Necroptosis, necrostatins and tissue injury.

Cell death is an integral part of the life of an organism being necessary for the maintenance of organs and tissues. If, however, cell death is allowed to proceed unrestricted, tissue damage and degenerative disease may ensue. Until recently, three morphologically distinct types of cell death were recognized, apoptosis (type I), autophagy (type II) and necrosis (type III). Apoptosis is a highly regulated, genetically determined mechanism designed to dismantle cells systematically (e.g. cells that are no longer functionally viable), via protease (caspase) action, and maintain homeostasis. Autophagy is responsible for the degradation of cytoplasmic material, e.g. proteins and organelles, through autophagosome formation and subsequent proteolytic degradation by lysosomes, and is normally considered in the context of survival although it is sometimes associated with cell death. Necrosis was formerly considered to be an accidental, unregulated form of cell death resulting from excessive stress, although it has been suggested that this is an over-simplistic view as necrosis may under certain circumstances involve the mobilization of specific transduction mechanisms. Indeed, recently, an alternative death pathway, termed necroptosis, was delineated and proposed as a form of 'programmed necrosis'. Identified with the aid of specific inhibitors called necrostatins, necroptosis shares characteristics with both necrosis and apoptosis. Necroptosis involves Fas/tumour necrosis factor-α death domain receptor activation and inhibition of receptor-interacting protein I kinase, and it has been suggested that it may contribute to the development of neurological and myocardial diseases. Significantly, necrostatin-like drugs have been mooted as possible future therapeutic agents for the treatment of degenerative conditions.

Leptin-induced cardioprotection involves JAK/STAT signaling that may be linked to the mitochondrial permeability transition pore.

Leptin-induced protection against myocardial ischemia-reperfusion (I/R) injury involves the activation of the reperfusion injury salvage kinase pathway, incorporating phosphatidylinositol 3-kinase-Akt/protein kinase B and p44/42 MAPK, and the inhibition of the mitochondrial permeability transition pore (MPTP). Recently published data indicate that the JAK/STAT signaling pathway, which mediates the metabolic actions of leptin, also plays a pivotal role in cardioprotection. Consequently, in the present study we considered the possibility that JAK/STAT signaling linked to the MPTP may be involved in modulating the cardioprotective actions of leptin. Employing rat in vitro models (Langendorff-perfused hearts and cardiomyocytes) of I/R injury, we investigated the actions of leptin (10 nM), administered at reperfusion, in the presence or absence of the JAK2 inhibitor, AG-490 (5 µM). Leptin reduced infarct size significantly (control, 60.05 ± 7.41% vs. leptin treated, 29.9 ± 3.24%, P < 0.05), protection being abolished by AG-490. Time course studies revealed that leptin caused a 171% (P < 0.001) increase in STAT3/tyrosine-705 phosphorylation at 2.5 min reperfusion; however, increases were not seen at 5, 10, 15, or 30 min reperfusion. Contrasting with STAT3, Akt/serine-473 phosphorylation was not significantly increased until 15 min into the reperfusion phase (140%, P < 0.05). AG-490 blocked the leptin-induced rise in STAT3 phosphorylation seen at 2.5 min reperfusion but did not influence Akt/serine-473 phosphorylation at 15 min. Leptin reduced the MPTP opening (P < 0.001), which was blocked by AG-490. This is the first study to yield evidence that JAK/STAT signaling linked to the MPTP plays a role in leptin-induced cardioprotection. Under the experimental conditions employed, STAT3 phosphorylation appears to have occurred earlier during reperfusion than that of Akt. Further research into the interactions between these two signaling pathways in the setting of I/R injury is, however, required.

The cannabinoid CB1 receptor antagonist, rimonabant, protects against acute myocardial infarction.

CB1 antagonism is associated with reduced doxorubicin-induced cardiotoxicity and decreased cerebrocortical infarction. Rimonabant, a selective CB1 receptor antagonist, was, before it was withdrawn, proposed as a treatment for obesity and reported to reduce cardiovascular risk by improving glucose and lipid profiles and raising adiponectin levels. The cardioprotective actions of rimonabant in 6-week-old C57BL/6J mice fed either high-fat (HFD) or standard diets (STD) for 8 weeks were investigated. At 14 weeks, mice received rimonabant (10 mg/kg/day, i.p.) or vehicle for 1 week and were then subjected to an in vivo acute myocardial infarction. The influence of rimonabant on infarct size (IS) in CB1 knockout (CB1-/-) and wild-type (CB1+/+) mice was also examined. C57BL/6J mice that had been maintained on STD or HFD exhibited 4.3 and 21.4% reductions in body weight following 7 days rimonabant treatment. Rimonabant reduced IS in both STD (29.6 +/- 3.5% vs. 49.8 +/- 6.9% in control, P < 0.05) and HFD (26.9 +/- 1.5% vs. 48.7 +/- 7% in control, P < 0.05) mice. In CB1-/- mice rimonabant failed to reduce body weight or IS (51.0 +/- 5.3% vs. 49.7 +/- 4.7% in control, P > 0.05), although significant reductions were seen in CB1+/+ mice (IS, 48.9 +/- 4.6% control vs. 30.5 +/- 3.1% rimonabant, P < 0.05). To exclude the possibility that weight loss alone induced cardioprotection, HFD mice were switched to STD for 7 days (HFD-STD), resulting in an 11.3 +/- 1.0% decrease in body weight compared to control (+2.1 +/- 1.1% in HFD). This, however, was not associated with IS reduction (39.1 +/- 3.9% HFD-STD vs. 40.0 +/- 5.3% HFD, P > 0.05). Serum and cardiac adiponectin levels were unaltered by rimonabant treatment. HL-1 cell death was not prevented by 1 or 7 days treatment with rimonabant. We conclude that rimonabant-induced infarct limitation may involve the CB1 receptor, although not necessarily cardiac CB1 receptors, and is unrelated to weight loss or altered adiponectin synthesis.

The cardioprotective actions of leptin are lost in the Zucker obese (fa/fa) rat.

Protection against myocardial ischemia-reperfusion injury, including that induced by leptin, involves activation of the reperfusion injury salvage kinase pathway and inhibition of the mitochondrial permeability transition pore. In the current study, we explored the mechanisms underlying leptin-induced cardioprotection further with reference to the leptin receptor (OB-R) and obesity. We examined hearts from Wistar and Zucker lean rats that express functional OB-R and Zucker obese (fa/fa) rats with nonfunctional OB-R. In Langendorff experiments, leptin (10 nM) caused significant reductions in infarct size in hearts from Wistar (leptin treated, 32.4% +/- 3.9% vs. control, 53.2% +/- 3.2%, P < 0.01) and Zucker lean animals (leptin treated, 25.2% +/- 3.7% vs. control, 53.9% +/- 11.3%, P < 0.01). By contrast, hearts from (fa/fa) did not exhibit significant decreases in infarct size. Leptin increased p44 and p42 phosphorylation in Wistar rat hearts by 103.9% (P < 0.05) and 157.3% (P < 0.001), respectively, and by 97.0% (P < 0.05) and 158.1% (P < 0.05) in hearts from Zucker lean rats. Akt/serine-473 phosphorylation was increased in Wistar hearts by 96.7% (P < 0.05), whereas Akt/threonine-308 phosphorylation was elevated by 43.9% (P < 0.05) in Zucker lean rat hearts. Leptin did not influence Akt or p44/42 phosphorylation in (fa/fa) animals. On leptin treatment, mitochondrial permeability transition pore opening was delayed by 43% (P < 0.01) and 30.9% (P < 0.01), respectively, in cardiomyocytes from Wistar and Zucker lean rat hearts but not in cardiomyocytes from (fa/fa). This study provides the first evidence that myocardial sensitivity to the tissue preserving actions of leptin is influenced by adiposity and OB-R status.

Platelet alpha- and beta-secretase activities: A preliminary study in normal human subjects.

beta-amyloid (Abeta) arises from an amyloid precursor protein (APP) via beta- and gamma-secretase proteolysis. Platelets are the primary depot of APP in the circulation and may be a source of cerebral Abeta. We measured the platelet activities of alpha- and beta-secretases in normal individuals. Platelet alpha- and beta-secretase activities were linear with respect to incubation time and quantity of platelet extract included in the assays. beta-secretase activity, calculated relative to platelet count (PC) and platelet protein (PP), varied between individuals 3.5-fold and 4.6-fold, respectively. Meanwhile, alpha-secretase varied 3.75-fold and 5.8-fold. beta-secretase/alpha-secretase ratios varied 4.1-fold (PC) or 10.5-fold (PP). beta-secretase correlated with age (r = 0.779, p < 0.02, PP) and mean platelet volume (MPV) (r = 0.702, p < 0.05, PC). alpha-secretase correlated negatively with high-density lipoprotein (HDL) (-0.76, p < 0.01, PP). beta-secretase/alpha-secretase ratios correlated with MPV (r = 0.67, p < 0.05, PC) and HDL (r = 0.77, p < 0.02, PP). These data could indicate that platelet secretase activity is related to aging, platelet size and plasma lipoprotein levels. Clearly, however, this is a preliminary study involving a limited number of individuals and further, more detailed, investigations are required to establish that such relationships are valid.

Apelin-13 and apelin-36 exhibit direct cardioprotective activity against ischemia-reperfusion injury.

Protection against myocardial ischemia-reperfusion (I/R) injury involves activation of phosphatidylinositol-3-OH kinase (PI3K)- Akt/protein kinase B and p44/42 mitogen-activated protein kinase (MAPK), components of the reperfusion injury salvage kinase (RISK) pathway. The adipocytokine, apelin, activates PI3K-Akt and p44/42 in various tissues and we, therefore, hypothesised that it might demonstrate cardioprotective activity. Employing both in vivo (open-chest) and in vitro (Langendorff and cardiomyocytes) rodent (mouse and rat) models ofmyocardial I/R injury we investigated if apelin administered at reperfusion at concentrations akin to pharmacological doses possesses cardioprotective activity. Apelin-13 and the physiologically less potent peptide, apelin-36, decreased infarct size in vitro by 39.6% (p<0.01) and 26.1% (p<0.05) respectively. In vivo apelin-13 and apelin-36 reduced infarct size by 43.1% (p<0.01) and 32.7% (p<0.05). LY294002 and UO126, inhibitors of PI3K-Akt and p44/42 phosphorylation respectively, abolished the protective effects of apelin-13 in vitro.Western blot analysis provided further evidence for the involvement of PI3K-Akt and p44/42 in the cardioprotective actions of apelin. In addition,mitochondrial permeability transition pore (MPTP) opening was delayed by both apelin- 13 (127%, p<0.01) and apelin-36 (93%, p<0.01) which, in the case of apelin-13, was inhibited by LY294002 and mitogen-activated protein kinase kinase (MEK) inhibitor 1. This is the first study to yield evidence that the adipocytokine, apelin, produces direct cardioprotective actions involving the RISK pathway and the MPTP.

Native and oxidized low-density lipoproteins modulate the vasoactive actions of soluble beta-amyloid peptides in rat aorta.

Cerebrovascular accumulation of Abeta (beta-amyloid) occurs in aging and AD (Alzheimer's disease). Hypercholesterolaemia, which is associated with raised plasma LDL (low-density lipoprotein), may predispose to AD. Soluble Abeta is found in the circulation and enhances vasoconstriction. Under conditions that may favour the formation of short Abeta oligomers, as opposed to more severe polymerization leading to Abeta fibrillogenesis, we investigated the influence of LDLs on the vasoactive actions of soluble Abeta. Thus the actions of Abeta40 and Abeta42 in combination with native or oxidized LDL on vasoconstriction to NA (noradrenaline) and vasodilatation to ACh (acetylcholine) were examined in rat aortic rings. LDL, particularly when oxidized, potentiated NA-induced constriction when combined with soluble Abeta40 and, especially, Abeta42. Soluble Abeta40 reduced relaxation induced by ACh, but Abeta42 was ineffective. Native and oxidized LDL also attenuated relaxation. Synergism occurred between oxidized LDL and Abeta with respect to ACh-induced relaxation, but not between native LDL and Abeta. We have shown for the first time that, under conditions that may result in Abeta oligomer formation, LDL, particularly when oxidized, modulates the vascular actions of soluble Abeta to extents greater than those reported previously for fibrillar Abeta preparations. Mechanisms whereby a treatable condition, namely hypercholesterolaemia, might contribute to the development of the cerebrovascular component of AD are indicated.

Exaggerated polymerisation of beta-amyloid 40 stimulated by plasma lipoproteins results in fibrillar Abeta preparations that are ineffective in promoting ADP-induced platelet aggregation.

The cytotoxic beta-amyloid peptide (Abeta) of Alzheimer's disease (AD) occurs in both plasma and platelets and may modulate platelet function. Its biological activity may relate to its fibril content and factors that promote Abeta fibrillogenesis, e.g., plasma lipoproteins could, therefore, have implications for Abeta action. We undertook a study in which structure-activity relationships were considered with respect to the actions of Abeta(1-40) on platelet function. Thus, the influence of soluble Abeta and various fibrillar Abeta preparations (0.1-10 microM) on platelet aggregation and endogenous 5-hydroxytryptamine (5-HT) efflux was investigated. Soluble Abeta(1-40) only enhanced platelet aggregation (+30%, P<0.05) and 5-HT release (+28%) stimulated by ADP (1 microM) at the highest concentration tested (10 microM). By contrast, fibrillar Abeta(1-40) at 1, 5 and 10 microM potentiated aggregation by 17.4%, 68.8% (P<0.05) and 99.5% (P<0.0001), respectively, and 5-HT efflux by 17.4%, 65% and 208% (P<0.001). Abeta(1-40) fibrils generated in the presence of native and oxidised very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) yielded platelet responses that did not differ from those seen with the lipoproteins alone. These responses were markedly lower than those obtained with homogeneous Abeta fibrils. Our data indicate that homogeneous Abeta(1-40) fibrils are more potent than soluble Abeta(1-40) in promoting platelet reactivity and that interactions with plasma lipoproteins result in the formation of Abeta fibrils that are ineffective. We suggest that lipoproteins may interfere with the recognition of Abeta by appropriate platelet receptors and/or cause Abeta to assume an "overaggregated" biologically inert state.

Potentiation of beta-amyloid polymerisation by low-density lipoprotein enhances the peptide's vasoactivity.

Alzheimer's disease (AD) is characterised by the accumulation of insoluble beta-amyloid (A beta) fibrils in the brain. Factors that promote A beta fibrillogenesis may influence the pathogenesis of AD and represent targets for therapeutic intervention. Some A beta deposited in AD may originate in the circulation and plasma factors could promote A beta deposition, particularly in the cerebrovasculature. We investigated the effects of plasma low-density lipoprotein (LDL), in both its native and oxidised forms, on A beta(1-40) fibrillogenesis and vasoactivity. LDL enhanced A beta fibrillogenesis in a process dependent on LDL concentration and the oxidative state of the lipoprotein, as indicated by measurements of thiobarbituric acid reactive substances (TBARS) and conjugated dienes. LDL's actions were inhibited by the iA beta 5 peptide, suggesting that LDL-induced A beta polymerisation involved beta-pleated sheet formation. Potentiated A beta polymerisation was reflected by enhanced A beta-mediated vascular responses. Human endothelial cells exposed to fibrillar A beta generated with LDL, especially oxidised LDL, exhibited decreased 20S proteasome activity. Rat aortic ring constriction induced by noradrenaline was enhanced by A beta fibrils generated with LDL, with oxidised LDL producing the more marked effects. Should plasma lipoproteins prove to play a role in cerebral A beta deposition their modification with statins or antioxidants may offer therapeutic benefit.

Nitric oxide as a mediator of delayed pharmacological (A(1) receptor triggered) preconditioning; is eNOS masquerading as iNOS?

BACKGROUND: Nitric oxide (NO), synthesised from the inducible isoform of nitric oxide synthase (iNOS), is implicated in mediating second window of protection (SWOP)/delayed ischemic preconditioning. However the role of NO and iNOS in delayed pharmacological protection remains unclear and is the subject of this investigation. METHODS: To test the hypothesis that iNOS is necessary for delayed pharmacological preconditioning, the adenosine A(1) receptor agonist, 2-chloro N(6) cyclopentyl adenosine (CCPA) (25 microg/kg i.v.) or saline was administered to wild type (WT) or iNOS gene knockout mice (KO). Twenty-four hours later, the hearts were isolated, Langendorff perfused and subjected to 35 min ischemia/30 min reperfusion prior to infarct size determination. RESULTS: WT and KO control hearts had identical infarct sizes of 37 +/- 3% and 37 +/- 2%, respectively. CCPA significantly reduced infarct size in WT hearts to 22 +/- 2% and also, unexpectedly, in KO hearts (27 +/- 2%). This protection was abrogated with the non-specific NOS inhibitor, N(omega) nitro L-arginine methyl ester (L-NAME, 100 microM), and could be mimicked in naïve hearts with the NO donor, donor S-nitroso N-acetyl DL penicillamine (SNAP, 1 microM). Delayed protection appeared to be mediated by NO synthesis in both WT and KO hearts. Additional studies using Western blot analysis demonstrated endothelial NOS (eNOS) upregulation and increased NO(x) release in both WT and KO hearts. CONCLUSIONS: This is the first study to demonstrate a role for eNOS in delayed A(1) receptor triggered (pharmacological) preconditioning, potentially representing a new pharmacological target for protecting the ischemic heart.

An investigation into the mechanisms mediating plasma lipoprotein-potentiated beta-amyloid fibrillogenesis.

The toxicity of the beta-amyloid (Abeta) peptide of Alzheimer's disease may relate to its polymerisation state (i.e. fibril content). We have shown previously that plasma lipoproteins, particularly when oxidised, greatly enhance Abeta polymerisation. In the present study the nature of the interactions between both native and oxidised lipoproteins and Abeta1-40 was investigated employing various chemical treatments. The addition of ascorbic acid or the vitamin E analogue, trolox, to lipoprotein/Abeta coincubations failed to inhibit Abeta fibrillogenesis, as did the treatment of lipoproteins with the aldehyde reductant, sodium borohydride. The putative lipid peroxide-derived aldehyde scavenger, aminoguanidine, however, inhibited Abeta-oxidised lipoprotein-potentiated polymerisation, but in a manner consistent with an antioxidant action for the drug. Lipoprotein treatment with the reactive aldehyde 4-hydroxy-2-trans-nonenal enhanced Abeta polymerisation in a concentration-dependent fashion. Incubation of Abeta with lipoprotein fractions from which the apoprotein components had been removed resulted in extents of polymerisation comparable to those observed with Abeta alone. These data indicate that the apoprotein components of plasma lipoproteins play a key role in promoting Abeta polymerisation, possibly via interactions with aldehydes.

Plasma beta-amyloid (A beta) 40 concentration, lipid status and age in humans.

The circulation constitutes a potential source of the beta-amyloid (A beta) protein deposited cerebrally in Alzheimer's disease (AD). Cardiovascular risk factors, including hyperlipidaemia, may be involved in the pathogenesis of AD. Plasma A beta 40 was measured by radioimmunoassay in normal and hyperlipidaemic subjects with the aim of determining if plasma lipid content and/or age correlated with circulating A beta 40 concentration. Plasma A beta 40 levels in hyperlipidaemics were elevated by 20.3% compared to normal subjects. A beta 40 did not correlate with plasma lipids in normal subjects. Age, however, correlated positively with A beta 40 in these individuals and with total cholesterol, low-density lipoprotein (LDL) and triglycerides. No correlations were observed in hyperlipidaemic patients or when the data for the two groups were combined. These data are consistent with ageing, the primary risk factor for AD, but not hyperlipidaemia influencing circulating A beta 40 levels.

Soluble beta-amyloid (A beta) 40 causes attenuation or potentiation of noradrenaline-induced vasoconstriction in rats depending upon the concentration employed.

Soluble beta-amyloid (A beta) 40 peptide (1 microM) has been reported to enhance phenylephrine and endothelin-1 induced contraction of rat aortic rings. We conducted similar experiments with aortic rings from Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats but employing noradrenaline (NA) as the vasoconstrictor. Unlike previous studies we found that, rather than enhancing agonist-induced contraction, 1 microM A beta 40 attenuated the vasoconstrictive responses to NA. With aortic rings from SD rats the attenuation of contractile responses was coupled with a 99% increase in NA EC(50) values. EC(50) values obtained for aortic rings from WKY and SHR, however, exhibited no changes. Contrasting with the effects observed with 1 microM A beta 40, treatment of SD aortic rings with 5 microM A beta 40 resulted in potentiation of NA-induced constriction and a 46% decrease in EC(50) values. We hypothesise that at low concentrations A beta 40 may cause attenuation of NA-induced constriction by dint of enhanced endothelial vasodilator (nitric oxide, prostacyclin) synthesis. By contrast, at higher concentrations A beta 40 may potentiate vasoconstriction via the generation of toxic A beta oligomers which act on the endothelium to reduce vasodilator output.

Evaluation of methods for the extraction of nitrite and nitrate in biological fluids employing high-performance anion-exchange liquid chromatography for their determination.

Measurements of nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in biological fluids are proposed as indices of cellular nitric oxide (NO) production. Determination of NO(2)(-) and NO(3)(-) in standard solutions is not difficult, however, determinations which reflect accurately cellular NO synthesis represent a considerable analytical challenge. Problems are often encountered arising from background NO(2)(-)/NO(3)(-) contamination in experimental solutions and laboratory hardware, and with methods for sample extraction. We investigated potential procedures for the extraction and determination of NO(2)(-) and NO(3)(-) in biological samples. Consequently, a protocol was devised which yielded acceptable results regarding extraction efficiency, assay reproducibility, sample throughput and contaminant minimisation. It entailed rigorous washing of all equipment with water of low NO(2)(-) and NO(3)(-) content, sample deproteinisation by centrifugal ultrafiltration through a 3K filter and analysis by high-performance anion-exchange liquid chromatography with UV detection. Retention times for NO(2)(-) and NO(3)(-) in standards and plasma were 4.4 and 5.6 min, respectively. Assay linearity for standards ranged between 31 nM and 1 mM. The limit of detection for NO(2)(-) and NO(3)(-) in standards was 3 pmol. Recoveries of NO(2)(-) and NO(3)(-) from spiked plasma (1-100 microM KNO(2)/KNO(3)) and from extracted standards (1-250 microM) were approximately 100%. Intra-assay and inter-assay RSDs for NO(2)(-) and NO(3)(-) in spiked and unspiked plasma were 10.6% or less. Assays on washed platelet supernatants demonstrated collagen-induced platelet generation of NO products and analysis of murine and rat cardiac perfusates was achieved. Our procedure may be suitable for routine determination of NO(2)(-) and NO(3)(-) in various biological fluids, e.g., plasma.

Adipocytokines, cardiovascular pathophysiology and myocardial protection.

Reducing myocardial damage resulting from ischaemia-reperfusion (I/R) is vital in ensuring patient recovery and survival. It relies upon the activation of the so-called Reperfusion Injury Salvage Kinase (RISK) pathway. Experimentally various treatments, both mechanical and chemical, have been shown to protect the myocardium against I/R injury. Chemical facilitators of myocardial preservation include endogenous factors such as insulin, erythropoietin and glucagon-like peptide 1. The adipocytokines, products of white adipose tissue, are important peptide hormones with respect to metabolic control and satiety, and were formerly considered in the context of obesity and metabolic disease. More recently, however, evidence has been presented indicating that the adipocytokines play significant roles in cardiac function and, as we have suggested, in myocardial protection. To date leptin, adiponectin, apelin and visfatin have all been shown to protect against I/R injury. Significantly, the protection afforded by these peptides involves the activation of kinases which are key elements of the mechanisms underlying tissue preservation, including the RISK pathway components PI3K-Akt and p44/42, and inhibition of the mitochondrial permeability transition pore (MPTP). In this article we examine the roles played by the adipocytokines in cardiovascular function and disease. In particular, we focus on the evidence that these peptides promote myocardial survival, much of it having been obtained in this laboratory. To conclude, we discuss some future directions in the field, including the prospects for some of the adipocytokines finding application as therapeutic agents in myocardial infarction.

Failure of the adipocytokine, resistin, to protect the heart from ischemia-reperfusion injury.

Experimental studies have linked the adipocytokines with acute cardioprotection. Whether the adipocytokine, resistin, confers protection is, however, debatable. In the current study, the actions of resistin, administered at reperfusion, were investigated in in vivo and in vitro rodent and in vitro human models of myocardial ischemia-reperfusion (I/R) injury. Resistin did not reduce infarct size in Langendorff-perfused rat hearts or murine hearts perfused in vivo. Resistin also did not protect human atrial muscle subjected to hypoxia-reoxygenation. Although cyclosporin A delayed mitochondrial permeability transition pore (MPTP) opening in murine cardiomyocytes, resistin was ineffective. Western blot analysis revealed that resistin treatment was associated with enhanced phosphorylation of Akt, at both the serine-473 (+ 51.9%, P = .01) and threonine-308 (+107%, P < .01) phosphorylation sites, although not to the extent seen with ischemic preconditioning (+132.5%, P = .002 and +389.1%, P < .01, respectively). We conclude that resistin administered at reperfusion at concentrations/doses equivalent to normal (upper end) and pathological serum levels does not protect against I/R injury or inhibit MPTP opening.

The novel adipocytokine visfatin exerts direct cardioprotective effects.

Visfatin is an adipocytokine capable of mimicking the glucose-lowering effects of insulin and activating the pro-survival kinases phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-extracellular signal-regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 x 10(-6) micromol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1+/-4.1% in control hearts to 27.3+/-4.0% (n>or= 6/group, P<0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8+/-5.5% and 45.9+/-8.4%, respectively, versus 27.3+/-4.0% with visfatin; n>or= 6/group, P<0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2+/-4.6% in control to 49.2+/-3.7%(n>200 cells/group, P<0.05), an effect that was abrogated by wortmannin and U0126 (68.1+/-5.2% and 59.7+/-6.2%, respectively; n>200 cells/group, P>0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2+/-4 sec. in control to 120+/-7 sec. (n>20 cells/group, P<0.05) in a PI3K- and MEK1/2-dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.

Necrostatin: a potentially novel cardioprotective agent?

BACKGROUND: Necrostatin-1 (Nec-1), a small tryptophan-based molecule, was recently reported to protect the cerebral cortex against ischemia-reperfusion (I/R) injury. We investigated the actions of Nec-1 and its so-called inactive analog, Nec-1i, in the setting of myocardial I/R injury. MATERIALS AND METHODS: The actions of Nec-1 and Nec-1i were examined in cultured C2C12 and H9c2 myocytes, cardiomyocytes isolated from male Sprague-Dawley rats, Langendorff isolated perfused C57Bl/6J mouse hearts and an in vivo open-chest C57Bl/6J mouse heart model. RESULTS: Nec-1 at 30 microM and 100 microM (but not 100 microM Nec-1i) reduced peroxide-induced cell death in C2C12 cells from 51.2 +/- 1.1% (control) to 26.3 +/- 2.9% (p < 0.01 vs control) and 17.8 +/- 0.9% (p < 0.001), respectively. With H9c2 cells cell death was also reduced from 73.0 +/- 0.4% (control) to 56.7 +/- 0% (30 microM Nec-1, p < 0.05) and 45.4 +/- 3.3% (100 microM Nec-1, p < 0.01). In the isolated perfused heart Nec-1 (30 microM) reduced infarct size (calculated as a percentage of the risk area) from 48.0 +/- 2.0% (control) to 32.1 +/- 5.4% (p < 0.05). Nec-1i (30 microM) also reduced infarct size (32.9 +/- 5.1%, p < 0.05). In anesthetized C57Bl/6J mice Nec-1 (1.65 mg/kg), given intraperitoneally to coincide with reperfusion following left anterior descending artery ligation (30 min), also reduced infarct size from 45.3 +/- 5.1% (control) to 26.6 +/- 4.0% (p < 0.05), whilst Nec-1i (1.74 mg/kg) was ineffective (37.8 +/- 6.0%). Stimulus-induced opening of the mitochondrial permeability transition pore (MPTP) in rat cardiomyocytes, as reflected by the time until mitochondrial depolarisation, was unaffected by Nec-1 or Nec-1i at 30 muM but increased at 100 muM i.e. 91% (p < 0.05 vs control) and 152% (p < 0.001) for Nec-1 and Nec-1i, respectively. CONCLUSION: This is the first study to demonstrate that necrostatins inhibit myocardial cell death and reduce infarct size, possibly via a mechanism independent of the MPTP.

Temporal changes in myocardial salvage kinases during reperfusion following ischemia: studies involving the cardioprotective adipocytokine apelin.

INTRODUCTION: Activation of the Reperfusion Injury Salvage Kinase (RISK) pathway, which incorporates phosphatidylinositol-3-OH kinase (PI3K)-Akt/protein kinase B (PKB) and p44/42 mitogen-activated protein kinase (MAPK), underlies protection against ischemia-reperfusion (I/R) injury. The temporal nature of the activation of these RISK pathway components during reperfusion is, however, uncertain. We examined Akt and p44/42 phosphorylation in hearts subjected to ischemia and varying periods of reperfusion in the absence or presence of the putative cardioprotectant, apelin-13. Akt activity was also measured. MATERIALS AND METHODS: Langendorff perfused C57Bl/6J mouse hearts were subjected to 35 min global ischemia followed by 0, 2.5, 5 or 10 min reperfusion with or without 1 microM apelin-13. Basal and apelin-induced phosphorylation of Akt (at both the threonine 308 and serine 473 phosphorylation sites) and p44/42 during the reperfusion phase was determined by Western blotting and Akt activity measured using an Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS: Basal phosphorylation of both Akt and p44/42 increased progressively with time of reperfusion. Apelin enhanced Akt and p44/42 phosphorylation at all reperfusion time points. Akt activity did not change under basal conditions but was increased by apelin at 5 min (NS) and 10 min (p<0.05) reperfusion. DISCUSSION: We conclude that under basal conditions Akt and p44/42 phosphorylation increases with time of reperfusion but that this is not accompanied by increased kinase (Akt) activity. On application of a cardioprotectant, however, kinase phosphorylation and activity are enhanced suggesting that it is the combination of these two mechanisms that may underly the tissue preserving actions of such agents.