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.     

低氧诱导因子-1α在后处理心肌保护效应中的作用

目的 探讨后处理减轻心肌缺血/再灌注损伤的效应与低氧诱导因子-1α（HIF-1α）的关系.方法 建立大鼠心肌缺血/再灌注及后处理模型,以心肌梗死面积、乳酸脱氢酶（LDH）活性来反映心肌的损伤程度,运用Western blot、real time-PCR等方法检测心肌组织中HIF-1α的表达情况,以探讨其是否参与了后处理的心肌保护效应.结果 后处理缩小了缺血/再灌注所致的心梗面积,降低了血清LDH活性,同时上调了心肌组织中HIF-1α的表达.预先给予HIF-1α脯氨酸羟化酶抑制剂DMOG使HIF-1α表达上调后,后处理减轻心肌损伤的效应进一步增强.结论 后处理减轻缺血/再灌注所致心肌损伤的效应可能与其上调心肌组织中HIF-1α的蛋白表达有关.     

Intracoronary acid infusion as an alternative to ischemic postconditioning in pigs

Previous studies suggested that prolongation of acidosis during reperfusion is protective and may be an important mechanism of postconditioning protection. The aim of this study was to analyze the therapeutic value of this intervention during in vivo coronary reperfusion, and to compare it with ischemic postconditioning. Pigs were submitted to 48 or 60 min of ischemia and 2 h of reperfusion. Animals were allocated to either intracoronary infusion of Krebs solution at dose and duration previously described as optimal in rat hears (pH 6.4 for the first 3 min of reperfusion), ischemic postconditioning (8 cycles of 30 s ischemia/reperfusion) or their respective control groups (n = 9–11 per group). Neither prolongation of acidosis nor postconditioning modified infarct size after 48 min of ischemia as compared to pooled controls. In contrast, in animals submitted to 60 min of coronary occlusion, infarct size was reduced both by infusion of acid Krebs and ischemic postconditioning (57.92 ± 18.15% and 56.91 ± 7.50 vs. 75.37 ± 9.29% in controls, P < 0.01), despite having similar areas at risk. However, an increased incidence of ventricular fibrillation was observed in pigs reperfused with acid Krebs as compared to ischemic postconditioning (11 out of 20 vs. 3 out of 19 pigs, P < 0.05). In conclusion, in pigs submitted to coronary occlusion, intracoronary acid infusion and postconditioning offered protection against cell death only after prolonged coronary occlusion. Both interventions were equally effective, but intracoronary acid infusion was associated with high risk of ventricular fibrillation. These results are strongly against translation of acidic reperfusion to patients with acute myocardial infarction.     

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.     

Long-term inhibition of myocardial infarction by postconditioning during reperfusion

Cardioprotection with postconditioning has been well demonstrated after a short period of reperfusion. This study tested the hypothesis that postconditioning reduces infarct size, vascular dysfunction, and neutrophil accumulation after a long-term reperfusion. Canines undergoing 60 min left anterior descending artery (LAD) occlusion were divided into two control groups of either 3 h or 24 h of full reperfusion and two postconditioning groups with three 30 s cycles of reperfusion and re-occlusion applied at the onset of either 3 h or 24 h of reperfusion. Size of the area at risk (AAR) and collateral blood flow during ischemia were similar among groups. In controls, infarct size as percentage of the AAR (30 ± 3 vs. 39 ± 2* %) by TTC staining, superoxide anion generation from the post-ischemic coronary arteries by lucigenin-enhanced chemiluminescence [(89 ± 5 vs. 236 ± 27* relative light units (RLU/mg)], and neutrophil (PMN) accumulation by immunohistochemical staining in the AAR (52 ± 11 vs. 84 ± 14* cells/mm² myocardium) significantly increased between 3 and 24 h of reperfusion. Postconditioning reduced infarct size (15 ± 4† and 27 ± 3.6† %), superoxide anion generation (24 ± 4† and 43 ± 11† RLU/mg), and PMN accumulation (19 ± 6† and 45 ± 8† cells/mm² myocardium) in the 3 and 24 h reperfusion groups relative to time-matched controls. These data suggest that myocardial injury increases with duration of reperfusion; reduction in infarct size and attenuation in inflammatory responses with postconditioning persist after a prolonged reperfusion. * p < 0.05 24 vs. 3 h control; † p < 0.05 postconditioning vs. time-matched control.     

Postconditioning cardioprotection against infarct size and post-ischemic systolic dysfunction is influenced by gender

Whether cardioprotection by postconditioning (PostC) is gender dependent is not clear. We studied the effect of PostC in terms of both infarct size (IS) and post-ischemic systolic dysfunction (PSD) reduction. Isolated male and female rat hearts were subjected to 10- or 30-min of global ischemia and 120-min of reperfusion, with or without PostC (i.e., 5 cycles of 10-s reperfusion/ischemia immediately after the ischemia). Surprisingly, after 10-min ischemia, IS and PSD were greater in female than male hearts (IS: 21 ± 2% Vs. 11 ± 2%; P < 0.01), while PostC attenuated IS and PSD in female hearts only. After 30-min ischemia IS was smaller in female than male hearts (52 ± 2% Vs. 61 ± 3%; P < 0.05), whereas PSD was similar in these two groups. PostC reduced IS in both genders, though the effect was smaller (P < 0.05) in females. Yet, PostC reduced PSD in female, but not in male hearts. Contracture development paralleled IS in all groups. To check the effects of buffer perfusion over heart function, additional hearts underwent 150-min buffer perfusion only. Contractile function of these hearts was not significantly affected over time. In conclusion IS, contracture and PSD are differently affected by gender, depending on ischemia duration. Yet, reduction of IS induced by PostC depends on the extension of IS induced by index-ischemia. While in female hearts reduction of PSD paralleled IS reduction, in male it does not occur. Results suggest that improvement of systolic function is mainly due to the anti-necrotic rather than to the anti-stunning effect exerted by PostC. Returned for 1. Revision: 20 August 2008 1. Revision received: 6 October 2008 Returned for 2. Revision: 20 October 2008 2. Revision received: 24 October 2008     

Long-term Follow-up of Patients Undergoing Postconditioning During ST-Elevation Myocardial Infarction

Reperfusion injury may offset the optimal salvage of myocardium achieved during primary coronary angioplasty. Thus, coronary reperfusion must be combined with cardioprotective adjunctive therapies in order to optimize myocardial salvage and minimize infarct size. Forty-three patients with their first ST-elevation myocardial infarction were randomized to myocardial postconditioning or standard of care at the time of primary coronary angioplasty. Postconditioning was performed immediately upon crossing the lesion with the guide wire and consisted of four cycles of 30 s occlusion followed by 30 s of reperfusion. End-points included infarct size, myocardial perfusion grade (MPG), left-ventricular ejection fraction (LVEF), and long-term clinical events (death and heart failure). Despite similar ischemic times (≅4.5 h) (p = 0.9) a reduction in infarct size was observed among patients treated with the postconditioning protocol. Peak creatine phosphokinase (CPK), as well as its myocardial band (MB) fraction, was significantly lower in the postconditioning group when compared with the control group (CPK—control, 2,444 ± 1,928 IU/L vs. PC, 2,182 ± 1,717 IU/L; CPK-MB—control, 242 ± 40 IU/L vs. PC, 195 ± 33 IU/L; p = 0.64 and p < 0.01, respectively). EF in the postconditioning group was improved when compared with the control group (control, 43% ± 15 vs. PC, 52% ± 9; p = 0.05). After a mean follow-up of 3.4 years, a 6-point absolute difference in LVEF was still evident in the postconditioning group (p = 0.18). MPG was better among patients treated with the postconditioning protocol compared with control (2.5 ± 0.5 vs. 2.1 ± 0.6; p = 0.02). Due to the small sample size no significant differences in clinical events were detected (p value for death = 0.9; p value for heart failure = 0.2). A simple postconditioning protocol applied at the onset of mechanical reperfusion, resulted in reduction of infarct size, better epicardial and myocardial flow, and improvement in left ventricular function. The beneficial effects of postconditioning on cardiac function persist beyond 3 years.     

One Hour Reperfusion is Enough to Assess Function and Infarct Size With TTC Staining in Langendorff Rat Model

Background  There is not general agreement concerning the optimal time of reperfusion necessary to assess myocardial function and necrosis on isolated perfused heart model. Nevertheless, the study of cardioprotection (especially, pre– and postconditioning) requires a reliable and standardized assessment of myocardial necrosis. Objective  The objective of this study was thus to evaluate whether 1 h of reperfusion was sufficient to assess rat heart viability on Langendorff preparation. Isolated rat hearts (n = 30) underwent 40 min of global normothermic ischemia followed by 60 or 120 min Langendorff reperfusion. In each group, hearts were also randomly assigned into the 2 following sub-groups: postconditioning (PostC, consisting in 2 episodes of 30 s ischemia and 30 s reperfusion at the onset of reperfusion), and control (no intervention). Coronary flow, heart rate, dP/dt and rate-pressure-product were measured. Myocardial necrosis was assessed by TTC staining and LDH, CK release analysis. Results  Our results indicated that heart function tended to slightly decrease between 60 min and 120 min reperfusion. Infarct size was identical at 60 min and 120 min reperfusion, averaging 33–34% of total LV area in controls versus 17% in PostC (p < 0.001 between control and PostC groups). Similarly, the maximum of enzymatic releases (CK and LDH) measured in coronary effluents was at 60 min of reperfusion, followed by a progressive decrease at 90 min and 120 min. As expected, postconditioning limited enzymatic releases whatever the studied time of reperfusion. Conclusion  In conclusion, we showed that prolonged reperfusion beyond 60 min was not useful for function assessment and did not change infarct size measurement, on Langendorff rat model of ischemia-reperfusion.     

Postconditioning and intermittent bradykinin induced cardioprotection require cyclooxygenase activation and prostacyclin release during reperfusion

Postconditioning (PostC), obtained with brief intermittent cycles of ischemia alternating with reperfusion applied after the ischemic event, has been shown to reduce infarct size. Recently, we have shown that PostC triggering includes B(2) receptor activation and its downstream pathway. Moreover, we showed that BK intermittent infusion induces a cardioprotection similar to PostC. The aim of this study was to investigate the involvement of cyclooxygenase-(COX)-derivated prostaglandins, such as prostacyclin (PGI(2)) pathway in the cardioprotective action mediated by intermittent BK infusion.Isolated rat hearts underwent 30 min ischemia and 120 min reperfusion. Myocardial damage was evaluated using nitro-blue-tetrazolium staining. The production of metabolite of PGI(2), 6-keto-PGF1alpha, was evaluated with EIA assay on the samples collected during reperfusion. The perfusion pressure and the left ventricular pressure were monitored. In Control hearts, the infarct size was 64% +/- 4% of risk area. PostC reduced significantly the infarct size (28% +/- 4% P < 0.001 Vs. Control). BK intermittent protocol to mimic PostC, attenuated infarct size (40% +/- 2% P < 0.01 Vs. Control). The BK-intermittent and PostC protections were abolished with COX-inhibition. Intermittent BK and PostC enhanced the release of prostacyclin metabolite, 6-keto-PGF1alpha, in the late phase of reperfusion (i.e., 6-keto-PGF1alpha peaked 30 min after protective maneuvers). Also the stable PGI(2) analogue, Iloprost, given in the early reperfusion reduced infarct size and improved post-ischemic heart function. In conclusion, protection by PostC and intermittent BK requires COX activation and PGI(2) release during late reperfusion. These data suggest that COX must not be inhibited to have PostC protection. This finding should be kept present by future clinical studies on PostC.     

Remote postconditioning

Objectives A series of brief coronary artery reperfusions and reocclusions applied during the early minutes of coronary artery reflow (“postconditioning”) attenuates reperfusion injury. However, it is not known whether brief ischemia–reperfusion applied to a distant organ at the onset of myocardial reperfusion (i.e. “remote postconditioning”, remote PostC) reduces infarct size in the reperfused myocardium. In an in vivo anesthetized rat model of myocardial infarction induced by coronary artery occlusion and reperfusion, this study tested the hypothesis that remote postC induced by a single 5 minute episode of renal artery (RA) occlusion and reperfusion applied immediately before the onset of coronary artery reperfusion protects the myocardium from reperfusion injury by mechanisms involving endogenous adenosine receptor activation. Methods All rats were subjected to a total of 30 minutes of left coronary artery occlusion (LCAO) and 3 hours of reperfusion. The rats were randomized to one of six groups: 1) Control: LCAO and reperfusion only with no other intervention; 2) Remote PostC: after 24 minutes of LCAO the RA was occluded for 5 minutes and released 1 min before coronary artery reperfusion; 3) Permanent RA occlusion: the RA was permanently occluded after 24 minutes LCAO continuing to the end of reperfusion; 4) Delayed Remote PostC: after 26 minutes LCAO the RA was occluded for 5 minutes, and its release was delayed until 1 min after coronary artery reperfusion; 5) CON + SPT: rats with LCAO and reperfusion received 10 mg/kg IV of the non–selective adenosine receptor antagonist 8–sulfophenyl theophylline [SPT] administered 5 minutes before coronary artery reperfusion; and 6) Remote PostC + SPT: after 24 minutes of LCAO the RA was occluded for 5 minutes and released 1 minute before coronary artery reperfusion in the presence of 10 mg/kg SPT given 5 min before coronary artery reperfusion. Results Myocardial infarct size (percentage necrosis/area at risk, mean ± SEM) was reduced by 50% in Remote PostC (25 ± 4%) compared to Control (49 ± 4%, p = 0.003), consistent with a reduction in plasma CK activity (44 ± 5 vs. 67 ± 6 U/ml, p = 0.023). In contrast, permanent RA occlusion before LCAO and reperfusion failed to reduce myocardial infarct size (47 ± 5%) vs Control. Delaying the release of the RA occlusion (delayed Remote PostC) abrogated the myocardial infarct reduction observed with Remote PostC (48 ± 6%). SPT alone had no effect on infarct size (47 ± 4% in CON + SPT vs. 49 ± 4% in CON); however, Remote PostC+SPT abrogated the myocardial infarct size reduction in Remote PostC (50 ± 3% in Remote PostC + SPT vs. 25 ± 4% in Remote PostC). Conclusions Remote renal postconditioning applied immediately before the onset of coronary artery reperfusion provides potent myocardial infarct size reduction likely exerted during the first minutes of coronary artery reperfusion. This inter–organ remote postconditioning phenomenon is likely mediated in part by release of adenosine by the ischemic–reperfused kidney and subsequent activation of adenosine receptors.     

Effects of a protocol of ischemic postconditioning and/or captopril in hearts of normotensive and hypertensive rats

Brief periods (a few seconds) of cyclic coronary occlusions applied early in reperfusion induce a cardioprotection against infarct size, called postconditioning (PostC) in which B₂-bradykinin receptors play a pivotal role. Since angiotensin-converting enzyme (ACE) inhibitors reduce degradation of kinins, we studied the effects of PostC on infarct size and postischemic myocardial dysfunction in both normotensive (WKY) and spontaneously hypertensive rats (SHR) acutely or chronically treated with the ACE inhibitor Captopril. Isolated hearts from SHR and WKY rats were subjected to the following protocols: (a) ischemia for 30- and 120-min reperfusion (I/R); (b) I/R + PostC protocol (5-cycles 10-s I/R); (c) pretreatment with Captopril for 4-weeks before to subject the hearts to I/R with or without PostC maneuvers. Some SHR hearts were treated with Captopril during the 20- or 40-min early reperfusion with or without PostC maneuvers. Cardiac function was assessed in vivo with echocardiography. Left ventricular pressure and infarct size were measured ex vivo. Chronic Captopril significantly reduced left ventricular hypertrophy in SHR, and reduced infarct size in both WKY and SHR hearts. PostC maneuvers significantly reduced infarct size in WKY, but not in SHR hearts. Yet, PostC slightly improved postischemic systolic function in untreated SHR. Captopril given in reperfusion was unable to limit I/R injury in SHR hearts. Data show that PostC protection against infarct size is blunted in SHR and that PostC is unable to add its protective effect to those of chronic Captopril, which per se reduces cardiac hypertrophy and heart susceptibility to I/R insult.     

后处理减轻心肌缺血/再灌注损伤与HIF-1α-iNOS-cGMP通路激活的关系

目的 探讨后处理减轻缺血/再灌注所致的心肌损伤效应是否与低氧诱导因子-1α （HIF-1α）及其下游通路有关.方法 建立大鼠心肌缺血/再灌注及后处理模型,检测心肌组织中HIF-1α及其下游基因诱导性一氧化氮合酶（iNOS）的表达情况及cGMP的含量.结果 后处理缩小缺血/再灌注所致的心梗面积[（27.30±4.16）%比 （36.00±5.29）%,P＜0.01],减少心肌细胞凋亡（Caspase 3比活性：（1.85±0.50）比（3.79±0.64）,P＜0.01）,上调心肌组织中HIF-1α表达[（5.76±0.55） 比（2.85±0.13）,P＜0.01）的同时,提高了iNOS的表达及其cGMP的含量.预先给予HIF-1α脯氨酸羟化酶抑制剂DMOG使后处理心肌组织中HIF-1α表达进一步上调后,iNOS的表达及cGMP含量随之增加,同时后处理减轻心肌损伤的效应[心梗面积：（17.95±2.00）% 比 （27.30±4.16）%,P＜0.01; Caspase3比活性：0.43±0.13比1.85±0.50,P＜0.01]也进一步增强.结论 后处理减轻缺血/再灌注所致心肌损伤的效应可能与其激活心肌组织中HIF-1α-iNOS-cGMP通路有关.     

Postconditioning inhibits mPTP opening independent of oxidative phosphorylation and membrane potential

Mitochondrial permeability transition pore (mPTP) inhibition plays a relevant role in postconditioning (PostC). Ischemia damages the electron transport chain, and the potential contribution of additional modifications in mitochondrial function caused by PostC remains unknown. We sought to determine which mitochondrial functions are involved in the inhibition of mPTP opening during the first minutes of reperfusion. Anesthetized New Zealand White rabbits underwent 30-min ischemia followed by 10-min reperfusion. At reperfusion, they received either no intervention (Control, C), PostC with 4 cycles of 1-min ischemia followed by 1-min reperfusion, or an IV injection of 5 mg/kg cyclosporine A (CsA: a powerful inhibitor of mPTP opening). Sham rabbits underwent no ischemia throughout the 40-min experiment. At the end of the 10-min reperfusion, mitochondria were isolated from the area at risk by differential centrifugations. Calcium retention capacity (CRC) and mitochondrial membrane potential (ΔΨ_m) were assessed by fluorimetry in subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Oxidative phosphorylation was assessed using a Clark-type electrode, and oxidative stress via protein carbonylation by Western blotting. PostC and CsA treatments improved CRC when compared to the C group. Control, PostC and CsA mitochondria exhibited a comparable significant dissipation of ΔΨ_m, together with a comparable significant decrease in state 3 and an increase in state 4 respiration, in both SSM and IFM. However, PostC but not CsA treatment reduced total heart oxidative stress. These data suggest that during the early minutes of reperfusion, PostC reduces oxidative stress and inhibits mPTP opening, independent of alteration of oxidative phosphorylation or of ΔΨ_m.     

Simvastatin in contrast to postconditioning reduces infarct size in hyperlipidemic rabbits: possible role of oxidative/nitrosative stress attenuation

Postconditioning (POC) reduces lethal reperfusion injury under normal conditions, but its effectiveness under certain pathological states is in dispute. In the present study, we sought to determine the effect of chronic simvastatin treatment in hyperlipidemic animals with or without POC. Anesthetized rabbits were randomized into eight groups, as follows, and were subjected to 30-min myocardial ischemia followed by 3-h reperfusion. Normally fed animals: a Control group with no additional intervention, a Sim group treated with simvastatin for 3 weeks at a dose of 3 mg kg⁻¹, a POC group subjected to POC with eight cycles of 30-s ischemia/reperfusion, a Sim-POC group treated with simvastatin, and POC. Cholesterol fed (6 weeks) animals: a Chol group with no additional interventions, a Chol–Sim group treated with simvastatin for 3 weeks, a Chol-POC group subjected to POC, and a Chol–Sim-POC group treated with simvastatin and POC. Infarct size and plasma levels of malondialdehyde (MDA), nitrotyrosine (NT), NO _x , total cholesterol, and LDL were evaluated. In a second series of experiments, heart tissue samples were taken for MDA, NT, and NO _x assessment. Infarct size, circulating MDA, NT, NO _x and cardiac MDA, NT, and NO _x levels declined in POC and all Sim groups compared with Control, Chol, and Chol-POC (p < 0.05). Simvastatin also reduced total cholesterol and LDL plasma levels. In conclusion, a 3-week simvastatin treatment limits the infarct size and attenuates the oxidative and nitrosative stress both in normo- and in hyper-cholesterolemic rabbits subjected to ischemia–reperfusion irrespective of the presence of POC, while POC is effective only in normocholesterolemic animals.     

Postconditioning Protects Against Reperfusion Injury in Hypertensive Dilated Cardiomyopathy by Activating MEK/ERK1/2 Signaling

BackgroundPostconditioning (PostC) cardioprotection has been related to up-regulation of survival kinases; however, the efficacy of PostC and the role of ERK1/2 (extracellular signal-regulated kinase 1/2) remain to be substantiated in hypertension states that may produce “pathologic remodeling.” Therefore, in this work we compared PostC effect and assessed the role of ERK1/2 activation in a model of hypertensive dilated cardiomyopathy (DCM), versus normal (Sham) and compensated hypertrophy (CH) models.Methods and ResultsRats were subjected to angiotensin II administration until development of cardiovascular diseases. Then, isolated hearts underwent ischemia followed by PostC and reperfusion. PostC maintained the double product in all groups. PostC reduced infarct size from 36.16 ± 3% to 9.8% ± 2.2 in Sham, from 37.5 ± 2.4% to 12 ± 3% in CH, and from 40 ± 2.4% to 11.55 ± 3% in DCM. Inhibition of the mitogen-activated protein kinase kinase (MEK)/ERK1/2 pathway had different effects on PostC-conferred cardioprotection in the evaluated groups. Interestingly, although phosphatidylinositol-3-kinase activation was negligible in PostC DCM hearts, we observed Akt activation.ConclusionsPostC confers cardioprotection through alternative survival pathways in normal and CH hearts, and cardiac function recovery in DCM relies mainly on MEK/ERK1/2 cascade. Down-regulation of phosphatidylinositide 3-kinase does not affect the cardioprotective response in DCM, because MEK/ERK1/2 cascade may convey direct Akt activation, strengthening downstream signaling.     

B-type natriuretic peptide at early reperfusion limits infarct size in the rat isolated heart

Natriuretic peptides are regulatory autacoids in the mammalian myocardium whose functions, mediated via particulate guanylyl cyclase/cGMP, may include cytoprotection against ischaemia-reperfusion injury. Previous work has identified that B-type natriuretic peptide (BNP) limits infarct size when administered prior to and during coronary occlusion through a K_ATP channel-dependent mechanism. The present study examined the hypothesis that the protection afforded by BNP is mediated specifically at reperfusion in a postconditioning-like manner. Langendorff-perfused rat hearts were subjected to 35 min coronary artery occlusion and 120 min reperfusion, and infarct size was determined by tetrazolium staining. Postconditioning was effected by applying six 10-second periods of global ischaemia at the onset of reperfusion.Treatment with either BNP 10 nM or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) 1–10 μM was commenced 5 min prior to reperfusion and continued until 10 min after reperfusion. Control infarct size (% of ischaemic risk zone) was 40.8 ± 3.7%.BNP at reperfusion induced a significant limitation of infarct size (BNP 22.9 ± 4.1% P<0.05 vs. control). Co-treatment at reperfusion with BNP and the K_ATP channel blockers 5-hydroxydecanote (5HD, 100 μM), glibenclamide (Glib; 10 μM) or HMR1098 (10 μM) abolished the infarct-limiting effect of BNP (BNP + 5HD 41.0 ± 3.9%, BNP + Glib 39.8 ± 5.6%, BNP + HMR 1098 46.0 ± 7.1%,P < 0.05 vs. BNP). BNP given together with L-NAME (100 μM) at reperfusion resulted in a marked loss of protection (BNP + L-NAME 53.1 ± 3.8% P < 0.001 vs. BNP). In a second series of experiments, SNAP (1–10 μM) given at reperfusion was found not to be protective (SNAP 1 μM 30.2 ± 4.9%, SNAP 2 μM 27.5 ± 9.5%, SNAP 5 μM 39.2 ± 5.7%, SNAP 10 μM 33.7 ± 6.4%, not significant vs. control). In a third series of experiments, postconditioning significantly limited infarct size (14.9 ± 3.6 % vs. control 34.5 ± 4.9%, P < 0.01) and this effect of postconditioning was abolished in the presence of isatin (100 μM), a non-specific blocker of particulate guanylyl cyclases (35.1 ± 6%, P < 0.05 vs. postconditioning). In conclusion, pharmacological activation of pGC by BNP can effectively induce protection against reperfusion injury, by mechanisms involving K_ATP channel opening and endogenous NO synthase activation. Furthermore, endogenous activation of pGC could play a role in the mechanism of postconditioning.     

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.     

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

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

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.     

Angiotensin II Receptor Antagonist EXP 3174 Reduces Infarct Size Comparable with Enalaprilat and Augments Preconditioning in the Pig Heart

There is no agreement on the effect of angiotensin II receptor blockade in the setting of ischemic reperfusion. Our aim was to assess the acute effects of angiotensin-converting enzyme (ACE) inhibition and angiotensin II AT1-subtype receptor blockade in pig heart. Five groups of open-chest pigs received 1 hour of left anterior descending (LAD) coronary artery occlusion and 2 hours of reperfusion. Left ventricular pressure was monitored by an intraventricular catheter, and regional segment shortening (%SS) in the LAD-supplied territory was measured by ultrasonic crystals implanted in the subendocardium. Group 1 (n = 6) served as the control; groups 2 (n = 6) and 3 (n = 6) received the angiotensin II receptor blocker, EXP 3174 (C₂₂H₂₁Cl₁ N₆O₂), and the ACE inhibitor, enalaprilat, respectively, prior to LAD occlusion; group 4 (n = 6) was preconditioned with two cycles of 10 minutes of coronary occlusion and 30 minutes of reperfusion; and group 5 (n = 6) underwent preconditioning with additional administration of EXP 3174 prior to the 60-minute occlusion period. Infarct sizes were measured by p-nitrobluetetrazolium staining and were expressed in percent of the ischemic area of risk. The angiotensin II receptor blocker EXP 3174 and enalaprilat reduced infarct sizes significantly (35.3 ± 17.1% and 40.1 ± 15.1%, respectively) compared with controls (71.2± 12.8%, P < 0.05), and EXP 3174 augmented the infarct size–limiting effects of preconditioning by ischemia (10.5 ± 6% vs. 28.6 ± 5.3%, P < 0.05). Regional contractile dysfunction during reperfusion demonstrated no changes after angiotensin II receptor blockade. Angiotensin II receptor blockade reduced infarct size comparable with that obtained with angiotensin converting-enzyme inhibition. The infarct size–limiting effects of ischemic preconditioning were augmented by administration of the angiotensin II receptor antagonist EXP3174. These data support the concept that blockade or inhibition of angiotensin II before coronary occlusion is protective in a swine model of acute ischemia and reperfusion. This revised version was published online in July 2006 with corrections to the Cover Date.