AMPK-Regulated and Akt-Dependent Enhancement of Glucose Uptake Is Essential in Ischemic Preconditioning-Alleviated Reperfusion Injury

Aims

Ischemic preconditioning (IPC) is a potent form of endogenous protection. However, IPC-induced cardioprotective effect is significantly blunted in insulin resistance-related diseases and the underlying mechanism is unclear. This study aimed to determine the role of glucose metabolism in IPC-reduced reperfusion injury.

Methods

Normal or streptozotocin (STZ)-treated diabetic rats subjected to 2 cycles of 5 min ischemia/5 min reperfusion prior to myocardial ischemia (30 min)/reperfusion (3 h). Myocardial glucose uptake was determined by ¹⁸F-fluorodeoxyglucose-positron emission tomography (PET) scan and gamma-counter biodistribution assay.

Results

IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (P<0.01) as evidenced by PET images and gamma-counter biodistribution assay in ischemia/reperfused rats. Meanwhile, myocardial translocation of glucose transporter 4 (GLUT4) to plasma membrane together with myocardial Akt and AMPK phosphorylation were significantly enhanced in preconditioned hearts. Intramyocardial injection of GLUT4 siRNA markedly decreased GLUT4 expression and blocked the cardioprotection of IPC as evidence by increased myocardial infarct size. Moreover, the PI3K inhibitor wortmannin significantly inhibited activation of Akt and AMPK, reduced GLUT4 translocation, glucose uptake and ultimately, depressed IPC-induced cardioprotection. Furthermore, IPC-afforded antiapoptotic effect was markedly blunted in STZ-treated diabetic rats. Exogenous insulin supplementation significantly improved glucose uptake via co-activation of myocardial AMPK and Akt and alleviated ischemia/reperfusion injury as evidenced by reduced myocardial apoptosis and infarction size in STZ-treated rats (P<0.05).

Conclusions

The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation in vivo. Augmented glucose uptake via co-activation of myocardial AMPK and Akt in reperfused myocardium is essential to IPC-alleviated reperfusion injury. This intrinsic metabolic modulation and cardioprotective capacity are present in STZ-treated hearts and can be triggered by insulin.     

Sphingosine kinase and sphingosine 1-phosphate in the heart: A decade of progress

Activation of sphingosine kinase/sphingosine 1-phosphate (SK/S1P)‐mediated signaling has emerged as a critical cardioprotective pathway in response to acute ischemia/reperfusion injury. S1P is released in both ischemic pre- and post-conditioning. Application of exogenous S1P to cultured cardiac myocytes subjected to hypoxia or treatment of isolated hearts either before ischemia or at the onset of reperfusion exerts prosurvival effects. Synthetic congeners of S1P such as FTY720 mimic these responses. Gene targeted mice null for the SK1 isoform whose hearts are subjected to ischemia/reperfusion injury exhibit increased infarct size and respond poorly either to ischemic pre- or postconditioning. Measurements of cardiac SK activity and S1P parallel these observations. Experiments in SK2 knockout mice have revealed that this isoform is necessary for survival in the heart. High density lipoprotein (HDL) is a major carrier of S1P, and studies of hearts in which selected S1P receptors have been inhibited implicate the S1P cargo of HDL in cardioprotection. Inhibition of S1P lyase, an endogenous enzyme that degrades S1P, also leads to cardioprotection. These observations have considerable relevance for future therapeutic approaches to acute and chronic myocardial injury. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

Ischemic preconditioning requires opening of pannexin-1/P2X7 channels not only during preconditioning but again after index ischemia at full reperfusion

Protection of the ex vivo rat heart from ischemia/reperfusion injury can be provided by ischemic preconditioning (IPC). Previous studies revealed that a complex of pannexin-1 with the P2X? receptor forms a channel during IPC that results in the release of cardioprotectants such as adenosine and sphingosine 1-phosphate (S1P) that bind to G-protein-coupled cell surface receptors triggering cardioprotective cell signaling pathways. Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X? receptors (brilliant blue G) are known to block IPC when administered at the time of preconditioning (Vessey et al. J Cardiovasc Pharmacol Ther 15:190, 2010). We now demonstrate that these same antagonists also block the cardioprotective effects of IPC when added after the index ischemia during full reperfusion. Likewise, addition at full reperfusion of binding antagonists to the endogenous cardioprotectants S1P (VPC) or adenosine (8-SPT) reduced the effectiveness of IPC. These data suggest that IPC has a component that requires the release of cardioprotectants via pannexin-1/P2X? channels not only during preconditioning phase but again during the early stages of reperfusion following the index ischemia. It was found that the level of cardioprotectant release required at reperfusion to achieve cardioprotection was lower when hearts had been preconditioned. Further, pharmacologic preconditioning with S1P or adenosine was also blocked at reperfusion by antagonists of the pannexin-1/P2X? channels indicating that pharmacologic preconditioning also requires opening of the channel at full reperfusion. In untreated hearts, key components of the PI3 kinase/Akt signaling pathway were revealed by western analysis to be lost during ischemia. This correlates with an inability to generate phospho-Akt at reperfusion. IPC prevents this loss and thereby primes the cell for response to cardioprotectants released at full reperfusion.     

Bakuchiol attenuates myocardial ischemia reperfusion injury by maintaining mitochondrial function: the role of silent information regulator 1

Ischemia reperfusion (IR) injury (IRI) is associated with poor prognoses in the settings of both cardiac surgery and ischemic heart disease and causes mitochondrial oxidative stress and cell death. Silent information regulator 1 (SIRT1), a member of the histone deacetylase family, exerts anti-IRI effects. Bakuchiol (BAK), an analog of resveratrol and a monoterpene phenol isolated from the seeds of Psoralea corylifolia (Leguminosae), protects tissues from injury. This study was designed to investigate the protective effects of BAK treatment in the setting of myocardial IRI and to elucidate the potential mechanism of those effects. Prior to induction of IR, isolated rat hearts or cardiomyocytes were exposed to BAK in either the absence or presence of the SIRT1 inhibitors Sirtinol and SIRT1 siRNA. BAK exerted cardioprotective effects, as evidenced by the improvements noted in cardiac function following ischemia, attenuated myocardial apoptosis, and changes in several biochemical parameters (including increases in the level of the anti-apoptotic protein Bcl2, decreases in the level of the pro-apoptotic protein Bax, and decreases in the cleaved Caspase 3 level). However, Sirtinol and SIRT1 siRNA each blocked BAK-induced cardioprotection by inhibiting SIRT1 signaling. Additionally, BAK significantly increased the activities of mitochondrial succinate dehydrogenase, cytochrome c oxidase, and mitochondrial superoxide dismutase and decreased the production of malondialdehyde. These findings suggested that BAK significantly attenuated IR-induced mitochondrial oxidative damage. However, Sirtinol and SIRT1 siRNA abolished BAK-dependent mitochondrial function. In summary, our results demonstrate that BAK treatment attenuates IRI by attenuating IR-induced mitochondrial oxidative damage via the activation of SIRT1/PGC-1α signaling.     

胰岛素保护缺血/再灌注心脏:PI3-K/Akt和JNKs信号通路间的交互作用

我们前期研究表明胰岛素可激活细胞内信号转导机制如磷脂酰肌醇3．激酶．蛋白激酶B．内皮型一氧化氮合酶．一氧化氮（P13-K-Akt-eNOS-NO）信号通路,减轻心肌缺血／再灌注（ischemia／reperfusion,I／R）损伤,改善缺血后心肌功能恢复。然而c-Jun氨基末端激酶（c-JunNH2-terminal kinase,JNK）信号通路在胰岛素保护I／R心肌中的作用尚不清楚,本研究旨在探讨JNK信号通路在胰岛素保护I／R心肌中的作用及其与P13．K／Akt信号通路间的相互关系。离体Sprague-Dawley大鼠心脏缺血30min后施行2h或4h的再灌注,缺血前用LY294002（15mmol／L）和SP600125（10mmol／L）灌注15min,分别阻断P13．K／Akt和磷酸化JNK（phosphorylated．JNK,p-JNK）活化,观测心脏功能、心肌梗死、细胞凋亡和蛋白磷酸化水平。与对照组相比,胰岛素再灌注2h后,心率、左心室发展压和左心室收缩／舒张最大速率均明显增加,梗死面积减少约16．1％[（28．9±2．0）％vs（45．0±4．0）％,n=6,P〈O．01],细胞凋亡指数从（27．6±113）％减少到（16．0±0．7）％（n=6,P〈O．01）,Akt的活性增加1．7倍（n=6,P〈0．05）,同时JNK活性增加1．5倍铆=6,P〈O．05）。用LY294002处理后,胰岛素对I／R心肌的保护作用消失;而用SP600125处理可增强胰岛素的保护作用,且可部分逆转LY294002的抑制作用。进一步观察发现SP600125减弱了Akt的磷酸化m=6,P〈0．05）。上述结果表明,在I／R心肌中,胰岛素可同时激活P13．K／Akt及JNK信号通路,且通过后者进一步增加Akt活化,从而减轻I／R损伤,改善心肌功能。这种P13．K／Akt与JNK信号通路交互机制对胰岛素保护I／R心肌有重要意义。     

SIRT1 activation by curcumin pretreatment attenuates mitochondrial oxidative damage induced by myocardial ischemia reperfusion injury

Ischemia reperfusion (IR) injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Silent information regulator 1 (SIRT1), a type of histone deacetylase, contributes to IRI. Curcumin (Cur) is a strong natural antioxidant and is the active component in Curcuma longa; Cur has protective effects against IRI and may regulate the activity of SIRT1. This study was designed to investigate the protective effect of Cur pretreatment on myocardial IRI and to elucidate this potential mechanism. Isolated and in vivo rat hearts and cultured neonatal rat cardiomyocytes were subjected to IR. Prior to this procedure, the hearts or cardiomyocytes were exposed to Cur in the absence or presence of the SIRT1 inhibitor sirtinol or SIRT1 siRNA. Cur conferred a cardioprotective effect, as shown by improved postischemic cardiac function, decreased myocardial infarct size, decreased myocardial apoptotic index, and several biochemical parameters, including the up-regulation of the antiapoptotic protein Bcl2 and the down-regulation of the proapoptotic protein Bax. Sirtinol and SIRT1 siRNA each blocked the Cur-mediated cardioprotection by inhibiting SIRT1 signaling. Cur also resulted in a well-preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase activity, and decreased formation of mitochondrial hydrogen peroxide and malondialdehyde. These observations indicated that the IR-induced mitochondrial oxidative damage was remarkably attenuated. However, this Cur-elevated mitochondrial function was reversed by sirtinol or SIRT1 siRNA treatment. In summary, our results demonstrate that Cur pretreatment attenuates IRI by reducing IR-induced mitochondrial oxidative damage through the activation of SIRT1 signaling.     

Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning

We have previously reported that the prolonged transient acidosis during early reperfusion mediates the cardioprotective effects in canine hearts. Recently, postconditioning has been shown to be one of the novel strategies to mediate cardioprotection. We tested the contribution of the prolonged transient acidosis to the cardioprotection of postconditioning. Open-chest anesthetized dogs subjected to 90-min occlusion of the left anterior descending coronary artery and 6-h reperfusion were divided into four groups: 1) control group; no intervention after reperfusion (n = 6); 2) postconditioning (Postcon) group; four cycles of 1-min reperfusion and 1-min reocclusion (n = 7); 3) Postcon + sodium bicarbonate (NaHCO(3)) group; four cycles of 1-min reperfusion and 1-min reocclusion with the administration of NaHCO(3) (n = 8); and 4) NaHCO(3) group; administration of NaHCO(3) without postconditioning (n = 6). Infarct size, the area at risk (AAR), collateral blood flow during ischemia, and pH in coronary venous blood were measured. The phosphorylation of Akt and extracellular signal-regulated kinase (ERK) in ischemic myocardium was assessed by Western blot analysis. Systemic hemodynamic parameters, AAR, and collateral blood flow were not different among the four groups. Postconditioning induced prolonged transient acidosis during the early reperfusion phase. Administration of NaHCO(3) completely abolished the infarct size-limiting effects of postconditioning. Furthermore, the phosphorylation of Akt and ERK in ischemic myocardium induced by postconditioning was also blunted by the cotreatment of NaHCO(3). In conclusion, postconditioning mediates its cardioprotective effects possibly via prolonged transient acidosis during the early reperfusion phase with the activation of Akt and ERK.     

Protective effect of ginsenoside Rb1 against myocardial ischemia/reperfusion injury in streptozotocin-induced diabetic rats

The objective of the current study is to investigate whether ginsenoside Rb1, a major pharmacological extract of ginseng that could attenuate myocardial ischemia reperfusion (MI/R) injury in non-diabetic myocardium, can attenuate MI/R injury in diabetes that are more vulnerable to ischemic insult. Rats were divided into seven groups: (i) diabetic sham, (ii) diabetic, (iii) normal, (iv) diabetic + ginsenoside Rb1, (v) diabetic + wortmannin, (vi) diabetic + wortmannin + ginsenoside Rb1, (vii) diabetic sham + wortmannin. Ginsenoside Rb1 and/or wortmannin were administered prior to inducing MI/R (30 min of coronary artery occlusion followed by 120 min reperfusion). At the end of the experiment, postischemic myocardial infarct size was significantly higher in the diabetic untreated group as compared to normal (P < 0.05), accompanied with increased myocardial apoptosis, elevated plasma CK-MB and LDH release and reduced blood pressure. Ginsenoside Rb1 reduced infarct size, cardiomyocyte apoptosis and caspase-3 activity compared to the diabetic group. The cardioprotective effects of ginsenoside Rb1 were cancelled by wortmannin. Ginsenoside Rb1 significantly upregulated phosphorylated Akt expression, which was attenuated by wortmannin. Ginsenoside Rb1 exerts cardioprotective effects against MI/R injury in diabetic rats, which is partly through activation of phosphatidylinositol 3-kinase (PI3 K)/Akt pathway. Thus this study shows a novel pharmacological preconditioning with ginsenoside Rb1 in the diabetic myocardium.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α₂-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α₂-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α₂-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α₂-adrenergic receptor stimulation.     

Akt activation induced by an antioxidant compound during ischemia-reperfusion

Molecular mechanisms of cardioprotection afforded by modified mexiletine compounds were investigated during ischemia-reperfusion (IR) in Langendorff perfused hearts. Rat hearts were subjected to a global 25 min ischemia followed by reperfusion, either untreated or treated with mexiletine, or three substituted mexiletine derivates (5 muM). A modified mexiletine derivative (H-2693) promoted best the recovery of myocardial energy metabolism (assessed by (31)P NMR spectroscopy) compared to untreated and mexiletine-treated hearts. H-2693 also preserved cardiac contractile function and attenuated the IR-induced lipid peroxidation (TBARS formation) and protein oxidation (carbonyl content). Western blot revealed that H-2693 propagated the phosphorylation of Akt (activation) and its downstream substrate glycogen synthase kinase-3beta (GSK-3beta, inactivation) compared to untreated IR. Parallel treatment with the phosphatidylinositol-3-kinase (upstream activator of Akt) inhibitor wortmannin (100 nM) abolished the beneficial effects of H-2693 on energetics and function, and reduced Akt and GSK-3beta phosphorylation. As a result of the antiapoptotic impacts of Akt activation, H-2693 decreased caspase-3 activity, which was neutralized by wortmannin. Here we first demonstrated that a free radical-entrapping compound could activate the prosurvival Akt pathway beyond its proven ability to scavenge reactive oxygen species. In conclusion, the favorable influence of H-2693 on signaling events during IR may have considerably contributed to its cardioprotective effect.     

Uncoupled eNOS annihilates neuregulin-1β-induced cardioprotection: a novel mechanism in pharmacological postconditioning in myocardial infarction

Myocardial infarct size can be limited by pharmacological postconditioning (pPC) with cardioprotective agents. Cardioprotective effects of neuregulin-1β (NRG) via activation of protein kinase B (Akt) and downstream pathways like endothelial nitric oxide synthase (eNOS) have been postulated based on results from cell culture experiments. The purpose of this study was to investigate if eNOS may be involved in pPC with NRG. NRG application in an ex vivo mouse model (C57Bl6) of ischemia–reperfusion injury was analyzed. Unexpectedly, the infarct size increased when NRG was infused starting 5 min prior to reperfusion, even though protective Akt and GSK3β phosphorylation were enhanced. In eNOS deficient mice, however, NRG significantly reduced the infarct size. Co-infusion of NRG and l-arginine (Arg) lead to a reduction in infarct size in wild type animals. Electron paramagnetic resonance measurements revealed that NRG treatment prior to reperfusion leads to an enhanced release of reactive oxygen species compared to controls and this effect is blunted by co-infusion of Arg. This study documents the cardioprotective mechanisms of NRG signaling to be mediated by GSK3β inactivation. This is the first study to show that this protection fails in situations with dysfunctional eNOS. In eNOS deficient mice NRG exerts its protective effect via the GSK3β pathway, suggesting that the eNOS can limit cardioprotection. As dysfunctional eNOS has been described in cardiovascular risk factors like diabetes, hypertension, and hypercholesterolemia these findings can help to explain lack of postconditioning performance in models of cardiovascular co-morbidities.     

Effects of increased accumulation of doxorubicin due to emodin on efflux transporter and LRP1 expression in lung adenocarcinoma and colorectal carcinoma cells

We investigated the eplerenone-induced, PI3K/Akt- and GSK-3β-mediated cardioprotection against ischemia/reperfusion (I/R) injury in diabetic rats. The study groups comprising diabetic rats were treated for 14 days with 150 mg/kg/day eplerenone orally and 1 mg/kg wortmannin (PI3K/Akt antagonist) intraperitoneally with eplerenone. On the 15th day, the rats were exposed to I/R injury by 20-min occlusion of the left anterior descending coronary artery followed by 30 min of reperfusion. The hearts were processed for biochemical, molecular, and histological investigations. The I/R injury in diabetic rats inflicted a significant rise in the oxidative stress and apoptosis along with a decrease in the arterial and ventricular function and the expressions of PI3K/Akt and GSK-3β proteins. Eplerenone pretreatment reduced the arterial pressure, cardiac inotropy, and lusitropy. It significantly reduced apoptosis and cardiac injury markers. The histology revealed cardioprotection in eplerenone-treated rats. Eplerenone up-regulated the PI3K/Akt and reduced the GSK-3β expression. The group receiving wortmannin with eplerenone was deprived eplerenone-induced cardioprotection. Our results reveal the eplerenone-induced cardioprotection against I/R injury in diabetic rats and substantiate the involvement of PI3K/Akt and GSK-3β pathways in its efficacy.     

The β subunit of soluble guanylyl cyclase GUCY1B3 exerts cardioprotective effects against ischemic injury via the PKCε/Akt pathway

The soluble form of guanylyl cyclase (sGC) is the main receptor for the signaling agent nitric oxide (NO), which regulates cardiomyocyte contractile function and attenuates cardiomyocyte hypertrophy. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), a regulator of vascular tone, and cardiac NO-sGC-cGMP signaling modulates cardiac stress responses, including ischemia and reperfusion (IR) injury. Here, we investigated the role of GUCY1B3 (the β subunit of sGC) in cardiomyocyte IR injury and myocardial infarction (MI) in vitro and in vivo. GUCY1B3 was upregulated in neonatal rat ventricular myocytes in response to IR injury, and GUCY1B3 overexpression restored IR-induced cell death and apoptosis. Treatment with specific inhibitors of PKCδ, PKCε, and Akt suggested that the protective effects of GUCY1B3 were mediated by PKCε/Akt signaling. In a mouse model of coronary artery ligation-induced MI, GUCY1B3 silencing aggravated MI-induced cardiac dysfunction and increased infarct size and exacerbated cardiomyocyte apoptosis in association with the inactivation of PKCε and Akt. Our results suggest that GUCY1B3 exerts cardioprotective effects through the modulation of the PKCε/Akt activity and identify a potential mechanism involved in NO-sGC-cGMP signaling in the heart.     

RISK pathway is involved in oxytocin postconditioning in isolated rat heart

The reperfusion injury salvage kinase (RISK) pathway is a fundamental signal transduction cascade in the cardioprotective mechanism of ischemic postconditioning. In the present study, we examined the cardioprotective role of oxytocin as a postconditioning agent via activation of the RISK pathway (PI3K/Akt and ERK1/2).Animals were randomly divided into 6 groups. The hearts were subjected under 30 minutes (min) ischemia and 100 min reperfusion. OT was perfused 15 min at the early phase of reperfusion. RISK pathway inhibitors (Wortmannin; an Akt inhibitor, PD98059; an ERK1/2 inhibitor) and Atosiban (an OT receptor antagonist) were applied either alone 10 min before the onset of the ischemia or in the combination with OT during early reperfusion phase. Myocardial infarct size, hemodynamic factors, ventricular arrhythmia, coronary flow and cardiac biochemical marker were measured at the end of reperfusion.OT postconditioning (OTpost), significantly decreased the infarct size, arrhythmia score, incidence of ventricular fibrillation, Lactate dehydrogenase and it increased coronary flow. The cardioprotective effect of OTpos was abrogated by PI3K/Akt, ERK1/2 inhibitors and Atosiban.Our data have shown that OTpost can activate RISK pathway mostly via the PI3K/Akt and ERK1/2 signaling cascades during the early phase of reperfusion.     

Sex differences in the mechanism of Met5-enkephalin-induced cardioprotection: role of PI3K/Akt

Met(5)-enkephalin (ME)-induced cardioprotection occurs via epidermal growth factor receptor (EGFR) transactivation with the subsequent activation of phosphatidylinositol 3-kinase (PI3K). In the present study, we investigated whether there is a sex difference in ME-elicited PI3K signaling. Neonatal murine cardiomyocytes were isolated by collagenase digestion and subjected to 90 min hypoxia and 180 min reoxygenation at 37 degrees C (n = 5 to 7 replicates). PI3K/Akt signaling was interrogated using pharmacological inhibitors and small interfering RNA (siRNA). Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. The data are presented as means +/- SE. There was not a sex difference in the basal content of total Akt. ME (100 microM) elicited comparable protection in both sexes. Wortmannin and the nonselective Akt inhibitor IV completely abolished ME-induced protection in male cardiomyocytes but only attenuated protection in female cardiomyocytes. Isoform-selective knockdown of Akt in males with siRNAs against Akt1/2 completely abolished ME-induced cardioprotection, whereas the siRNAs against Akt3 only attenuated protection of approximately 40%. In contrast, in females the siRNAs against Akt1/2 attenuated and against Akt3 eliminated ME-induced cardioprotection. There is not a sex difference in the degree of ME-induced protection, and there is a sex difference in the cardioprotective signaling pathways after the administration of ME; ME-induced cardioprotection in males primarily utilizes a PI3K/Akt1/2 pathway and in females primarily utilizes a PI3K/Akt3 pathway. The incomplete loss of protection in females following the blockade of PI3K suggests that additional factors may facilitate the maintenance or function of activated Akt.     

Akt and Erk1/2 activate the ornithine decarboxylase/polyamine system in cardioprotective ischemic preconditioning in rats: the role of mitochondrial permeability transition pores

Ornithine decarboxylase (ODC) is the first rate-limiting enzyme in polyamine biosynthesis, which is essential for cell survival. We hypothesized that the ODC/polyamine system is involved in ischemic preconditioning (IPC)-mediated cardioprotection through the activation of Erk1/2 and Akt and through the inhibition of the mitochondrial permeability transition (mPT). Isolated rat hearts were subjected to 40 min of ischemia either with or without IPC (3 cycles of 5-min global ischemia), and ODC protein expression, polyamine content, and Akt and Erk1/2 phosphorylation were evaluated after 30 min of reperfusion. IPC significantly upregulated the ODC/polyamine pathway, promoted Erk1/2 and Akt phosphorylation, and reduced the infarct size and heart dysfunction after reperfusion. An inhibitor of ODC, α-difluoromethylornithine (DFMO), abolished the IPC-induced cardioprotection. Moreover, the inhibition of the IPC-induced activation of Erk1/2 and Akt using PD98059 or wortmannin downregulated the ODC/polyamine system. In separate studies, the Ca²⁺ load required to open the mPT pore was significantly lower in DFMO-treated cardiac mitochondria than in mitochondria from IPC hearts. Furthermore, spermine or spermidine significantly inhibited the mPT induced by CaCl₂. These results suggest that IPC upregulates the ODC/polyamine system and mediates preconditioning cardioprotection, which may depend on the phosphorylation/activation of Erk1/2 and Akt and on the inhibition of the mPT during reperfusion.     

Sirtuin 1 (SIRT1) Activation Mediates Sildenafil Induced Delayed Cardioprotection against Ischemia-Reperfusion Injury in Mice

Background

It has been well documented that phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. SIRT1 is part of the class III Sirtuin family of histone deacetylases that deacetylates proteins involved in cellular stress response including those related to I-R injury.

Objective/Hypothesis

We tested the hypothesis that SIL-induced cardioprotection may be mediated through activation of SIRT1.

Methods

Adult male ICR mice were treated with SIL (0.7 mg/kg, i.p.), Resveratrol (RSV, 5 mg/kg, a putative activator of SIRT1 used as the positive control), or saline (0.2 mL). The hearts were harvested 24 hours later and homogenized for SIRT1 activity analysis.

Results

Both SIL- and RSV-treated mice had increased cardiac SIRT1 activity (P<0.001) as compared to the saline-treated controls 24 hours after drug treatment. In isolated ventricular cardiomyocytes, pretreatment with SIL (1 µM) or RSV (1 µM) for one hour in vitro also upregulated SIRT1 activity (P<0.05). We further examined the causative relationship between SIRT1 activation and SIL-induced late cardioprotection. Pretreatment with SIL (or RSV) 24 hours prior to 30 min ischemia and 24 hours of reperfusion significantly reduced infarct size, which was associated with a significant increase in SIRT1 activity (P<0.05). Moreover, sirtinol (a SIRT1 inhibitor, 5 mg/kg, i.p.) given 30 min before I-R blunted the infarct-limiting effect of SIL and RSV (P<0.001).

Conclusion

Our study shows that activation of SIRT1 following SIL treatment plays an essential role in mediating the SIL-induced cardioprotection against I-R injury. This newly identified SIRT1-activating property of SIL may have enormous therapeutic implications.     

Activation of the JNK pathway is important for cardiomyocyte death in response to simulated ischemia

Multiple signaling pathways, including the c-Jun N-terminal kinase (JNK) pathway, are activated in myocardial ischemia and reperfusion (MI/R) and correlate with cell death. However, the role of the JNK pathway in MI/R-induced cell death is poorly understood. In a rabbit model, we found that ischemia followed by reperfusion resulted in JNK activation which could be detected in cytosol as well as in mitochondria. To address the functional role of the JNK activation, we examined the consequences of blockade of JNK activation in isolated cardiomyocytes under conditions of simulated ischemia. The JNK activity was stimulated approximately sixfold by simulated ischemia and reperfusion (simulated MI). When a dominant negative mutant of JNK kinase-2 (dnJNKK2), an upstream regulator of JNK, and JNK-interacting protein-1 (JIP-1) were expressed in myocytes by recombinant adenovirus, the activation of JNK by simulated MI was reduced 53%. Furthermore, the TNFalpha-activated JNK activity in H9c2 cells was completely abolished by dnJNKK2 and JIP-1. In correlation, when dnJNKK2 and JIP-1 were expressed in cardiomyocytes, both constructs significantly reduced cell death after simulated MI compared to vector controls. We conclude that activation of the JNK cascade is important for cardiomyocyte death in response to simulated ischemia.