磷脂酰肌醇3激酶信号通路在缺血后适应中对大鼠心肌保护机制的研究

目的研究缺血后适应(IPost)对大鼠心肌保护作用及磷脂酰肌醇3激酶(PI3K/Akt)信号通路机制。方法将32只雄性Wistar大鼠随机分为缺血再灌注组(A组),IPost组(B组),IPost+Wortmannin组(C组)和缺血再灌注+SB216763组(D组),每组8只。测定各组左心室收缩压(LVSP)和再灌注30 min冠状动脉流出液中乳酸脱氢酶和肌酸激酶含量。并测定心肌梗死面积,对心肌进行免疫组织化学染色,观察Akt磷酸化和GSK-3β磷酸化的表达。结果与B组比较,A组LVSP明显降低,乳酸脱氢酶和肌酸激酶含量明显升高(P0.05);同时IPost干预减小了心肌梗死面积(47.3% vs 29.5%),B组Akt磷酸化和GSK-3β磷酸化表达增加。结论 IPost对体外大鼠缺血再灌注损伤有明确的保护作用。Wortmannin可削弱IPost的保护作用,SB216763具有模拟IPost的心肌保护作用,Akt和GSK-3β的磷酸化水平在IPost的心肌保护作用信号通道传导机制中具有重要地位。     

The RIPOST-MI study, assessing remote ischemic perconditioning alone or in combination with local ischemic postconditioning in ST-segment elevation myocardial infarction

Local ischemic postconditioning (IPost) and remote ischemic perconditioning (RIPer) are promising cardioprotective therapies in ST-elevation myocardial infarction (STEMI). We aimed: (1) to investigate whether RIPer initiated at the catheterization laboratory would reduce infarct size, as measured using serum creatine kinase-MB isoenzyme (CK-MB) release as a surrogate marker; (2) to assess if the combination of RIPer and IPost would provide an additional reduction. Patients (n = 151) were randomly allocated to one of the following groups: (1) control group, percutaneous transluminal coronary angioplasty (PTCA) alone; (2) RIPer group, PTCA combined with RIPer, consisting of three cycles of 5-min inflation and 5-min deflation of an upper-arm blood-pressure cuff initiated before reperfusion; (3) RIPer+IPost group, PTCA combined with RIPer and IPost, consisting of four cycles of 1-min inflation and 1-min deflation of the angioplasty balloon. The CK-MB area under the curve (AUC) over 72 h was reduced in RIPer, and RIPer+IPost groups, by 31 and 29 %, respectively, compared to the Control group; however, CK-MB AUC differences between the three groups were not statistically significant (p = 0.06). Peak CK-MB, CK-MB AUC to area at risk (AAR) ratio, and peak CK-MB level to AAR ratio were all significantly reduced in the RIPer and RIPer+IPost groups, compared to the Control group. On the contrary, none of these parameters was significantly different between RIPer+IPost and RIPer groups. To conclude, starting RIPer therapy immediately prior to revascularization was shown to reduce infarct size in STEMI patients, yet combining this therapy with an IPost strategy did not lead to further decrease in infarct size.     

Diabetes mellitus abrogates erythropoietin-induced cardioprotection against ischemic-reperfusion injury by alteration of the RISK/GSK-3β signaling

Recent studies reported cardioprotective effects of erythropoietin (EPO) against ischemia–reperfusion (I/R) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been reported to be impaired in diabetes and insulin resistance syndrome, we examined whether EPO-induced cardioprotection was maintained in rat models of type 1 diabetes and insulin resistance syndrome. Isolated hearts were obtained from three rat cohorts: healthy controls, streptozotocin (STZ)-induced diabetes, and high-fat diet (HFD)-induced insulin resistance syndrome. All hearts underwent 25 min ischemia and 30 min or 120 min reperfusion. They were assigned to receive either no intervention or a single dose of EPO at the onset of reperfusion. In hearts from healthy controls, EPO decreased infarct size (14.36 ± 0.60 and 36.22 ± 4.20% of left ventricle in EPO-treated and untreated hearts, respectively, p < 0.05) and increased phosphorylated forms of Akt, ERK1/2, and their downstream target GSK-3β. In hearts from STZ-induced diabetic rats, EPO did not decrease infarct size (32.05 ± 2.38 and 31.88 ± 1.87% in EPO-treated and untreated diabetic rat hearts, respectively, NS) nor did it increase phosphorylation of Akt, ERK1/2, and GSK-3β. In contrast, in hearts from HFD-induced insulin resistance rats, EPO decreased infarct size (18.66 ± 1.99 and 34.62 ± 3.41% in EPO-treated and untreated HFD rat hearts, respectively, p < 0.05) and increased phosphorylation of Akt, ERK1/2, and GSK-3β. Administration of GSK-3β inhibitor SB216763 was cardioprotective in healthy and diabetic hearts. STZ-induced diabetes abolished EPO-induced cardioprotection against I/R injury through a disruption of upstream signaling of GSK-3β. In conclusion, direct inhibition of GSK-3β may provide an alternative strategy to protect diabetic hearts against I/R injury.     

Reperfusion injury salvage kinase signalling: taking a RISK for cardioprotection

Following an acute myocardial infarction (AMI), early coronary artery reperfusion remains the most effective means of limiting the eventual infarct size. The resultant left ventricular systolic function is a critical determinant of the patient’s clinical outcome. Despite current myocardial reperfusion strategies and ancillary antithrombotic and antiplatelet therapies, the morbidity and mortality of an AMI remain significant, with the number of patients developing cardiac failure increasing, necessitating the development of novel strategies for cardioprotection which can be applied at the time of myocardial reperfusion to reduce myocardial infarct size. In this regard, the Reperfusion Injury Salvage Kinase (RISK) Pathway, the term given to a group of pro-survival protein kinases (including Akt and Erk1/2), which confer powerful cardioprotection, when activated specifically at the time of myocardial reperfusion, provides an amenable pharmacological target for cardioprotection. Preclinical studies have demonstrated that an increasing number of agents including insulin, erythropoietin, adipocytokines, adenosine, volatile anesthetics natriuretic peptides and ‘statins’, when administered specifically at the time of myocardial reperfusion, reduce myocardial infarct size through the activation of the RISK pathway. This recruits various survival pathways that include the inhibition of mitochondrial permeability transition pore opening. Interestingly, the RISK pathway is also recruited by the cardioprotective phenomena of ischemic preconditioning (IPC) and postconditioning (IPost), enabling the use of pharmacological agents which target the RISK pathway, to be used at the time of myocardial reperfusion, as pharmacological mimetics of IPC and IPost. This article reviews the origins and evolution of the RISK pathway, as part of a potential common cardioprotective pathway, which can be activated by an ever-expanding list of agents administered at the time of myocardial reperfusion, as well as by IPC and IPost. Preliminary clinical studies have demonstrated myocardial protection with several of these pharmacological activators of the RISK pathway in AMI patients undergoing PCI. Through the use of appropriately designed clinical trials, guided by the wealth of existing preclinical data, the administration of pharmacological agents which are known to activate the RISK pathway, when applied as adjuvant therapy to current myocardial reperfusion strategies for patients presenting with an AMI, should lead to improved clinical outcomes in this patient group.     

Interplay Between SAFE and RISK Pathways in Sphingosine-1-Phosphate–Induced Cardioprotection

Purpose

We studied the role of two powerful molecular signalling mechanisms involved in the cardioprotective effect of sphingosine-1-phosphate (S1P), a major component of high density lipoprotein (HDL) against myocardial ischaemic-reperfusion injury, namely the RISK pathway (Akt/Erk), including its downstream target FOXO-1 and, the SAFE pathway (TNF/STAT-3).

Methods

Control hearts from wildtype, TNF deficient (TNF^?/?) or cardiomyocyte STAT-3 deficient (STAT-3^?/?) male mice were perfused on a Langendorff apparatus (35?min global ischaemia and 45?min reperfusion). S1P (10 nM) was given at the onset of reperfusion for the first 7?min, with/without STAT-3 or Akt inhibitors, AG490 and wortmannin (W), respectively.

Results

S1P reduced myocardial infarct size in wildtype hearts (39.3?±?4.4% in control vs 17.3?±?3.1% in S1P-treated hearts; n????6; p??/? or TNF^?/? mice (34.2?±?4.3% in STAT-3^?/? and 34.1?±?2.0% in TNF^?/? mice; n????6; p?=?ns vs. their respective control). Both STAT-3 and Akt inhibitors abolished the protective effects of S1P (33.7?±?3.3% in S1P?+?AG490 and 36.6?±?4.9% in S1P?+?W; n?=?6; p?=?ns vs. their respective control). Increased nuclear levels of phosphorylated STAT-3 (pSTAT-3), Akt and FOXO-1 were observed at 15?min reperfusion in wildtype mice with Western Blot analysis (53% STAT-3, 47% Akt, 41% FOXO-1; p?Conclusions In conclusion, S1P activates both the SAFE and RISK pathways, therefore suggesting a dual protective signalling in S1P-induced cardioprotection.     

The reperfusion injury salvage kinase pathway: a common target for both ischemic preconditioning and postconditioning

Novel treatment approaches, as potential adjunctive therapy to current reperfusion strategies (such as thrombolysis, primary coronary angioplasty, and cardiac surgery), are required to provide further cardioprotection in the setting of an acute myocardial infarction to effect further reductions in morbidity and mortality. In this regard, the activation of prosurvival kinases, such as Akt and Erk1/2 (which we have termed the reperfusion injury salvage kinase [RISK] pathway), at the time of reperfusion, has been demonstrated to confer powerful cardioprotection against myocardial ischemia-reperfusion injury. In this review, we present evidence suggesting that the cardioprotective phenomena of ischemic preconditioning and the recently described ischemic postconditioning exert their cardioprotective effects through the recruitment of the RISK pathway, at the time of reperfusion, and that the protection in these settings is mediated through the inhibition of mitochondrial permeability transition pore (mPTP) opening at this time. Therefore, the pharmacologic manipulation of the RISK pathway at the time of reperfusion may enable one to harness the powerful cardioprotective benefits of both ischemic preconditioning and postconditioning, and provide a novel approach to cardioprotection.     

Postconditioning protects human atrial muscle through the activation of the RISK pathway

Ischemic postconditioning (IPost) has been demonstrated to reduce myocardial injury in patients undergoing primary coronary angioplasty for an acute myocardial infarction.Pre-clinical animal studies suggest that pro-survival protein kinases of the Reperfusion Injury Salvage Kinase (RISK) pathway such as Akt and Erk1/2 mediate the cardioprotective effect of IPost.Whether IPost can protect human myocardial tissue ex vivo and whether it recruits the RISK pathway in human myocardium are both not known. To investigate this, atrial appendages were harvested from patients undergoing cardiac surgery. From these samples atrial trabeculae were isolated and mounted on a superperfusion apparatus and subjected to 90 min of hypoxia followed by 120 min of reoxygenation at the end of which function expressed as a percentage of the recovery of baseline contractile function was determined.Atrial trabeculae were randomized to control, hypoxic preconditioning (HPre), hypoxic postconditioning comprising either four 30-s (HPost-30) or 60-s (HPost-60) episodes of alternating hypoxia and reoxygenation, and HPost in the presence or absence of UO126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor).HPre and HPost-60 improved the recovery of baseline contractile function (45.4±3.2% with HPre and 45.2±2.2% with HPost-60 vs 26.7±2.1 % in control: N≥ 6/group: P<0.05), whereas HPost-30 failed to cardioprotect (28.3±3.4% with HPost-30 vs 26.7±2.1 % in control: N≥ 6/group: P>0.05). The cardioprotective effect of HPost-60 was abolished in the presence of either LY (28.1±2.5% with HPost-60+LY vs 45.2±2.2% with HPost-60: N≥ 6/group: P<0.05) or UO (32.7±1.8% with HPost-60+UO vs 45.2±2.2% with HPost-60:N=7/group: P<0.05). The kinase inhibitors alone had no effect on functional recovery (28.2±3.6% with LY and 30.1±4.8% with UO vs 26.7±2.1 % in control: N≥ 5/group: P>0.05). In conclusion, we demonstrate for the first time that postconditioning protects human myocardium ex vivo and that this effect is dependent on the activation of the RISK pathway.     

Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.     

Attenuation of myocardial injury by postconditioning: role of hypoxia inducible factor-1α

Postconditioning (PostC) has regenerated interest as a mechanical intervention against myocardial ischemia/reperfusion injury, but its molecular mechanisms remain elusive. This study tested the hypothesis that hypoxia inducible factor-1α (HIF-1α) plays a role in PostC-induced cardioprotection. Male Wistar rats were subjected to 30 min ischemia followed by 3 h of reperfusion (Control). PostC with 3 cycles of 10 s reperfusion and 10 s re-occlusion was applied at the onset of reperfusion. Relative to the Sham group, HIF-1α protein level was increased by 2.9-fold in the Control group, but its level was enhanced by 5.8-fold with PostC (P < 0.01 vs. Control). However, HIF-1α protein level was further augmented by 2.0-fold and 3.3-fold, respectively, when the prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG, 40 mg/kg, i.p.) was given at 24 h before ischemia in both Control and PostC groups. PostC reduced infarct size by 24% compared with the Control (27 ± 4.2% vs. 36 ± 5.2%, P < 0.01), consistent with significant lower levels of plasma creatine kinase activity, index of cardiomyocyte apoptosis and caspase-3 activity. Although pretreatment with DMOG significantly reduced infarct size relative to the Control, the infarct-sparing effect of PostC was remarkably enhanced when DMOG was given before PostC (18 ± 2.0% vs. 27 ± 4.2% in PostC alone, P < 0.05). There was a significant linear inverse relationship between HIF-1α protein level and infarct size (r = −0.799, P < 0.01) among all groups. Furthermore, along with up-regulated HIF-1α expression, the levels of iNOS mRNA and protein were significantly increased in the PostC alone and DMOG plus PostC groups. In conclusion, these data suggest that HIF-1α is involved in cardioprotection by PostC and pharmacological augmentation of HIF-1α expression that enhances the infarct-sparing effect of PostC; iNOS, the downstream gene of HIF-1α, may participate in signaling pathways in mediating PostC’s protection.     

Ginsenoside Rb1 Preconditioning Protects Against Myocardial Infarction After Regional Ischemia and Reperfusion by Activation of Phosphatidylinositol-3-kinase Signal Transduction

Background  Ginsenoside Rb1, a major bioactive component of Panax ginseng, bears various beneficial effects on the cardiovascular system. This study investigated whether ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia–reperfusion injury and its potential mechanism. Methods  Rats subjected to 45 min of myocardial ischemia followed by 120 min of reperfusion were assigned to the following groups: sham-operated, ischemia–reperfusion (I/R), ginsenoside Rb1+I/R, wortmannin(a specific PI3K inhibitor)+I/R, wortmannin drug vehicle (dimethyl sulfoxide, DMSO), wortmannin+sham, ginsenoside Rb1+ wortmannin +I/R. Infarct size was assessed by triphenyltetrazolium chloride staining. Plasma creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), lactate dehydrogenase (LDH), and troponin T levels were also measured. Akt phosphorylation expression was assessed by immunoblotting. Results  Ginsenoside Rb1 preconditioning reduced infarct size compared with that in the I/R group: 30 ± 2.6% versus 51 ± 2.7% (p < 0.01). Ginsenoside Rb1 preconditioning also markedly reduced the plasma CK, CK-MB, LDH and troponin T levels in blood. Akt phosphorylation expression increased after ginsenoside Rb1 preconditioning. These effects of ginsenoside Rb1 preconditoning were significantly inhibited by wortmannin. Conclusion  This is the first study to demonstrate that ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia and reperfusion injury, partly by mediating the activation of the PI3K pathway and phosphorylation of Akt. Project supported by the National Basic Research Program of China (a.k.a. 973 Program) (No.2005CB523305).     

Ischemia/reperfusion injury is increased and cardioprotection by a postconditioning protocol is lost as cardiac hypertrophy develops in nandrolone treated rats

We hypothesized that nandrolone (ND)-abuse induces cardiac hypertrophy, increases myocardial susceptibility to ischemia/reperfusion (I/R) injury, and reduces responsiveness to postconditioning (PostC) cardioprotection. Wistar-rats were ND treated for 2 weeks (short_ND) or 10 weeks (long_ND). Vehicle-treated rats served as controls. Hearts were retrogradely perfused and left ventricular pressure (LVP) was measured before and after 30-min global ischemia. In subgroups of hearts, to induce cardioprotection a PostC protocol (five cycles of 10-s reperfusion and 10-s ischemia) was performed. β-adrenoreceptors, kinases (Akt and GSK-3β) and phosphatases (PP2A sub A and PP2A sub B) were examined by Western blot before and after ischemia. After 120-min reperfusion, infarct size was measured. Short_ND slightly increased cardiac/body weight ratio, but did not affect cardiac baseline nor post-ischemic contractile function or infarct size when compared to vehicle hearts. However, PostC limited cardiac dysfunction much more in short_ND hearts than the other groups. Although cardiac/body weight ratio markedly increased after long_ND, baseline LVP was not affected. Yet, post-ischemic contracture and infarct size were exacerbated and PostC was unable to reduce infarct size and ventricular dysfunction. While short_ND increased phosphatases, non-phosphorylated and phosphorylated Akt, long_ND reduced phosphatase-expression and Akt phosphorylation. Both short_ND and long_ND had no effect on the GSK-3β-phosphorylation but increased the expression of β₂-adrenoreceptors. In reperfusion, PostC increased Akt phosphorylation regardless of protective effects, but reduced phosphatase-expression in protected hearts only. In conclusion, short_ND improves post-ischemic myocardial performance in postconditioned hearts. However, long_ND increases myocardial susceptibility to I/R injury and abolishes cardioprotection by PostC. This increased susceptibility might be related to steroid-induced hypertrophy and/or to altered enzyme expression/phosphorylation.     

RISK信号转导通路与肾缺血后处理的心肌保护作用

目的通过在体兔心肌缺血再灌注模型,研究再灌注损伤补救酶(RISK)信号转导通路是否参与肾缺血后处理对急性心肌缺血再灌注损伤的保护作用。方法32只兔随机分为4组,每组8只。对照组:结扎冠状动脉前降支1h,再灌注5h。实验A组于再灌注同时对左侧肾动脉行结扎30s、开通30s的3次反复循环,余同对照组;实验B组于再灌注15min后对左侧肾动脉行结扎30s、开通30s的3次反复循环,余同对照组;药物组于再灌注前5min耳缘静脉注射磷脂酰肌醇3激酶(PI3K)抑制剂LY294002(0.3mg/kg),余同实验A组。分别于再灌注3h、再灌注5h取兔血,测定各组血超氧化物歧化酶(SOD)和丙二醛(MDA)的水平。实验终末,取结扎血管支配部位心肌进行免疫组化处理,观察心肌组织蛋白激酶B(Akt)和内皮一氧化氮合酶(eNOS)含量以及心肌组织结构的变化。结果实验A组血SOD含量高于其它组(P<0.01),MDA含量低于其它组(P<0.01),实验A组心肌组织Akt和eNOS含量明显高于其它组(P<0.01);其余组间各项观察指标无显著差异。结论RISK信号转导通路参与肾缺血后处理对急性心肌缺血再灌注损伤的保护作用;肾缺血后处理必须在心肌再灌注后数分钟内立刻进行才能发挥其心肌保护作用。     

磷酸肌酸后适应联合缺血后适应减轻大鼠心肌缺血再灌注损伤

目的探讨磷酸肌酸后适应联合缺血后适应对大鼠心肌缺血再灌注损伤的影响。方法取健康雄性Wistar大鼠40只,随机分成假手术组(Sham组)、缺血再灌注组(I/R组)、缺血后适应组(IPost组)、磷酸肌酸后适应+缺血后适应组(Pcr+IPost组)各10只,均给予心肌缺血30 min,再灌注120 min处理。再灌注2 h后用比色法测量各组血清肌酸激酶(CK)、乳酸脱氢酶(LDH)、髓过氧化物酶(MPO),ELISA方法检测核因子κB(NF-κB),TTC染色测定心肌梗死面积。结果 IPost组血清CK、LDH、MPO活性和NF-κB以及心肌梗死面积显著低于I/R组,Pcr+IPost组较IPost组各项指标进一步降低(P均<0.05)。结论磷酸肌酸后适应联合缺血后适应可以明显减轻大鼠心肌缺血再灌注损伤,其机制之一可能与抑制NF-κB参与的炎症反应有关。     

Erythropoietin (EPO) Affords More Potent Cardioprotection by Activation of Distinct Signaling to Mitochondrial Kinases Compared with Carbamylated EPO

Purpose

Erythropoietin (EPO) and its non-erythrogenic derivative, carbarmylated EPO (CEPO), have been reported to activate different receptors (homomeric EPO receptor vs. heteromeric receptor consisting of EPO receptor monomer and common β-subunit). The aim of this study was to examine differences between EPO and CEPO in efficacy of cardioprotection against infarction and in activation of pro-survival kinases.

Methods

In isolated rat hearts, infarction was induced by global ischemia followed by reperfusion. Infarct size was determined 2 h after reperfusion, and ventricular tissues for immunoblotting were sampled at 5 min after reperfusion.

Results

Pretreatment with EPO (10 units/ml) before ischemia reduced infarct size (% of risk area; %IS/AR) from 47.0?±?2.1% of the control after 20-min ischemia to 24.7?±?4.3% and from 62.0?±?3.0% after 25-min ischemia to 45.5?±?4.1%. Desialylated EPO (asialoEPO, 100 ng/ml) mimicked the protection by EPO. However, CEPO (100 ng/ml) failed to reduce infarct size after 20-min ischemia (%IS/AR?=?47.5?±?5.9%) and that after 25-min ischemia (%IS/AR?=?56.1?±?4.2%). The infarct size-limiting effect of CEPO was not shown either by increasing CEPO dose to 500 ng/ml or by shortening ischemia to 15 min. Both EPO and CEPO enhanced phosphorylation of cytosolic GSK-3β upon reperfusion. In contrast, phosphorylation of GSK-3β, Akt, and PKC-ε in mitochondria upon reperfusion was significantly enhanced by EPO but not by CEPO.

Conclusion

EPO affords more potent protection against infarction than does CEPO by distinct activation of signaling leading to phosphorylation of pro-survival protein kinases in mitochondria upon reperfusion.     

Postconditioning with levosimendan reduces the infarct size involving the PI3K pathway and KATP-channel activation but is independent of PDE-III inhibition

Reperfusion injury is strongly involved in the loss of functional heart tissue in patients after acute myocardial infarction. Various signal transduction pathways to reduce infarct size during reperfusion have been characterized. However, so far in the clinical setting no standard therapies are applied due to the lack of suitable drugs. Levosimendan, a calcium sensitizer, has been shown to improve survival in cardiogenic shock after infarction. Focus of the present study was to address the question, whether a bolus application of levosimendan prior to reperfusion is able to reduce the infarct size. A well-characterized model, the in vivo rat model, was used and levosimendan applied 5 min prior to reperfusion after 30-min occlusion of the left coronary artery followed by a 30-min reperfusion period. This pharmacological postconditioning was compared to the ischemic postconditioning with three times occlusion/reperfusion periods of 30 s each. To further address the question if in this in vivo model the phosphatidylinositol 3-kinase (PI3K) pathway may be involved, the PDE-III inhibiting property of levosimendan was compared to the PDE-III inhibitor enoximone. Ischemic postconditioning significantly reduced the infarct size from 48 ± 2 to 32 ± 1% of the area at risk (P < 0.05). Similarly, levosimendan decreased infarct size down to 29 ± 3%. The combination of ischemic postconditioning and pharmacological postconditioning using levosimendan did not result in a further reduction of the infarct size. Both, the mitochondrial KATP-channel blocker 5-hydroxydecanoate (5-HD) and the PI3K inhibitor wortmannin abolished the protection afforded by levosimendan completely, while the inhibitors alone did not influence the infarct size in control hearts. Pharmacological postconditioning with enoximone did not result in any infarct size reduction. Postconditioning with levosimendan significantly increased the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β) at 5 min of reperfusion, an effect which could be blocked completely by the additional administration of wortmannin. In conclusion, levosimendan applied prior to reperfusion in acute myocardial infarction significantly reduces the infarct size in an in vivo rat model. This protection involves the PI3K pathway and the activation of mitochondrial KATP-channels, but is independent of PDE-III inhibition. This finding may open new possibilities for the treatment of patients with acute myocardial infarction using levosimendan, which is an already established therapy in cardiogenic shock. Whether the reduction of mortality in cardiogenic shock by levosimendan may in part be based on this postconditoning effect remains to be elucidated in clinical setting.     

Hypercholesterolemia blocked sevoflurane-induced cardioprotection against ischemia–reperfusion injury by alteration of the MG53/RISK/GSK3β signaling

Background

Recent studies have demonstrated that volatile anesthetic preconditioning confers myocardial protection against ischemia–reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats.

Methods

Normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane during three 5 min periods with and without PI3K antagonist wortmannin (10 μg/kg, Wort) or the ERK inhibitor PD 98059 (1 mg/kg, PD). The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3β were determined.

Results

Two hundred and six rats were analyzed in the study. In the healthy rats, sevoflurane significantly reduced infarct size by 42%, a phenomenon completely reversed by wortmannin and PD98059 and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3β. In the hypercholesterolemic rats, sevoflurane failed to reduce infarct size and increase the phosphorylated Akt, ERK1/2 and GSK3β. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts.

Conclusions

Hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3β and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.     

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 μM 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.