Balance of nitric oxide and reactive oxygen species in myocardial reperfusion injury and protection.

Depending on their concentrations, both nitric oxide (NO) and reactive oxygen species (ROS) take part either in myocardial ischemia reperfusion injury or in protection by ischemic and pharmacological preconditioning (Ipre) and postconditioning (Ipost). At the beginning of reperfusion, a transient release of NO is promptly scavenged by ROS to form the highly toxic peroxynitrite, which is responsible for a further increase of ROS through endothelial nitric oxide synthase uncoupling. The protective role of NO has suggested the use of NO donors to mimic Ipre and Ipost. However, NO donors have not always given the expected protection, possibly because they are responsible for the production of different amounts of ROS that depend on the amount of released NO. This review is focused on the role of the balance of NO and ROS in myocardial injury and its prevention by Ipre and Ipost and after the use of NO donors given with or without antioxidant compounds to mimic Ipre and Ipost.

A lipophilic nitric oxide donor and a lipophilic antioxidant compound protect rat heart against ischemia-reperfusion injury if given as hybrid molecule but not as a mixture.

Low concentrations of a hydrophilic nitric oxide donor (NOD) are reported to reduce myocardial reperfusion injury only when combined with a lipophilic antioxidant (AOX) to form a hybrid molecule (HYB). Here we tested whether liposoluble NOD requires to be combined with AOX to be protective. Isolated rat hearts underwent 30 minutes of ischemia and 120 minutes of reperfusion. To induce postconditioning, 1 µM solutions of the following liposoluble compounds were given during the first 20 minutes of reperfusion: NOD with weak (w-NOD) or strong NO-releasing potency (s-NOD); weak HYB built up with w-NOD and a per se ineffective AOX lead; strong HYB built up with s-NOD and the same AOX; mixtures of w-NOD plus AOX or s-NOD plus AOX. A significant reduction of infarct size with improved recovery of cardiac function was obtained only with weak HYB. We suggest that w-NOD requires the synergy with a per se ineffective AOX to protect. The synergy is possible only if the 2 moieties enter the cell simultaneously as a hybrid, but not as a mixture. It seems that strong HYB was ineffective because an excessive intracellular NO release produces a large amount of reactive species, as shown from the increased nitrotyrosine production.

Apelin-13 limits infarct size and improves cardiac postischemic mechanical recovery only if given after ischemia.

We studied whether apelin-13 is cardioprotective against ischemia/reperfusion injury if given as either a pre- or postconditioning mimetic and whether the improved postischemic mechanical recovery induced by apelin-13 depends only on the reduced infarct size or also on a recovery of function of the viable myocardium. We also studied whether nitric oxide (NO) is involved in apelin-induced protection and whether the reported ischemia-induced overexpression of the apelin receptor (APJ) plays a role in cardioprotection. Langendorff-perfused rat hearts underwent 30 min of global ischemia and 120 min of reperfusion. Left ventricular pressure was recorded. Infarct size and lactate dehydrogenase release were determined to evaluate the severity of myocardial injury. Apelin-13 was infused at 0.5 µM concentration for 20 min either before ischemia or in early reperfusion, without and with NO synthase inhibition by N(G)-nitro-l-arginine (l-NNA). In additional experiments, before ischemia also 1 µM apelin-13 was tested. APJ protein level was measured before and after ischemia. Whereas before ischemia apelin-13 (0.5 and 1.0 µM) was ineffective, after ischemia it reduced infarct size from 54 ± 2% to 26 ± 4% of risk area (P < 0.001) and limited the postischemic myocardial contracture (P < 0.001). l-NNA alone increased postischemic myocardial contracture. This increase was attenuated by apelin-13, which, however, was unable to reduce infarct size. Ischemia increased APJ protein level after 15-min perfusion, i.e., after most of reperfusion injury has occurred. Apelin-13 protects the heart only if given after ischemia. In this protection NO plays an important role. Apelin-13 efficiency as postconditioning mimetic cannot be explained by the increased APJ level.

Intermittent activation of bradykinin B2 receptors and mitochondrial KATP channels trigger cardiac postconditioning through redox signaling.

OBJECTIVE: Postconditioning (PostC) maneuvers allow post-ischemic accumulation of autacoids, which trigger protection. We tested if PostC-triggering includes bradykinin (BK) B2 receptor activation and its downstream pathway. METHODS AND RESULTS: Isolated rat hearts underwent 30 min ischemia and 120 min reperfusion. Infarct size was evaluated using nitro-blue tetrazolium staining. In Control hearts infarct size was 61+/-5% of risk area. PostC (5 cycles of 10 s reperfusion/ischemia) reduced infarct size to 22+/-4% (p<0.01). PostC protection was abolished by B2 BK receptor-antagonists (HOE140 or WIN64338), nitric oxide synthase-inhibitor (L-nitro-arginine-methylester), protein kinase G (PKG)-blocker (8-bromoguanosine-3',5'-cyclic-monophosphorothioate), and mitochondrial K(ATP) (mK(ATP))-blocker (5-hydroxydecanoate) each given for 3 min only. Since 3 min of BK-infusion (100 nM) did not reproduce PostC protection, protocols with Intermittent-BK infusion were used to mimic PostC: a) 5 cycles of 10 s oxygenated-no-BK/oxygenated+BK buffer; b) 5 cycles of 10 s oxygenated-no-BK/hypoxic+BK buffer. Both protocols with Intermittent-BK attenuated infarct size (36+/-5% and 38+/-4%, respectively; p<0.05 vs Control and NS vs PostC for both; NS vs each other). Intermittent-BK protection was abolished by the same antagonists used to prevent PostC protection. Intermittence of re-oxygenation only (5 cycles of 10 s oxygenated/hypoxic buffer) did not reproduce PostC. Yet, cardioprotection was triggered by intermittent mK(ATP) activation with diazoxide, but not by intermittent reactive oxygen species (ROS) generation with purine/xanthine oxidase. ROS scavengers (N-acetyl-L-cysteine or 2-mercaptopropionylglycine), given for 3 min only, abolished PostC-, Intermittent BK-and diazoxide-induced protection. CONCLUSIONS: Intermittent targeting of specific cellular sites (i.e. BK B2 receptors and mK(ATP) channels) during early reperfusion triggers PostC protection via ROS signaling. Since neither intermittent oxygenation nor exogenous ROS generators can trigger protection, it is likely that intermittent autacoid accumulation and ROS compartmentalization may play a pivotal role in PostC-triggering.

In vitro and in vivo studies of F(0)F(1)ATP synthase regulation by inhibitor protein IF(1) in goat heart.

A method has been developed to allow the level of F(0)F(1)ATP synthase capacity and the quantity of IF(1) bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF(1) content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF(1) antibodies. Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF(1) content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF(1). In addition, both in vivo and in vitro, 1.3-1.4 mol of IF(1) was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF(1) in the F(0) sector.

[Activity of apelin and APJ receptors on myocardial contractility and vasomotor tone]. / Azione dell'apelina e del recettore APJ sulla contrattilità miocardica e sul tono vasomotore.

Apelin, an endogenous peptide, is the ligand of APJ receptors. Although initially it was identified in the gastrointestinal tract, later its presence was found in several organs and tissues. On the cardiovascular system apelin induces an increase in myocardial contractility and a reduction of vasomotor tone. While the increase in contractility seems to depend on an activation of Na+/H+ and Na+/Ca2+ exchangers, vasodilation is attributed to a release of nitric oxide from the vascular endothelial cells. Apelin-induced vasodilation leads to a reduction of mean filling pressure which in turn causes a decrease of afterload and preload. When apelin is given acutely, the decrease in preload favors the reduction of stroke volume and cardiac output in spite of an increased contractility. On the contrary, when the peptide is administered for 2 weeks, cardiac output increases significantly without the occurrence of cardiac hypertrophy. It is not excluded that hypertrophy might occur after a longer administration.

Ischemic preconditioning changes the pattern of coronary reactive hyperemia regardless of mitochondrial ATP-sensitive K(+) channel blockade.

Ischemic preconditioning increases the velocity of vasodilatation and reduces the total hyperemic flow (THF) of a subsequent coronary reactive hyperemia (CRH). The increase in the velocity of vasodilatation has been shown to depend on an up-regulation of the endothelial release of nitric oxide, while the reduction of THF is attributed to an adenosine A(1) receptor-mediated mechanism. We investigated whether the changes in CRH induced by preconditioning ischemia (PI) can still be obtained after blockade of mitochondrial ATP-sensitive K(+) channels by sodium 5-hydroxydecanoate (5-HD), and whether the blockade per se affects the pattern of CRH. In anesthetized goats, flow was recorded from the left circumflex coronary artery (LCCA). CRH was obtained with the occlusion of LCCA for 15 s. PI was obtained by 2 cycles of 2.5 min of LCCA occlusion with a 5 min interval of reperfusion between the two occlusions. CRH was studied before and after i.v. administration of 5-HD (20 mg/kg), as well as in the presence of 5-HD after PI. Following 5-HD, the pattern of CRH remained unchanged. After 5-HD and PI, velocity of vasodilatation and total hyperemic flow of CRH showed the same changes as in previous studies after PI alone. It was concluded that the blockade of mitochondrial ATP-sensitive K(+) channels, which is reported to prevent myocardial protection, does not affect CRH and does not prevent PI from increasing the velocity of vasodilatation and reducing THF. These results demonstrate that the changes induced in CRH by preconditioning are independent of the opening of the mitochondrial ATP-sensitive K(+) channels.

Coronary endothelial dysfunction after ischemia and reperfusion and its prevention by ischemic preconditioning.

In the coronary circulation, when reperfusion follows ischemia, endothelial dysfunction occurs. This is characterized by a reduced endothelial release of nitric oxide and by an increased release of reactive oxygen species and endothelin. The reduced availability of nitric oxide leads to the adhesion of neutrophils to the vascular endothelium, platelet aggregation and, with the contribution of endothelin, vasoconstriction, which are responsible for the "no-reflow" phenomenon. Neutrophil adhesion is followed by the release of the superoxide anion from neutrophils and endothelial cells. Preconditioning limits the endothelial damage by ischemia-reperfusion. A relevant role is attributed to the increased endothelial release of nitric oxide, while that of adenosine is controversial. Another effect of preconditioning on the coronary vasculature is the acceleration of vasodilation in reactive hyperemia after a brief coronary occlusion. The acceleration is prevented if myocardial protection is achieved by means of the activation of the mitochondrial adenosine triphosphate sensitive potassium channels by diazoxide and persists when ischemic preconditioning is induced after blockade of the same channels by 5-hydroxydecanoate.

Effect of apelin-apelin receptor system in postischaemic myocardial protection: a pharmacological postconditioning tool?

In the heart, a great part of ischaemia and reperfusion injuries occurs mainly during the first minutes of reperfusion. The opening of the mitochondrial permeability transition pores is the end point of the cascade to myocardial damage. Also, oxidative stress contributes to cell death. Postconditioning is a protective maneuver that can be selectively timed at the beginning of reperfusion. It is hypothesized that it acts via the reperfusion injury salvage kinase pathway, which includes nitric oxide-dependent and nitric oxide-independent cascades. Apelin is an endogenous peptide that can protect the heart from reperfusion injury if given at the beginning of reperfusion but not before ischaemia. It is hypothesized that it may trigger the reperfusion injury salvage kinase pathway via a specific apelin receptor. Apelin can also limit the oxidative stress by the activation of superoxide dismutase. Apelin and apelin receptor expression increase early after ischaemia and at the beginning of an ischaemic heart failure. These observations suggest that the endogenous release of the peptide can limit the severity of an infarction and ameliorate myocardial contractility compromised by the appearance of the failure. Due to its protective activities, apelin could be a therapeutic tool if administered with the same catheter used for angioplasty or after the maneuvers aimed at bypassing a coronary occlusion.

The chromogranin A- derived N-terminal peptide vasostatin-I: In vivo effects on cardiovascular variables in the rabbit.

This study is the first to report on vascular effect of the chromogranin A derived Vasostatin-I (CgA(1-76)) in vivo. Cardiovascular parameters were recorded in 29 rabbits with sympathetically decentralized right carotid vascular bed. The recombinant human STA CgA(1-78) (VS-1) was infused at 480 µg/kg over 25 min. Group I was kept awake while groups II-V were anesthetized with Ketamine-xylazine. VS-1 was given alone in groups I-II while in presence of either phentolamine, phentolamine plus propranolol or hexamethonium in groups III-V. Serum VS-1 peaked at 2 µg/ml (200 nM) before onset of vascular effects and declined rapidly to ~200 ng/ml within 30 min. In all groups but III and IV VS-1 induced a brief vasoconstriction, being larger in intact than in sympathetically decentralized beds. The VS-1 induced vasoconstriction was not altered by hexamethonium but was abolished by phentolamine. In presence of the α-adrenergic blocker a long lasting vasodilatation, unaffected by propranolol, was apparent on both innervated and decentralized sides. In conclusion, VS-1 induced an α-adrenoceptor-mediated vasoconstriction presumably brought about by noradrenaline release from sympathetic nerves when infused at a dose giving an initial serum concentration of ~200 nM. This initial vasoconstriction masked a persistent adrenoceptor-independent vasodilatation, consistent with previous reports from in vitro models.

Early homing of adult mesenchymal stem cells in normal and infarcted isolated beating hearts.

Little is known on the early homing features of transplanted mesenchymal stem cells (MSCs). We used the isolated rat heart model to study the homing of MSCs injected in the ventricular wall of a beating heart. In this model all types of cells and matrix elements with their interactions are represented, while external interferences by endothelial/neutrophil interaction and neurohormonal factors are excluded. We studied the morphology and marker expression of MSCs implanted in normal hearts and in the border-zone of infarcted myocardium. Early morphological adaptation of MSC homing differs between normal and infarcted hearts over the first 6 hrs after transplantation. In normal hearts, MSCs migrate very early through the interstitial milieu and begin to show morphological changes. Yet, in infarcted hearts MSCs remain in the site of injection forming clusters of round-shaped cells in the border-zone of the infarcted area. Both in normal and infarcted hearts, immuno-histochemistry and confocal imaging showed that, besides the proliferative marker proliferating cell nuclear agent (PCNA), some transplanted cells early express myoblastic maker GATA-4, and some of them show a VWF immunopositivity. Moreover, a few hours after injection connexin-43 is well evident between cardiomyocytes and injected cells. This study indicates for the first time that the isolated beating heart is a good model to study early features of MSC homing without external interferences. The results show (i) that MSCs start to change marker expression few hours after injection into a beating heart and (ii) that infarcted myocardium influences transplanted MSC morphology and mobility within the heart.

Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes.

In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST-BODIPY-Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X-Rhod-1. Immunofluorescence experiments showed the presence of connexin-43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage-dependent calcium signaling in cocultured MSCs. MSCs showed a time-dependent increase of labeling of ST-BODIPY-Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non-fluorescent DHP, Nifedipine, completely abolished ST-BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization-induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage-dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte-like phenotype.

Human recombinant chromogranin A-derived vasostatin-1 mimics preconditioning via an adenosine/nitric oxide signaling mechanism.

The acidic protein chromogranin A (CgA) is the precursor of several regulatory peptides generated by specific proteolytic processes. Human recombinant CgA NH(2)-terminal fragment STA-CgA(1-78) (hrSTA-CgA(1-78)), containing vasostatin-1 (CgA(1-76)) domain, exerts a negative inotropic effect and counteracts the beta-adrenergic positive inotropic effect on the rat heart. We hypothesized an involvement of nitric oxide (NO)-dependent pathway in both cardiodepression and cardioprotection by hrSTA-CgA(1-78). We also hypothesized an involvement of adenosine A(1) receptor and protein kinase C (PKC) in cardioprotection by hrSTA-CgA(1-78). Therefore, we evaluated whether 1) the cardioinhibition mediated by hrSTA-CgA(1-78) involves the G(i/o) proteins/NO-dependent signal transduction cascade, 2) hrSTA-CgA(1-78) induces ischemic preconditioning-like protective effects on the myocardium, and 3) inhibition of NO synthase (NOS), adenosine A(1) receptor, or PKC affects hrSTA-CgA(1-78) protection. Using the isolated rat heart, we found that the reduction of left ventricular pressure (LVP), rate-pressure product, and maximal values of the first derivative of LVP elicited by hrSTA-CgA(1-78) at 33 nM is abolished by blocking G(i/o) proteins with pertussis toxin, scavenging NO with hemoglobin, and blocking NOS activity with N(G)-monomethyl-l-arginine or N(5)-(iminoethyl)-l-ornithine, soluble guanylate cyclase with 1H-[1,2,4]oxadiazole-[4,4-a]quinoxalin-1-one, and protein kinase (PKG) with KT5823. Data suggest the involvement of the G(i/o) proteins/NO-cGMP-PKG pathway in the hrSTA-CgA(1-78)-dependent cardioinhibition. When given before 30 min of ischemia, hrSTA-CgA(1-78) significantly reduced the size of the infarct from 64 +/- 4 to 32 +/- 3% of the left ventricular mass. This protective effect was abolished by either NOS inhibition or PKC blockade and was attenuated, but not suppressed, by the blockade of A(1) receptors. These results suggest that hrSTA-CgA(1-78) activity triggers two different pathways: one of these pathways is mediated by A(1) receptors, and the other is mediated by NO release. As with repeated brief preconditioning ischemia, hrSTA-CgA(1-78) may be considered a stimulus strong enough to trigger both pathways, which may converge on PKC.

Post-conditioning reduces infarct size in the isolated rat heart: role of coronary flow and pressure and the nitric oxide/cGMP pathway.

We aimed to assess the role of the nitric oxide (NO)-cGMP pathway in cardioprotection by brief intermittent ischemias at the onset of reperfusion (i.e., post-conditioning (Post-con)). We also evaluated the role of coronary flow and pressure in Post-con. Rat isolated hearts perfused at constant- flow or -pressure underwent 30 min global ischemia and 120 min reperfusion. Post-con obtained with brief ischemias of different duration (modified, MPost-con) was compared with Post-con obtained with ischemias of identical duration (classical, C-Post-con) and with ischemic preconditioning (IP). Infarct size was evaluated using nitro-blue tetrazolium staining and lactate dehydrogenase (LDH) release. In the groups, NO synthase (NOS) or guanylyl-cyclase (GC) was inhibited with LNAME and ODQ, respectively. In the subgroups, the enzyme immunoassay technique was used to quantify cGMP release. In the constant-flow model, M-Post-con and C-Post-con were equally effective, but more effective than IP in reducing infarct size. The cardioprotection by M-Post-con was only blunted by the NOS-inhibitor, but was abolished by the GC-antagonist. Post-ischemic cGMP release was enhanced by MPost-con. In the constant-pressure model IP, M-Post-con and C-Post-con were equally effective in reducing infarct size. Post-con protocols were more effective in the constant-flow than in the constant-pressure model. In all groups, LDH release during reperfusion was proportional to infarct size. In conclusion, Post-con depends upon GC activation, which can be achieved by NOS-dependent and NOS-independent pathways. The benefits of M- and CPost-con are similar. However, protection by Post-con is greater in the constant-flow than in the constant-pressure model.

[Ischemic postconditioning: an effective strategy of myocardial protection?]. / II postcondizionamento ischemico: un'efficace strategia di protezione miocardica?

Post-ischemic reperfusion worsens myocardial injury. Ischemic preconditioning limits the damage by ischemia and reperfusion. Both adenosine and nitric oxide (NO) pathways are involved in protection. Preconditioning, however, is of little, if any, practical use as the onset of an infarction is usually unpredictable. Recently, it has been shown that the heart can be protected against the extension of reperfusion injuries if 3 or 4 brief (10-30 s) coronary occlusions are performed just at the beginning of the reperfusion. This procedure has been called post-conditioning. Post-conditioning reduces the oxidant-induced injury; moreover, it attenuates the local inflammatory response to reperfusion. Post-conditioning also activates triggers, signaling pathways and effectors implicated in other cardioprotective maneuvers, such as ischemic and pharmacological preconditioning. Post-conditioning seems to trigger the up-regulation of survival kinases principally known to attenuate the pathogenesis of apoptosis and possibly necrosis. As regards the possibility of pharmacological post-conditioning, several agents have been tested. We are testing NO donor(s), which can reduce infarct size in the rat in the absence of post-conditioning. Since during reperfusion there is a large production of reactive oxygen species, also the effect of administration of an antioxidant compound during reperfusion was studied. In the rat, such a procedure reduced the infarct size to a greater extent than post-conditioning. Moreover, an additive effect of NO donors and antioxidant compounds is possible.

Effect of endothelins on the cardiovascular system.

Endothelins (ETs) exert a persistent constrictor effect on the vessels via an increase in intracellular Ca2+ concentration due to the activation of Na+/H+ and Na+/Ca2+ exchangers of the vascular smooth muscle fibres. They also produce a transient dilator effect via the activation of endothelial nitric oxide synthase mediated by protein kinase B/Akt. ETA and ETB2 receptors are involved in vasoconstriction, whereas transient vasodilatation depends on the activation of ETB1 receptors. Depending on animal species and experimental conditions, ETs can also play a role in cardiac muscle contraction and induce either an increase or a decrease in contractility. It is likely that only ETA, and not ETB, receptors are involved in the ET-induced increase in myocardial contractility. As in the case of vasoconstriction, this inotropic effect depends on an increase in intracellular Ca2+ concentration induced by Na+/H+ and Na+/Ca2+ exchangers. Activation of the Na+/H+ exchanger is stimulated by protein kinase C, which is activated by diacylglycerol released in response to ET activity. It has also been proposed that the positive inotropic effect can occur without the contribution of the Na+/Ca2+ exchanger, if the cell alkalinisation produced by the Na/H exchanger improves myofibrillar Ca2+ sensitivity. A reduction in contractility has been attributed to the involvement of the Gi protein/protein kinase G pathway or to the activation of protein kinase C without an increase in intracellular Ca2+ concentration or in myofibrillar Ca2+ sensitivity. The chronic effect of ETs on the myocardium results in hypertrophy and prevention of apoptosis, two processes that are together responsible for the contradictory effect of ETs in heart failure.

Post-conditioning induced cardioprotection requires signaling through a redox-sensitive mechanism, mitochondrial ATP-sensitive K+ channel and protein kinase C activation.

Post-conditioning (Post-C) induced cardioprotection involves activation of guanylyl-cyclase. In the ischemic preconditioning scenario, the downstream targets of cGMP include mitochondrial ATP-sensitive K(+) (mK(ATP)) channels and protein kinase C (PKC), which involve reactive oxygen species (ROS) production. This study tests the hypothesis that mK(ATP), PKC and ROS are also involved in the Post-C protection. Isolated rat hearts underwent 30 min global ischemia (I) and 120 min reperfusion (R) with or without Post-C (i.e., 5 cycles of 10 s R/I immediately after the 30 min ischemia). In 6 groups (3 with and 3 without Post-C) either mK(ATP) channel blocker, 5- hydroxydecanoate (5-HD), or PKC inhibitor, chelerythrine (CHE) or ROS scavenger, N-acetyl-cysteine (NAC), were given during the entire reperfusion (120 min). In other 6 groups (3 with and 3 without Post-C), 5-HD, CHE or NAC were infused for 117 min only starting after 3 min of reperfusion not to interfere with the early effects of Post-C and/or reperfusion. In an additional group NAC was given during Post-C maneuvers (i.e., 3 min only). Myocardial damage was evaluated using nitro-blue tetrazolium staining and lactate dehydrogenase (LDH) release. Post-C attenuated myocardial infarct size (21 +/- 3% vs. 64 +/- 5% in control; p < 0.01). Such an effect was abolished by 5-HD or CHE given during either the 120 or 117 min of reperfusion as well as by NAC given during the 120 min or the initial 3 min of reperfusion. However, delayed NAC (i.e., 117 min infusion) did not alter the protective effect of Post- C (infarct size 32 +/- 5%; p < 0.01 vs. control, NS vs. Post-C). CHE, 5-HD or NAC given in the absence of Post-C did not alter the effects of I/R. Similar results were obtained in terms of LDH release. Our data show that Post-C induced protection involves an early redox-sensitive mechanism as well as a persistent activation of mK(ATP) and PKC, suggesting that the mK(ATP)/ROS/PKC pathway is involved in post-conditioning.

Platelet-activating factor induces cardioprotection in isolated rat heart akin to ischemic preconditioning: role of phosphoinositide 3-kinase and protein kinase C activation.

Ischemic preconditioning (IP) is a cardioprotective mechanism against myocellular death and cardiac dysfunction resulting from reperfusion of the ischemic heart. At present, the precise list of mediators involved in IP and the pathways of their mechanisms of action are not completely known. The aim of the present study was to investigate the role of platelet-activating factor (PAF), a phospholipid mediator that is known to be released by the ischemic-reperfused heart, as a possible endogenous agent involved in IP. Experiments were performed on Langendorff-perfused rat hearts undergoing 30 min of ischemia followed by 2 h of reperfusion. Treatment with a low concentration of PAF (2 x 10(-11) M) before ischemia reduced the extension of infarct size and improved the recovery of left ventricular developed pressure during reperfusion. The cardioprotective effect of PAF was comparable to that observed in hearts in which IP was induced by three brief (3 min) periods of ischemia separated by 5-min reperfusion intervals. The PAF receptor antagonist WEB-2170 (1 x 10(-9) M) abrogated the cardioprotective effect induced by both PAF and IP. The protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M) or the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (5 x 10(-5) M) also reduced the cardioprotective effect of PAF. Western blot analysis revealed that following IP treatment or PAF infusion, the phosphorylation of PKC-epsilon and Akt (the downstream target of PI3K) was higher than that in control hearts. The present data indicate that exogenous applications of low quantities of PAF induce a cardioprotective effect through PI3K and PKC activation, similar to that afforded by IP. Moreover, the study suggests that endogenous release of PAF, induced by brief periods of ischemia and reperfusion, may participate to the triggering of the IP of the heart.