Blockade of electron transport during ischemia protects cardiac mitochondria

Subsarcolemmal mitochondria sustain progressive damage during myocardial ischemia. Ischemia decreases the content of the mitochondrial phospholipid cardiolipin accompanied by a decrease in cytochrome c content and a diminished rate of oxidation through cytochrome oxidase. We propose that during ischemia mitochondria produce reactive oxygen species at sites in the electron transport chain proximal to cytochrome oxidase that contribute to the ischemic damage. Isolated, perfused rabbit hearts were treated with rotenone, an irreversible inhibitor of complex I in the proximal electron transport chain, immediately before ischemia. Rotenone pretreatment preserved the contents of cardiolipin and cytochrome c measured after 45 min of ischemia. The rate of oxidation through cytochrome oxidase also was improved in rotenone-treated hearts. Inhibition of the electron transport chain during ischemia lessens damage to mitochondria. Rotenone treatment of isolated subsarcolemmal mitochondria decreased the production of reactive oxygen species during the oxidation of complex I substrates. Thus, the limitation of electron flow during ischemia preserves cardiolipin content, cytochrome c content, and the rate of oxidation through cytochrome oxidase. The mitochondrial electron transport chain contributes to ischemic mitochondrial damage that in turn augments myocyte injury during subsequent reperfusion.     

Neonatal cardiac mitochondria and ischemia/reperfusion injury

Postnatal maturation of the heart is characterized by decreasing tolerance to ischemia/reperfusion (I/R) injury associated with significant changes in mitochondrial function. The aim of this study is to test the hypothesis that the role of the mitochondrial membrane permeability transition pore (MPTP) in the I/R injury differs in the neonatal and in the adult heart. For this purpose, the effect of blockade of MPTP on the degree of I/R injury and the sensitivity of MPTP to swelling-inducing agents was compared in hearts from neonatal (7 days old) and adult (90 days old) Wistar rats. It was found that the release of NAD⁺ from the perfused heart induced by I/R can be prevented by sanglifehrin A (SfA) only in the adult myocardium; SfA had no protective effect in the neonatal heart. Furthermore, the extent of Ca-induced swelling of mitochondria from neonatal rats was significantly lower than that from the adult animals; mitochondria from neonatal rats were more resistant at higher concentrations of calcium. In addition, not only the extent but also the rate of calcium-induced swelling was about twice higher in adult than in neonatal mitochondria. The results support the idea that lower sensitivity of the neonatal MPTP to opening may be involved in the mechanism of the higher tolerance of the neonatal heart to I/R injury.     

Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x(L) were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x(L) binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.     

AGGF1 protects from myocardial ischemia/reperfusion injury by regulating myocardial apoptosis and angiogenesis

Angiogenic factor with G patch and FHA domains 1 (AGGF1) is a newly identified proangiogenic protein, which plays an important role in vascular disease and angiogenesis. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. This study investigated whether AGGF1 is involved in the pathogenesis of mouse myocardial I/R injury and the underlying mechanisms. Wild-type (WT) C57BL/6 J mice were treated at 30 min prior to I/R injury with anti-AGGF1 neutralizing antibody (3 mg/kg) or recombinant human AGGF1 (rhAGGF1, 0.25 mg/kg). After I/R injury, the infarct size, the number of TUNEL-positive cardiomyocytes, Bax/Bcl2 ratio, inflammatory cytokine expression and angiogenesis were markedly increased as compared with sham control. Treatment of WT mice with anti-AGGF1 neutralizing antibody resulted in exaggeration of myocardial I/R injury but reducing angiogenesis. In contrast, administration of rhAGGF1 markedly reversed these effects. Furthermore, anti-AGGF1- or rhAGGF1-mediated effects on I/R-induced cardiac apoptosis, inflammation and angiogenesis were dose dependent. In addition, the protective effects of AGGF1 on cardiomyocyte apoptosis and inflammation were confirmed in cultured cardiomyocytes after I/R. Finally, these effects were associated with activation of ERK1/2, Stat3 and HIF-1α/VEGF pathways and inhibition of activation of NF-κB, p53 and JNK1/2 pathways. In conclusion, we report the first in vivo and in vitro evidence that AGGF1 reduces myocardial apoptosis and inflammation and enhances angiogenesis, leading to decreased infarct size after I/R injury. These results may provide a novel therapeutic approach for ischemic heart diseases.     

Biphasic modulation of the mitochondrial electron transport chain in myocardial ischemia and reperfusion

Mitochondrial electron transport chain (ETC) is the major source of reactive oxygen species during myocardial ischemia-reperfusion (I/R) injury. Ischemic defect and reperfusion-induced injury to ETC are critical in the disease pathogenesis of postischemic heart. The properties of ETC were investigated in an isolated heart model of global I/R. Rat hearts were subjected to ischemia for 30 min followed by reperfusion for 1 h. Studies of mitochondrial function indicated a biphasic modulation of electron transfer activity (ETA) and ETC protein expression during I/R. Analysis of ETAs in the isolated mitochondria indicated that complexes I, II, III, and IV activities were diminished after 30 min of ischemia but increased upon restoration of flow. Immunoblotting analysis and ultrastructural analysis with transmission electron microscopy further revealed marked downregulation of ETC in the ischemic heart and then upregulation of ETC upon reperfusion. No significant difference in the mRNA expression level of ETC was detected between ischemic and postischemic hearts. However, reperfusion-induced ETC biosynthesis in myocardium can be inhibited by cycloheximide, indicating the involvement of translational control. Immunoblotting analysis of tissue homogenates revealed a similar profile in peroxisome proliferator-activated receptor-γ coactivator-1α expression, suggesting its essential role as an upstream regulator in controlling ETC biosynthesis during I/R. Significant impairment caused by ischemic and postischemic injury was observed in the complexes I- III. Analysis of NADH ferricyanide reductase activity indicated that injury of flavoprotein subcomplex accounts for 50% decline of intact complex I activity from ischemic heart. Taken together, our findings provide a new insight into the molecular mechanism of I/R-induced mitochondrial dysfunction.     

Calpain and mitochondria in ischemia/reperfusion injury

Studies of ischemia/reperfusion (I/R) injury and preconditioning have shown that ion homeostasis, particularly calcium homeostasis, is critical to limiting tissue damage. However, the relationship between ion homeostasis and specific cell death pathways has not been investigated in the context of I/R. Previously we reported that calpain cleaved Bid in the absence of detectable caspase activation (1). In this study, we have shown that an inhibitor of the sodium/hydrogen exchanger prevented calpain activation after I/R. Calpain inhibitors prevented cleavage of Bid as well as the downstream indices of cell death, including DNA strand breaks, creatine kinase (CK) release, and infarction measured by triphenyl tetrazolium chloride (TTC) staining. In contrast, the broad spectrum caspase inhibitor IDN6734 was not protective in this model. To ascertain whether mitochondrial dysfunction downstream of these events was a required step, we utilized a peptide corresponding to residues 4-23 of Bcl-x(L) conjugated to the protein transduction domain of HIV TAT (TAT-BH4), which has been shown to protect mitochondria against Ca2+-induced deltaPsi(m) loss (2). TAT-BH4 attenuated CK release and loss of TTC staining, demonstrating the role of mitochondria and a pro-apoptotic Bcl-2 family member in the process leading to cell death. We propose the following pathway. (i) Reperfusion results in sodium influx followed by calcium accumulation. (ii) This leads to calpain activation, which in turn leads to Bid cleavage. (iii) Bid targets the mitochondria, causing dysfunction and release of pro-apoptotic factors, resulting in DNA fragmentation and death of the cell. Ischemia/reperfusion initiates a cell death pathway that is independent of caspases but requires calpain and mitochondrial dysfunction.     

Zinc and myocardial ischemia/reperfusion injury

As an important trace element, zinc is required for the normal cellular structure and function, and impairment of zinc homeostasis is associated with a variety of health problems including cardiovascular disease. Zinc homeostasis is regulated through zinc transporters, zinc binding molecules, and zinc sensors. Zinc also plays a critical role in cellular signaling. Studies have documented that zinc homeostasis is impaired by ischemia/reperfusion in the heart and zinc dyshomeostasis may play a role in the pathogenesis of myocardial ischemia/reperfusion injury. Both exogenous and endogenously released zinc may play an important role in cardioprotection against ischemia/reperfusion injury. The goal of this review is to summarize the current understanding of the roles of zinc homeostasis and zinc signaling in myocardial ischemia/reperfusion injury.     

Enhancing AMPK activation during ischemia protects the diabetic heart against reperfusion injury

AMPK activation during ischemia helps the myocardium to cope with the deficit of energy production. As AMPK activity is considered to be impaired in diabetes, we hypothesized that enhancing AMPK activation during ischemia above physiological levels would protect the ischemic diabetic heart through AMPK activation and subsequent inhibition of mitochondrial permeability transition pore (mPTP) opening. Isolated perfused hearts from normoglycemic Wistar or diabetic Goto-Kakizaki (GK) rats (n ≥ 6/group) were subjected to 35 min of ischemia in the presence of 10, 20, and 40 μM of A-769662, a known activator of AMPK, followed by 120 min of reperfusion with normal buffer. Myocardial infarction and AMPK phosphorylation were assessed. The effect of A-769662 on mPTP opening in adult cardiomyocytes isolated from both strains was also determined. A-769662 at 20 μM reduced infarct size in both Wistar (30.5 ± 2.7 vs. 51.8 ± 3.9% vehicle; P < 0.001) and GK hearts (22.7 ± 3.0 vs. 48.5 ± 4.7% vehicle; P < 0.001). This protection was accompanied by a significant increase in AMPK and GSK-3β phosphorylation. In addition, A-769662 significantly inhibited mPTP opening in both Wistar and GK cardiomyocytes subjected to oxidative stress. We demonstrate that AMPK activation during ischemia via A-769662 reduces myocardial infarct size in both the nondiabetic and diabetic rat heart. Furthermore, this cardioprotective effect appears to be mediated through inhibition of mPTP opening. Our findings suggest that improving AMPK activation during ischemia can be another mechanism for protecting the ischemic heart.     

Hydrogen sulfide post-conditioning preserves interfibrillar mitochondria of rat heart during ischemia reperfusion injury

Cardiac mitochondrial dysfunction is considered to be the main manifestation in the pathology of ischemia reperfusion injury, and by restoring its functional activity, hydrogen sulfide (H₂S), a novel endogenous gaseotransmitter renders cardioprotection. Given that interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria are the two main types in the heart, the present study investigates the specific H₂S-mediated action on IFM and SSM during ischemic reperfusion in the Langendorff rat heart model. Rats were randomly divided into five groups, namely normal, ischemic control, reperfusion control (I/R), ischemic post-conditioning (POC), and H₂S post-conditioning (POC_H₂S). In reperfusion control, cardiac contractility decreased, and lactate dehydrogenase, creatine kinase, and infracted size increased compared to both normal and ischemic group. In hearts post-conditioned with H₂S and the classical method improved cardiac mechanical function and decreased cardiac markers in the perfusate and infarct size significantly. Both POC and POC_H₂S exerts its cardioprotective effect of preserving the IFM, as evident by significant improvement in electron transport chain enzyme activities and mitochondrial respiration. The in vitro action of H₂S on IFM and SSM from normal and I/R rat heart supports H₂S and mediates cardioprotection via IFM preservation. Our study indicates that IFM play an important role in POC_H₂S mediated cardioprotection from reperfusion injury.     

Inhibition of endoplasmic reticulum stress by neuregulin-1 protects against myocardial ischemia/reperfusion injury

Neuregulin-1 (NRG-1), an endogenously produced polypeptide, is the ligand of cardiomyocyte ErbB receptors, with cardiovascular protective effects. In the present study, we explored whether the cardioprotective effect of NRG-1 against I/R injury is mediated by inhibiting myocardial endoplasmic reticulum (ER) stress. In vitro, NRG-1 directly inhibited the upregulation of ER stress markers such as glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12 induced by the ER stress inducers tunicamycin or dithiothreitol in both neonatal and adult ventricular myocytes. Attenuating ErbB signals by an ErbB inhibitor AG1478 or ErbB4 knockdown and preincubation with phosphoinositide 3-kinase inhibitors all reversed the effect of NRG-1 inhibiting ER stress in cultured neonatal rat cardiomyocytes. Concurrently, cardiomyocyte ER stress and apoptosis induced by hypoxia-reoxygenation were decreased by NRG-1 treatment in vitro. Furthermore, in an in vivo rat model of myocardium ischemia/reperfusion (I/R), intravenous NRG-1 administration significantly decreased ER stress and myocardial infarct size induced by I/R. NRG-1 could protect the heart against I/R injury by inhibiting myocardial ER stress, which might be mediated by the phosphoinositide 3-kinase/Akt signaling pathway.     

Ion transport systems as targets of free radicals during ischemia reperfusion injury

Oxidative stress is a recognized pathogenic factor in ischemia/reperfusion injury (IRI). Iron induced generation of reactive oxygen species (ROS) in vitro reduces both the Na+K+-ATPase activity and Na+-Ca2+ exchanger of synaptosomal membranes, concomitantly with alteration of physical state of membranes. Oxidative insult also leads to the loss of ability of endoplasmic reticular membranes (ER) to sequester Ca2+ as well as to the increase of Ca2+ permeability. Furthermore, ROS induces both lipid peroxidation and lipid-independent modifications of membrane proteins. Acute in vivo ischemia alters kinetic parameters of Na+K+-ATPase affecting mainly the dephosphorylation step of ATPase cycle with parallel changes of Na+-Ca2+ exchanger and alterations of physical membrane environment. Subsequent reperfusion after ischemia is associated with decrease of immuno signal for PMCA 1 isoform in hippocampus. In addition, incubation of non-ischemic membranes with cytosol from ischemic hippocampus decreases level of PMCA 1 in non-ischemic tissues. Loss of PMCA 1 protein is partially protected both by calpain- and by non-specific protease inhibitors which suggest possible activation of proteases in the reperfusion period. On the other hand, ischemia does not affect the level of Ca2+ pump (SERCA 2b) and calreticulin of intracellular Ca2+ stores. However, IRI resulted in a decrease of IP3 receptor I and altered active Ca2+ accumulation into the ER. A non-specific alteration of physical properties of total membranes such as the oxidative modifications of proteins as well as the content of lipoperoxidation products can also be detected after IRI. ROS can alter physical and functional properties of neuronal membranes. We discuss our results suggesting that ischemia-induced disturbation of ion transport systems may participate in or follow delayed death of neurons after ischemia.     

C5aR-mediated myocardial ischemia/reperfusion injury

The complement system activation can mediate myocardial ischemia and reperfusion (I/R). Inhibition of C5a activity reveals attenuation of I/R-induced myocardial infarct size. However, the contribution of C5a receptor (C5aR) to I/R injury remains to be unknown. Here, we reported that both mRNA and protein for the C5aR were constitutively expressed on cardiomyocytes and upregulated as a function of time after I/R-induced myocardial cell injury in mice. Blockade of C5aR markedly decreased microvascular permeability in ischemic myocardial area and leukocyte adherence to coronary artery endothelium. Importantly, the blocking of C5aR with an anti-C5aR antibody was associated with inhibition in activation of protein kinase C delta (PKC-delta) and induction of PKC-mediated mitogen-activated protein kinase phosphatases-1 (MKP-1) leading to the increased activity of p42/p44 mitogen-activated protein (MAP) kinase cascade. These data provide evidence that C5aR-mediated myocardial cell injury is an important pathogenic factor, and that C5aR blockade may be useful therapeutic targets for the prevention of myocardial I/R injury.     

Gallium-desferrioxamine protects the cat retina against injury after ischemia and reperfusion

This study sought to determine whether gallium-desferrioxamine (Ga/DFO) can curb free radical formation and mitigate biochemical and electrophysiological parameters of injury in the cat retina subjected to ischemia followed by reperfusion.For the biochemical studies, cat eyes were subjected to 90 min of retinal ischemia followed by 5 min of reperfusion, and enucleation of one eye of each cat was used to measure retinal reperfusion injury. Before enucleation of fellow eyes, 2.5 mg/kg Ga/DFO was injected intravenously 5 min before reperfusion. The flux of hydroxyl radicals, as measured directly by conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA), was significantly lower in Ga/DFO-treated eyes. The mean normalized level of 2,3-DHBA (considered a specific marker of hydroxyl radicals) was 3.5 times higher in untreated eyes. Ga/DFO caused a significant reduction, by 2.56-fold, in lipid peroxidation, as reflected by levels of malondialdehyde. Ascorbic acid, a natural antioxidant present in the retina, is severely depleted in untreated eyes. In contrast, in Ga/DFO-treated eyes, levels were 10 times higher than the control. Energy charge was 2.38 times higher in treated eyes. Levels of purine catabolites (hypoxanthine, xanthine, and uric acid) that reflect excessive metabolism of purine nucleotides were approximately twice higher in untreated retinas. Electroretionographic studies, performed on a different subset of animals, substantiated the biochemical results. In Ga/DFO-treated eyes the amplitude of the mixed cone-rod response b-wave (as compared with fellow nonischemic eyes) fully recovered within 24 h after ischemia (b-wave ratio 1.04 +/- 0.09, [mean +/- SEM]) whereas ischemic/reperfused and nontreated eyes recovered to only 0.33 +/- 0. 05. The results show that severe biochemical and functional retinal injury occurs in cat eyes subjected to ischemia and reperfusion. These severe changes were significantly reduced by a single administration of Ga/DFO just before reperfusion. We hypothesize that the protection afforded by Ga/DFO is due to a combined effect of "Push-Pull" mechanisms interfering with transition metal-dependent and free radical-mediated injurious processes.     

黑木耳多糖对抗离体心脏缺血/再灌注损伤的研究

目的:探讨黑木耳多糖(AAP)对离体大鼠心脏缺血/再灌注(I/R)损伤的防护作用及其机制。方法:健康雄性SD大鼠灌胃黑木耳多糖(50,100,200mg/(kg.d))4周后,采用离体心脏Langendorff灌流方法,全心停灌30min,复灌120min建立I/R模型。测定左心室动力学指标和再灌注各时间点冠脉流出液中乳酸脱氢酶(LDH)含量;实验结束测定心肌组织甲月赞(formazan)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性的变化。结果:与单纯I/R组相比,AAP预处理明显提高心肌细胞的formazan含量,降低再灌注期间冠脉流出液中LDH含量,明显增强左室发展压、左心室内压最大上升速率和心率与发展压乘积的恢复,缓解冠脉流量的减少;高剂量AAP改善I/R心肌功能的作用要好于丹参预处理(4ml/(kg.d),gastricperfusion)组。中剂量AAP(100mg/(kg.d))预处理4周后明显抑制I/R心肌MDA的增加和SOD活性的减弱(P0.01),其效果要好于丹参阳性对照组。结论:在大鼠离体心脏灌流模型上,黑木耳多糖预处理具有抗心脏I/R损伤的作用,这种保护作用可能与其增加心肌SOD活性,减少脂质过氧化损伤有关。     

吸入一氧化碳抗肢体缺血/再灌注损伤的实验研究

目的：观察吸入适量一氧化碳（CO）对大鼠肢体缺血/再灌注（I/R）损伤的防治作用。方法：SD大鼠44只,随机分为假手术（S）、I/R、I/R吸入CO（RC）组;通过夹闭股动脉4h、再开放48h,复制肢体I/R损伤模型;RC组行再灌注时,使动物吸入含有CO的医用空气（CO的体积分数为0.05%）,其余两组呼吸正常空气;对比观测缺血肢体大体及骨骼肌组织病理学、缺血肢体湿干重比值（W/D）的变化,流式细胞仪检测肌组织中Bax、Bcl-2的表达水平及细胞凋亡百分比,全自动生化分析仪检测血清乳酸脱氢酶（LDH）和肌酸激酶（CK）的变化。结果：与I/R组比较,RC组动物W/D、血清LDH及CK含量、肌组织中Bax表达水平及细胞凋亡百分比均显著降低,肌组织Bcl-2表达水平显著升高,缺血肢体大体观及肌组织病理学明显改善。结论：吸入适量浓度的外源性CO对肢体I/R损伤有防治作用。     

Effects of myocardial ischemia/reperfusion injury on plasma metabolomic profile during aging

BackgroundHeart disease is a frequent cause of hospitalization and mortality for elderly patients. A common feature of both heart disease and aging itself is the involvement of metabolic organ alterations ultimately leading to changes in circulating metabolite levels. However, the specific contribution of aging and ischemic injury to the metabolic dysregulation occurring in older adults with ischemic heart disease is still unknown.AimTo evaluate the effects of aging and ischemia/reperfusion (I/R) injury on plasma metabolomic profiling in mice.MethodsYoung and aged mice were subjected to a minimally invasive model of I/R injury or sham operation. Complete evaluation of cardiac function and untargeted plasma metabolomics analysis were performed.ResultsWe confirmed that aged mice from the sham group had impaired cardiac function and augmented left ventricular (LV) dimensions compared to young sham‐operated mice. Further, we found that ischemic injury did not drastically reduce LV systolic/diastolic function and dyssynchrony in aged compared to young mice. Using an untargeted metabolomics approach focused on aqueous metabolites, we found that ischemic injury does not affect the plasma metabolomic profile either in young or old mice. Our data also demonstrate that age significantly affects circulating metabolite levels (predominantly amino acids, phospholipids and organic acids) and perturbs several pathways involved in amino acid, glucid and nucleic acid metabolism as well as pyridoxal‐5′‐phosphate salvage pathway in both sham and ischemic mice.ConclusionsOur approach increases our understanding of age‐associated plasma metabolomic signatures in mice with and without heart disease excluding confounding factors related to metabolic comorbidities.     

Plasma endothelin levels during myocardial ischemia and reperfusion

Endothelin, an endothelium-derived vasoconstrictive peptide, has a strong potency of coronary artery constriction. However, the role of endogeneous endothelin under pathophysiological conditions has not yet been known. In this study, we examined plasma endothelin concentration in dogs with myocardial ischemia and reperfusion. Anesthetized open-chest dogs underwent either 45 minutes occlusion of the left anterior descending coronary artery followed by 3 hours reperfusion, or 4-10 hours of continuous occlusion. Plasma concentration of endothelin from the central vein was measured by the highly sensitive enzyme-immunoassay. Plasma endothelin concentration increased 2.2-fold with the peak level at 60 minutes after release of the ligated artery, but occlusion per se caused no remarkable change. These data suggest that reperfusion of the occluded artery might be needed to increase the plasma concentration of endothelin in case of myocardial infarction.