 min reperfusion compared with the controls. SPT abolished the improvement in myocardial energy metabolism and the reduction in myocardial IS caused by MPC or RPC. Myocardial IS in the CNT (n = 8), MPC (n = 9), RPC (n = 9), SPT (n = 6), MPC + SPT (n = 8) and RPC + SPT (n = 8) groups averaged 42.8 ± 3.5%, 18.2 ± 1.8%∗, 19.6 ± 1.3%∗, 44.9 ± 5.0%, 35.6 ± 2.7% and 34.8 ± 3.6% of the area at risk (∗p < 0.05 vs. CNT), respectively.ConclusionsPC in a remote organ, similar to MPC, improved myocardial energy metabolism during ischemia and reperfusion and reduced IS in vivo by an adenosine-dependent mechanism in rabbits.

Cardioprotective Effect of the Ischemic Preconditioning: Its Relation to Activation of Protein Kinase C

Background:We tested recent evidences that IP triggers selective activation of protein kinase C (PKC isozymes using isolated Langendorff-perfused rabbit heart with PKC activator, phorbol ester (PMA, 0.01 nM or inhibitor (calphostin C, 200 nM. Methods:After stabilization of baseline hemodynamics, the hearts were subjected to 45 min global ischemia (Ⅰ followed by 120 min reperfusion (R with IP (IP group, n=18 or without IP (ischemic control group, n=16. IP was induced by single episode of 5 min Ⅰ and 10 min R. In the PMA-treated group (n=19 and calphostin C-treated preconditioned group (n=15, PMA and calphostin C was given for 5 and 15 min before 45 min Ⅰ, respectively. Myocardial cytosolic and membrane PKC activities were measured by 32P-γ-ATP incorporation into PKC-specific pepetide;PKC isozymes were analyzed by Western blot with monoclonal antibodies. Results:IP significantly increased the recovery of the LV function including LVDP and coronary flow (p<0.05; however, enhancement of the functional recovery disappeared by calphostin C or PMA treatment. Cytosolic PKC activity decreased to 82-76% in the IP and PMA-treated group (p<0.05; membrane PKC activity increased to 218-272% (p<0.01. However, both fraction of PKC activity was not changed in the calphostin C-treated preconditioned group. In addition, Western blot revealed that PKC-α and e, especially e, were selectively translocated during subsequent sustained ischemia after IP or PMA administration. IP and PMA also reduced infarct size (frim 38 to 10-20%, p<0.05. However, calphostin C blocked infarct reduction effect of IP. Conclusion:These results indicate that in isolated rabbit heart model, cardioprotective effect of IP may be related, at least in part, to trigger selective translocation of PKC, especially e isotype. (Korean Circulation J 1999;29(6 :602-611

Ischemic preconditioning suppresses the noradrenaline turnover in the rat heart.

The mechanism by which ischemic preconditioning protects the heart is presumed to be related to the reduction of energy consumption during a subsequent myocardial infarction. Since the sympathetic nervous system enhances cardiac function and energy consumption, we investigated the relation between ischemic preconditioning and the turnover rate of noradrenaline (NA) in the rat heart. The effect of 3 cycles of 5-min occlusions of the rat left coronary artery on changes in arterial blood pressure and heart rate provoked by a subsequent 30 min of ischemia were examined until 60 min after reperfusion. The effect of 3 cycles of occlusions on the infarct size was also evaluated 60 min after reperfusion by comparing the infarcted area with the area at risk in these animals (6 per preconditioned and sham-operated group). The tissue concentration of NA during sustained ischemia was determined in the left ventricle, the intraventricular septum, and the right ventricle in the preconditioned and sham-operated groups. Changes in the turnover rate of NA after 3 cycles of occlusions were also evaluated by assessing the alpha-methyl-p-tyrosine-induced depletion of NA (n = 7 per group). A series of transient occlusions reduced the infarct size 60 min after a sustained ischemia for 30 min. Arterial pressure and heart rate were not affected. The concentration of NA was decreased in the left ventricle 60 min after the onset of sustained ischemia in both the preconditioned and sham-operated groups. The treatment with alpha-methyl-p-tyrosine decreased the NA concentration in all regions of the heart in the sham-operated group after 60 min. However, the treatment with alpha-methyl-p-tyrosine did not deplete the NA concentration in both the occluded and nonoccluded regions in the preconditioned group. Transient ischemia ameliorated the heart injury induced by a subsequent sustained ischemia, as assessed histologically. The activity of the sympathetic nervous system in all regions of the heart was reduced by transient ischemia in the left coronary vascular bed. These findings suggest that the inhibition of the sympathetic nervous system by the treatment of ischemic preconditioning takes part in the cardiac protection.