Simulated Ischemia Research Articles

Abstract 16479: Remote Ischemic Preconditioning: Do Exosomes Play a Requisite Role in Evoking Cardioprotection?

Remote ischemic preconditioning (RIPC) is the phenomenon whereby brief episodes of ischemia-reperfusion applied in distant tissues or organs render the heart resistant to a subsequent sustained ischemic insult. The hallmark of RIPC is inter-organ communication, with recent evidence suggesting that exosomes may serve as the vector for the transport of protective factor(s) from the site of the distant trigger to the cardiomyocytes. To test this hypothesis, rats underwent a standard RIPC stimulus: i.e., 4 5-min episodes of bilateral femoral artery occlusion. Serum (~3 mL per rat) was harvested immediately after RIPC; 1 mL was stored at -80°C, and 2 mL was processed for exosome isolation by prolonged ultracentrifugation (12 hours at 300,000 xg ). Aliquots of the pellet and supernatant were probed (by immunoblotting) for expression of exosome markers including HSP60 and flotillin. Most importantly, the cardioprotective efficacy of: 1) RIPC serum, 2) the pellet, reconstituted in buffer, 3) supernatant-bottom layer, 4) supernatant-top layer and 5) buffer alone was assessed by transfer of volume-matched aliquots to cultured HL-1 cardiomyocytes. After 1 hour of pretreatment, cells were subjected to 2.5 hours simulated ischemia + 24 hours reoxygenation and % cell death was quantified by Trypan blue staining. As expected, RIPC serum was protective: mean cell death was 34 + 5%* vs 48 + 5% in buffer-controls (*p<0.05). As further expected, the exosome-depleted top layer of supernatant failed to confer protection. However, in contrast to the ‘exosome hypothesis’, RIPC with exosome-rich aliquots did not yield consistent cardioprotection: both the pellet and the supernatant-bottom layer had robust expression of HSP60 and flotillin, yet only the supernatant-bottom attenuated cell death (32 + 3%*; Figure). Thus, while transport of protective factor(s) via exosomes may play a role in RIPC, our results reveal that exosomes per se are not sufficient to evoke remote cardioprotection.

Abstract 16115: Rapamycin Protects Diabetic Heart Against Ischemia-reperfusion Injury by Inhibiting JNK and STAT4

Background: Persistent activation of the mammalian target of rapamycin (mTOR) is implicated in diabetic complications and plays a critical role in myocardial injury following ischemia/reperfusion. Recently we reported that mTOR inhibition with rapamycin improves cardiac function in type 2 diabetic (T2D) mice through attenuation of oxidative stress and alteration of antioxidants and contractile protein expression. Since increased JNK and STAT4 activities have been strongly associated with T2D-induced cardiovascular complications, we hypothesized that rapamycin would reduce myocardial infarction (MI) in T2D mice through inhibition of JNK and STAT4. Methods and Results: Adult male wild type (C57) or db/db T2D mice were treated daily for 28 days with rapamycin (0.25 mg/kg, i.p.). Following treatment, the hearts were subjected to global I (30 min) and R (60 min) in Langendorff mode. In addition, cardiomyocytes were isolated from treated mice and subjected to 40 min simulated ischemia (SI) and 1 hr or 18 hr reoxygenation (RO) to assess necrosis (by trypan blue staining) or apoptosis (by TUNEL assay), respectively. Diabetes exacerbated ischemic infarct size (measured by tetrazolium chloride staining) in db/db mice as compared to C57 mice (Fig. A), and also enhanced cardiomyocyte necrosis (Fig. B) and apoptosis (Fig. C). Rapamycin treatment reduced infarct size following I/R and protected adult cardiomyocytes against necrosis and apoptosis following SI/RO. Rapamycin also improved post-ischemic contractile function in db/db mice (Fig. D). Western blot analysis revealed a marked increase in phosphorylation of JNK and STAT4 in diabetic hearts, which were reduced in rapamycin-treated diabetic hearts (Fig. E). Conclusion: Rapamycin treatment reduces MI possibly by inhibiting JNK and STAT4 in T2D heart. We propose that inhibition of JNK and STAT4 with rapamycin therapy may be an attractive strategy to improve prognosis in diabetics following MI.

Activation of mitochondrial STAT-3 and reduced mitochondria damage during hypothermia treatment for post-cardiac arrest myocardial dysfunction.

While therapeutic hypothermia improves the outcomes of individuals in cardiac arrest, the hemodynamic responses and mechanisms which underlie hypothermia-induced cardioprotection are not fully understood. Therefore, we investigated the mechanism by which induced hypothermia preserves cardiac function and protects against mitochondrial damage following cardiac arrest. Cardiac arrest was induced in adult male Wistar rats by asphyxiation for 8.5min. Following resuscitation, the animals were randomly assigned to a hypothermia (32°C) or normothermia (37°C) group. Monitoring results showed that cardiac output at the fourth hour after resuscitation was significantly better in rats treated with hypothermia when compared to rats treated with normothermia (P<0.01). Examinations by transmission electron microscopy showed that mitochondria in the left ventricle of rats in the hypothermia group were significantly less swollen compared to such mitochondria in the normothermia group (P<0.001). Additionally, opening of mitochondrial permeability transition pores occurred less frequently in the hypothermic group. While complex I/III activity in the electron transport reaction was damaged after cardiac arrest and resuscitation, the degree of injury was ameliorated by hypothermia treatment (P<0.05). The amount of STAT-3 phosphorylated at tyrosine 705 and its expression in mitochondria were significantly higher under hypothermia treatment compared to normothermia treatment. In vitro studies showed that inhibition STAT-3 activation abolished the ability of hypothermia to protect H9C2 cardiomyocytes against injury produced by simulated ischemia and reperfusion. Therapeutic hypothermia treatment can ameliorate cardiac dysfunction and help preserve both mitochondrial integrity and electron transport activity.

Insulin/NFκB protects against ischemia-induced necrotic cardiomyocyte death

In the heart, insulin controls key functions such as metabolism, muscle contraction and cell death. However, all studies have been focused on insulin action during reperfusion. Here we explore the cardioprotective action of this hormone during ischemia. Rat hearts were perfused ex vivo with an ischemia/reperfusion Langendorff model in absence or presence of insulin. Additionally, cultured rat cardiomyocytes were exposed to simulated ischemia in the absence or presence of insulin. Cytoprotective effects were measured by myocardial infarct size, trypan blue exclusion, released LDH and DNA fragmentation by flow cytometry. We found that insulin protected against cardiac ischemia ex vivo and in vitro. Moreover, insulin protected cardiomyocytes from simulated ischemia by reducing necrotic cell death. Protective effects of insulin were dependent of Akt and NFκB. These novel results show that insulin reduces ischemia-induced cardiomyocyte necrosis through an Akt/NF-κB dependent mechanism. These novel findings clarify the role of insulin during ischemia and further support its use in early GIK perfusion to treat myocardial infarction.

Estimation of thiocetam influence on glutathione link of thiol disulfide cerebral system in conditions of experimental chronic ischemia

Aim. Pharmacological remodeling of pathologic changes when cerebral ischemia takes place is actual task for today. With the purpose of thiocetam neuroprotective properties studying in conditions of experimental chronic cerebral ischemia the investigations of 45 white rats with simulated chronic cerebral ischemia have been carried out.Methods and results. A number of biochemical, enzyme immunoassay, pharmacological and statistic methods of investigation were used. Positive thiocetam influence on molecular and biochemical changes in brain of laboratory animals subjected to ischemia is established. This influence is showed in decreasing processes of oxidative and nitrosative stresses: increasing of reduced glutathione contents, activity of antioxidant enzymes of glutathione link, recreation of thiol disulfide state of a cell, decreasing of neurotoxic compound contents of nitrotyrosine in cerebral tissue.Conclusion. A positive influence of thiocetam on remembering processes among laboratory animals with simulated chronic cerebral ischemia has been noted.

Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro.

Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.

Pre-Analytical Determination of the Effect of Extended Warm or Cold Ischaemia on RNA Stability in the Human Ileum Mucosa.

The use of banked human tissue, obtained with informed consent after elective surgical procedures, represents a powerful model for understanding underlying mechanisms of diseases or therapeutic interventions and for establishing prognostic markers. However, donated tissues typically have varying times of warm ischaemia in situ due to blood arrest or cold ischaemia due to procurement and transportation. Hence, before using these tissues, it is important to carry out pre-analytical studies to ensure that they are representative of the in vivo state. In particular, tissues of the gastrointestinal tract have been thought to have low RNA stability. Therefore, this study aimed to determine if extended warm or cold ischaemia times and snap-freezing or banking in RNA stabilization solution affects RNA integrity or gene expression in human ileum mucosa. In short, ileum mucosa was collected for up to 1.5 h and 6 h of simulated warm or cold ischaemia respectively. Subsequently, RNA integrity and gene expressions were determined. It was found that RNA integrity remained high over the course of warm and cold ischaemia examined and there were in general no significant differences between snap-freezing and banking in RNA stabilization solution. Following the same trend, there were in general no significant changes in gene expressions measured (MYC, HIF1α, CDX, HMOX1 and IL1β). In conclusion, RNA in the ileum mucosa is maintained at a high integrity and has stable gene expression over the examined time course of warm or cold ischaemia when banked in RNA stabilization solution or snap-frozen in liquid nitrogen. As the average warm and cold ischaemia times imposed by surgery and the process of tissue banking are shorter than the time period examined in this study, human ileum mucosa samples collected after surgeries could be used for gene expression studies.

Gadd45γ regulates cardiomyocyte death and post-myocardial infarction left ventricular remodelling.

Post-infarction remodelling is accompanied and influenced by perturbations in mitogen-activated protein kinase (MAPK) signalling. The growth arrest and DNA-damage-inducible 45 (Gadd45) proteins are small acidic proteins involved in DNA repair and modulation of MAPK activity. Little is known about the role of Gadd45 in the heart. Here, we explored the potential contribution of Gadd45 gamma (γ) isoform to the acute and late phase of heart failure (HF) after myocardial infarction (MI) and determined the mechanisms underlying Gadd45γ actions. The Gadd45γ isoform is up-regulated in murine cardiomyocytes subjected to simulated ischaemia and in the mouse heart during MI. To mimic the situation observed during MI, we enhanced Gadd45γ content in cardiomyocytes with a single injection of an adeno-associated viral (AAV9) vector encoding Gadd45γ under the cTNT promoter. Gadd45γ overexpression induces cardiomyocyte apoptosis, fibrosis, left ventricular dysfunction, and HF. On the other hand, genetic deletion of Gadd45γ in knockout mice confers resistance to ischaemic injury, at least in part by limiting cardiomyocyte apoptosis. Mechanistically, Gadd45γ activates receptor-interacting protein 1 (RIP1) and caspase-8 in a p38 MAPK-dependent manner to promote cardiomyocyte death. This work is the first to demonstrate that Gadd45γ accumulation during MI promotes the development and persistence of HF by inducing cardiomyocyte apoptosis in a p38 MAPK-dependent manner. We clearly identify Gadd45γ as a therapeutic target in the development of HF.

Abstract 243: The E3 Ubiquitin Ligase Parkin Mediates Clearance of Damaged Mitochondria via Two Distinct Pathways

Damaged mitochondria release reactive oxygen species and pro-death factors which can lead to loss of cardiac myocytes. To protect against such damage, myocytes have developed several mechanisms of quality control that act both on the protein and organelle levels. We have previously identified the E3 ubiquitin ligase Parkin as an important regulator of mitochondrial clearance via autophagy in the myocardium. Here, we report that Parkin can also mediate clearance of mitochondria via the endosomal-lysosomal pathway. We found that Parkin promotes clearance of damaged mitochondria in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Mitochondrial damage leads to rapid activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. We further observed increased activation of Rab5, a protein involved in early endosome formation, in both WT and Atg5-/- MEFs after treatment with FCCP. In addition, we observed sequestration of damaged mitochondria in Rab5+ and Rab7+ early and late endosomes, respectively. Mitochondria also colocalized with Lamp2+ vesicles in Atg5-/- MEFs indicating that the mitochondria are ultimately being delivered to the lysosomes for degradation. Overexpression of Rab5S34N, a dominant negative of Rab5, reduces FCCP-mediated clearance and increases cell death in Atg5-/- MEFs. Pharmacological inhibition of the endosomal-lysosomal pathway also results in increased FCCP-mediated cell death. Furthermore, we confirmed that FCCP treatment or simulated ischemia reperfusion exposure induces Rab5 activation with subsequent mitochondrial sequestration in early endosomes in neonatal myocytes. Interestingly, the activation of Rab5 is abrogated in the presence of the mitochondrial targeted antioxidant Mito-Tempo, suggesting that mitochondrial ROS is involved in the activation the endosomal pathway. Mitochondrial clearance via this pathway is also dependent on Parkin, as FCCP treatment fails to activate Rab5 and induce mitochondrial clearance in both WT and Atg5-/- MEFS in the absence of Parkin. Thus, our data suggest that Parkin can mediate clearance of damaged mitochondria via two distinct pathways in cells.

Cardioprotective Effect of Ranolazine in the Process of Ischemia-reperfusion in Adult Rat Cardiomyocytes

Introduction and objectivesRanolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. MethodsThe effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. ResultsWe found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. ConclusionsRanolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia.

Efecto cardioprotector de la ranolazina en el proceso de isquemia-reperfusión en cardiomiocitos de rata adultos

Introduction and objectivesRanolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. MethodsThe effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. ResultsWe found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. ConclusionsRanolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia.Full English text available from:www.revespcardiol.org/en

Hydrogen sulfide mediates the cardioprotective effects of gene therapy with PKG-Iα.

Cyclic GMP-dependent protein kinase (PKG) is a serine-threonine kinase that mediates the cardioprotective effect of ischemic and pharmacologic preconditioning. Since hydrogen sulfide (H2S) has been implicated in mediating the cardioprotective effects of the cGMP modulators tadalafil and cinaciguat, we tested the hypothesis that myocardial gene therapy with PKG exerts cardioprotection against ischemia/reperfusion (I/R) injury through a mechanism involving H2S. Adult rat cardiomyocytes were infected with adenoviral vector encoding PKGIα or inactive mutant PKGIαK390A (K390A) for 24 h. Necrosis and apoptosis (n = 6/group) were determined after 90 min of simulated ischemia and 1 or 18 h of reoxygenation, respectively. To study the effect of PKGIα in vivo, mice received intramyocardial injections of adenoviral PKGIα or K390A. Four days later, the hearts were subjected to 30 min of ischemia followed by reperfusion for 24 h. The inhibitor of H2S-producing enzyme, cystathionine-γ-lyase (CSE), dl-propargylglycine (PAG, 50 mg/kg, ip) was given 30 min before ischemia. PKGIα overexpression induced CSE expression, whereas cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase expression was not changed. PKGIα overexpression increased H2S in the heart and cardiomyocytes in relation to control and PKGIαK390A. Moreover, PAG abolished protection with PKGIα in vitro by increasing necrosis (35.2 ± 1.7%, P < 0.05) and apoptosis (23.5 ± 1.8 %, P < 0.05) as compared to PKGIα-overexpressing cells (necrosis: 17.2 ± 0.9% and apoptosis: 13.2 ± 0.8%). In vivo, PKGIα overexpression reduced infarct size and preserved left ventricular fractional shortening as compared with K390A (P < 0.05) and PAG abolished the cardioprotective effect of PKGIα. The protective effect of myocardial gene therapy with PKGIα against I/R injury is mediated through a mechanism involving H2S signaling.

Human epicardial cell-conditioned medium contains HGF/IgG complexes that phosphorylate RYK and protect against vascular injury.

The aim of this study was to evaluate the paracrine activity of human epicardial-derived cells (hEPDCs) to screen for secreted vasoprotective factors and develop therapeutics to treat vascular reperfusion injury. Epicardial cells support cardiac development, repair, and remodelling after injury in part, through paracrine activity. We hypothesized that secreted ligands from hEPDCs would protect vascular integrity after myocardial infarction (MI) with reperfusion. During simulated ischaemia in culture (24-48 h), concentrated hEPDC-conditioned medium (EPI CdM) increased survival of primary cardiac endothelial cells. In a rat MI model, EPI CdM treatment reduced vascular injury in vivo after reperfusion. By phospho-receptor tyrosine kinase (RTK) arrays, ELISA, and neutralizing antibody screens, we identified hepatocyte growth factor (HGF) as a key vasoprotective factor in EPI CdM. Unexpectedly, we observed that some of the HGF in EPI CdM formed complexes with polyclonal IgG. Following reperfusion, preparations of HGF/IgG complexes provided greater vascular protection than free HGF with IgG. HGF/IgG complexes localized to blood vessels in vivo and increased HGF retention time after administration. In subsequent screens, we found that 'related to tyrosine kinase' (RYK) receptor was phosphorylated after exposure of cardiac endothelial cells to HGF/IgG complexes, but not to free HGF with IgG. The enhanced protection conferred by HGF/IgG complexes was lost after antibody blockade of RYK. Notably, the HGF/IgG complex is the first 'ligand' shown to promote phosphorylation of RYK. Early treatment with HGF/IgG complexes after myocardial ischaemia with reperfusion may rescue tissue through vasoprotection conferred by c-Met and RYK signalling.

Molecular mechanisms underlying oxytocin-induced cardiomyocyte protection from simulated ischemia–reperfusion

Oxytocin (OT) stimulates cardioprotection. Here we investigated heart-derived H9c2 cells in simulated ischemia–reperfusion (I–R) experiments in order to examine the mechanism of OT protection. I–R was induced in an anoxic chamber for 2 hours and followed by 2 h of reperfusion. In comparison to normoxia, I–R resulted in decrease of formazan production by H9c2 cells to 63.5 ± 1.7% (MTT assay) and in enhanced apoptosis from 1.7 ± 0.3% to 2.8 ± 0.4% (Tunel test). Using these assays it was observed that treatment with OT (1–500 nM) exerted significant protection during I–R, especially when OT was added at the time of ischemia or reperfusion. Using the CM-H2DCFDA probe we found that OT triggers a short-lived burst in reactive oxygen species (ROS) production in cells but reduces ROS production evoked by I–R. In cells treated with OT, Western-blot revealed the phosphorylation of Akt (Thr 308, p-Akt), eNOS and ERK 1/2. Microscopy showed translocation of p-Akt and eNOS into the nuclear and perinuclear area and NO production in cells treated with OT. The OT-induced protection against I–R was abrogated by an OT antagonist, the Pi3K inhibitor Wortmannin, the cGMP-dependent protein kinase (PKG) inhibitor, KT5823, as well as soluble guanylate cyclase (GC) inhibitor, ODQ, and particulate GC antagonist, A71915. In conditions of I–R, the cells with siRNA-mediated reduction in OT receptor (OTR) expression responded to OT treatment by enhanced apoptosis.In conclusion, the OTR protected H9c2 cells against I–R, especially if activated at the onset of ischemia or reperfusion. The OTR-transduced signals include pro-survival kinases, such as Akt and PKG.

Inhibition of endoplasmic reticulum stress by ethyl pyruvate attenuates simulated ischemia reperfusion injury of neonatal rat cardiomyocyte

Objective To explore the effect of ethyl pyruvate(EP)on the simulated ischemia reperfusion(SIR)injury of neonatal rat cardiomyocyteand the role of endoplasmic reticulum stress(ERS)in this procedure. Methods The cultured neonatal rat cardiomyocytes, pretreated with EP(5 or 10 mmol/L)for 2 h, were subjected to SIR injury(hypoxia for 2 h, reoxygenation for 4 h). Methyl thiazolyl tetrazolium (MTT)method was used to detect the cell viability,special kits were used to detected the levels of malondialdehyde(MDA)and superoxide dismutase(SOD)in culture medium.Western blotting was used to detect the expression of ERS related molecules glucose regulated protein 78(GRP78) and CCAAT/enhancerbinding protein homologous protein(CHOP).Further, EP treatment was added with the ERS activator thapsigargin(THA), the effect of THA on the protection of EP against SIR was observed. Results Both 5 and 10 mmol/LEP treatment dramatically increased the cell viability(0.424±0.016 and 0.517± 0.020), downregulated the increased MDA level[(16.17±1.75)and(11.45±1.20)μmol/L]and decreased SOD level[(58.61±4.70)and(72.60±4.35) U/L]in the culture medium(vs.the SIR group, P<0.05).Compared with the control group, the level of GRP78 and CHOP increased significantly in the SIR group(P< 0.05), while EP treatment significantly decreased the level of GRP78 and CHOP(vs.the SIR group, P<0.05).Further, the ERSactivator THA reversed the protection of EP, decreased the cell viability(vs.the EP+ SIR group, P< 0.05), upreguleted the expression of GRP78 and CHOP(vs.the EP+ SIR group, P<0.05). Conclusion EP treatment can protect against SIR injury of neonatal rat cardiomyocyte, this effect is associated with inhibition of ERS. Key words: Ethyl pyruvate; Cardiomyocyte; Simulated ischemia reperfusion; Endoplasmic reticulum stress

Inhibition of mammalian target of rapamycin protects against reperfusion injury in diabetic heart through STAT3 signaling.

Diabetic patients suffer augmented severity of myocardial infarction. Excessive activation of the mammalian target of rapamycin (mTOR) and decreased activation of STAT3 are implicated in diabetic complications. Considering the potent cardioprotective effect of mTOR inhibitor, rapamycin, we hypothesized that reperfusion therapy with rapamycin would reduce infarct size in the diabetic hearts through STAT3 signaling. Hearts from adult male db/db or wild type (WT) C57 mice were isolated and subjected to 30 min of normothermic global ischemia and 60 min of reperfusion in Langendorff mode. Rapamycin (100 nM) was infused at the onset of reperfusion. Myocardial infarct size (IS) was significantly reduced in rapamycin-treated mice (13.3 ± 2.4 %) compared to DMSO vehicle control (35.9 ± 0.9 %) or WT mice (27.7 ± 1.1 %). Rapamycin treatment restored phosphorylation of STAT3 and enhanced AKT phosphorylation (target of mTORC2), but significantly reduced ribosomal protein S6 phosphorylation (target of mTORC1) in the diabetic heart. To determine the cause and effect relationship of STAT3 in cardioprotection, inducible cardiac-specific STAT3-deficient (MCM TG:STAT3(flox/flox)) and WT mice (MCM TG:STAT3(flox/flox)) were made diabetic by feeding high fat diet (HFD). Rapamycin given at reperfusion reduced IS in WT mice but not in STAT3-deficient mice following I/R. Moreover, cardiomyocytes isolated from HFD-fed WT mice showed resistance against necrosis (trypan blue staining) and apoptosis (TUNEL assay) when treated with rapamycin during reoxygenation following simulated ischemia. Such protection was absent in cardiomyocytes from HFD-fed STAT3-deficient mice. STAT3 signaling plays critical role in reducing IS and attenuates cardiomyocyte death following reperfusion therapy with rapamycin in diabetic heart.

Pin1 promotes neuronal death in stroke by stabilizing Notch intracellular domain.

Stroke is a leading cause of mortality and disability. The peptidyl-prolyl cis/trans isomerase Pin1 regulates factors involved in cell growth. Recent evidence has shown that Pin1 plays a major role in apoptosis. However, the role of Pin1 in ischemic stroke remains to be investigated. We used Pin1 overexpression and knockdown to manipulate Pin1 expression and explore the effects of Pin1 in cell death on ischemic stress in vitro and in a mouse stroke model. We also used Pin 1 inhibitor, γ-secretase inhibitor, Notch1 intracellular domain (NICD1)-deleted mutant cells, and Pin1 mutant cells to investigate the underlying mechanisms of Pin1-NICD1-mediated cell death. Our findings indicate that Pin1 facilitates NICD1 stability and its proapoptotic function following ischemic stroke. Thus, overexpression of Pin1 increased NICD1 levels and enhanced its potentiation of neuronal death in simulated ischemia. By contrast, depletion or knockout of Pin1 reduced the NICD1 level, which in turn desensitized neurons to ischemic conditions. Pin1 interacted with NICD1 and increased its stability by inhibiting FBW7-induced polyubiquitination. We also demonstrate that Pin1 and NICD1 levels increase following stroke. Pin1 heterozygous (+/-) and knockout (-/-) mice, and also wild-type mice treated with an inhibitor of Pin1, each showed reduced brain damage and improved functional outcomes in a model of focal ischemic stroke. These results suggest that Pin1 contributes to the pathogenesis of ischemic stroke by promoting Notch signaling, and that inhibition of Pin1 is a novel approach for treating ischemic stroke.

Abstract W P96: Evidence of In Vivo Ischemic LTP Caused by Cardiac Arrest and Cardiopulmonary Resuscitation

Objective: Experience-dependent memory acquisition, in vitro tetanic stimulation and simulated ischemia results in long-term potentiation (LTP) of hippocampal CA1 synapses. The goal of this study was to determine whether in vivo cerebral ischemia results in iLTP of CA1 synapses. We hypothesized that iLTP resulting from CA/CPR prevents physiological LTP. Methods: Cardiac arrest (8 minutes) followed by cardiopulmonary resuscitation (CA/CPR) or sham surgery was performed on adult (8-12 week) male mice. Electrophysiology was performed in hippocampal slices from sham and 7 and 30 days after CA/CPR. Field excitatory postsynaptic potentials (fEPSP) resulting from Schaffer collateral stimulation were recorded and rising slope was analyzed. Theta burst stimulation (TBS) was used for LTP induction and low frequency stimulation for reversal of LTP (depotentiation). Western blot analysis of synaptic isolations were performed from hippocampi collected from shams and 7 and 30 days after CA/CPR. Results: TBS resulted in an increased fEPSP to 164±9.8% (n=7, P&lt;0.05) of baseline in sham controls, however no increase was observed at 7 (106±15.9% n=4) and 30 days (109±13.9% n=7) after CA/CPR. A depotentiation stimulus had no effect in sham controls, but caused a reduction in fEPSP in control slices that received LTP induction and slices from CA/CPR mice. Importantly, LTP induction protocol applied subsequent to depotentiation stimulus resulted in physiological LTP to 126.9±11.8% (n=3) of the original baseline, an effective increase of approximately 40%. Glutamate receptor subunit GluR1 phosphorylation and expression in synaptic fractions were increased at 7 and 30 days after CA/CPR. Conclusions: Depotentiation in slices from CA/CPR mice suggests CA1 synapses are in a chronically potentiated state. Increased GluR1 phosphorylation and expression after CA/CPR also suggest in vivo ischemia causes iLTP. Our ability to induce physiological LTP after depotentiation in slices from CA/CPR suggests that reversing iLTP allows for normal synaptic plasticity. The results of this study are significant as they demonstrate in vivo iLTP and suggest a physiological stimulus has the potential to reverse synaptic impairments in the chronic phase after cerebral ischemia.

Sepsis protects the myocardium and other organs from subsequent ischaemic/reperfusion injury via a MAPK-dependent mechanism.

BackgroundSepsis has been shown to precondition the intact heart against ischaemia/reperfusion (IR) injury, and prior endotoxin exposure of cells in in vitro models has shown evidence of protection against subsequent simulated ischaemia. Our aim in this study is to validate these findings and further investigate the signaling pathways involved.MethodsAdult male Sprague Dawley rats were randomised to control (n = 7) or caecal ligation and perforation (CLP)-induced sepsis (n = 7). Hearts were harvested at 48 h, suspended in Langendorff mode and subjected to 30-min global ischaemia followed by 90-min reperfusion. In subsequent experiments, designed to determine the mechanisms by which sepsis protected against ischaemic injury, endotoxin-stimulated isolated cardiomyocytes, pulmonary A549 cells and renal HK2 cells were subjected to normoxic and hypoxic conditions. The roles of key pathways, including mitogen-activated protein (MAP) kinases extracellular-regulated protein kinase (ERK) 1/2, p38 MAPK (p38), c-Jun NH2-terminal protein kinase (JNK)), and nuclear factor-kappaB (NF-κB) were examined.ResultsSystemic sepsis protected isolated hearts from subsequent ischaemic/reperfusion-induced injury, enhancing functional recovery on reperfusion [developed left ventricular pressure ((d)LVP) mean(SE) 66.63(±10.7) mmHg vs. 54.13(±9.9) mmHg; LVPmax at 60 min 67.29(±11.9) vs. 72.48(±9.3), sepsis vs. control] despite significantly reduced baseline LV function in CLP animals (p < 0.001). Septic preconditioning significantly reduced infarct size after IR injury (p < 0.05). Endotoxin exposure protected isolated cardiomyocytes against hypoxia-induced cell death (p < 0.001). This effect appeared mediated in part via the p38, JNK and NF-κB pathways, but was independent of the ERK pathway, and did not appear to be mediated via HMGB1. The preconditioning effect of endotoxin was also demonstrated in isolated kidney and lung cells, suggesting that this preconditioning effect of sepsis is not confined to the myocardium.ConclusionsSepsis preconditions the isolated rat heart against myocardial IR injury. These effects appeared to be mediated in part via the p38, JNK and NF-κB and pathways, but were independent of the ERK and HMGB pathways.

Hydrostatic pressure does not cause detectable changes in survival of human retinal ganglion cells.

PurposeElevated intraocular pressure (IOP) is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP). The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC) survival in the human retina was investigated.MethodsA chamber was designed to expose cells to increased HP (constant and fluctuating). Accurate pressure control (10-100mmHg) was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs) from donor eyes (<24h post mortem) were cultured in serum-free DMEM/HamF12. Increased HP was compared to simulated ischemia (oxygen glucose deprivation, OGD). Cell death and apoptosis were measured by LDH and TUNEL assays, RGC marker expression by qRT-PCR (THY-1) and RGC number by immunohistochemistry (NeuN). Activated p38 and JNK were detected by Western blot.ResultsExposure of HORCs to constant (60mmHg) or fluctuating (10-100mmHg; 1 cycle/min) pressure for 24 or 48h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100mmHg; 1 cycle/min) for 15, 30, 60 and 90min durations, whereas OGD (3h) increased activation of p38 and JNK, remaining elevated for 90min post-OGD.ConclusionsDirectly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina.