Acetaminophen in the post-ischemia reperfused myocardium

Acetaminophen was administered acutely at the onset of reperfusion after 20 min of low-flow, global myocardial ischemia in isolated, perfused guinea pig hearts (Langendorff) to evaluate its influence in the postischemia, reperfused myocardium. Similarly prepared hearts were treated with vehicle or with uric acid (another phenol for comparison). Functionally, acetaminophen-treated hearts (0.35 mM) achieved significantly greater recovery during reperfusion. For example, left ventricular developed pressures at 40 min reperfusion were 38 +/- 3, 27 +/- 3, and 20 +/- 2 in the presence of acetaminophen (P < 0.05, relative to the other two groups), vehicle, and uric acid, respectively. Coronary perfusion pressures and calculated coronary vascular resistances, in the acetaminophen-treated hearts, were significantly lower at the same time (e.g., coronary perfusion pressures in the three groups, respectively, were 40 +/- 2 [P < 0.05], 51 +/- 3, and 65 +/- 12 mm Hg). Under baseline, control conditions, creatine kinase ranged from 12-15 units/liter in the three groups. It increased to 35-40 units/liter (P < 0.05) during ischemia but was significantly reduced by acetaminophen during reperfusion (e.g., 5.3 +/- 0.8 units/liter at 40 min). Oxidant-mediated chemiluminescence in all three treatment groups during baseline conditions and ischemia was similar (i.e., approximately 1.5-2.0 min for peak luminescence to reach its half maximal value). It took significantly more time during reperfusion for the oxidation of luminol in the presence of acetaminophen (>20 min, P < 0.05) than in its absence (3-8 min in uric acid- and vehicle-treated hearts). These results suggest that administration of acetaminophen (0.35 mM), at the onset of reperfusion, provides anti-oxidant-mediated cardioprotection in the postischemia, reperfused myocardium.     

Ultrastructure, peroxisomes and lipid peroxidation in reperfused myocardium

Reperfusion injury was studied in dog myocardium using a transmission electron microscope and 3,3'-diaminobenzidine (DAB) to locate areas of peroxidatic activity. Dark electron dense DAB reaction product was observed in peroxisomes and damaged mitochondria. These results suggest attack by reactive oxygen species on mitochondrial membranes, which might result in the formation of lipid peroxides and prostaglandin-like compounds. It is suggested that the release of lipid peroxide or prostaglandins from the injured cells may contribute to reactive hyperaemia, ventricular fibrillation and angina.     

Cerebroprotective action of the phospholipase inhibitor quinacrine in the ischemia/reperfused gerbil hippocampus

The phospholipase inhibitor quinacrine (mepacrine; 5 mg/kg, i.p.) was tested for cerebroprotective activity in a gerbil stroke model. Quinacrine significantly reduced stroke injury assessed by locomotor activity monitoring and by histopathological measurement of hippocampal CA1 pyramidal cell loss. It is proposed that phospholipase activation, resulting from elevated levels of intracellular calcium, may contribute to ischemia-evoked neuronal injury.     

Alterations in fatty acid oxidation in ischemic and reperfused myocardium

Summary The focus of this review centered on describing the effects of excess fatty acids on myocardial recovery during reperfusion following ischemic stress. Effects on mechanical function were modest in our studies and are likely to remain difficult/impossible to measure due to the independent phenomenon of stunning which obfuscates and no doubt dominates the influences of other mechanical determinants. Mitochondria appear capable of again using long-chain fatty acids as a preferred substrate and in the presence of restored oxygen delivery can produce normal levels of CO₂. These changes in oxidative metabolism are not mirrored by equal recoveries in mitochondrial energetics. Because of inefficiencies in electron transport and oxidative phosphorylation together with moderate uncoupling of electron transport from oxidative phosphorylation, ATP resynthesis is blunted. This explains in part the absolute decrease in contents of exchangeable nucleotides noted both in cytosol and mitochondria. Further impairments in recovery reside in the inability of the mitochondria to exchange adenine nucleotides into cytosol through the adenine nucleotide translocase antiport. These findings contribute to our understanding of mechanical stunning and may be of value in designing future strategies to optimize the handling of substrates during myocardial reperfusion.Visiting scientist from the Shang Hai Second Medical University, Peoples Republic of China.     

Purine and oxypurine production in mitochondria of ischemic and reperfused myocardium

The present study was undertaken to determine whether significant breakdown of adenine nucleotides to purine bases and oxypurines occurred in mitochondria following myocardial ischemia and ischemia followed by reperfusion, and whether allopurinol prevented this effect. The adenine nucleotides adenosine, hypoxanthine, xanthine and uric acid were measured in the mitochondria and the results suggest that breakdown did occur. Malondialdehyde concentration was determined to gauge lipid peroxidation. This substance did not increase during ischemia or reperfusion, but did so in the presence of allopurinol. Xanthine dehydrogenase was converted to xanthine oxidase during reperfusion and the activity of both enzymes were inhibited by allopurinol. The results also suggested the presence of a mitochondrial 5'-nucleotidase. We conclude that significant breakdown of adenine nucleotide took place in myocardial mitochondria during ischemia and ischemia followed by reperfusion and that allopurinol may have a protective effect.     

Postischemic cardiac recovery in heme oxygenase‐1 transgenic ischemic/reperfused mouse myocardium

Heme oxygenase-1 (HO-1) transgenic mice (Tg) were created using a rat HO-1 genomic transgene. Transgene expression was detected by RT-PCR and Western blots in the left ventricle (LV), right ventricle (RV) and septum (S) in mouse hearts, and its function was demonstrated by the elevated HO enzyme activity. Tg and non-transgenic (NTg) mouse hearts were isolated and subjected to ischemia/reperfusion. Significant post-ischemic recovery in coronary flow (CF), aortic flow (AF), aortic pressure (AOP) and first derivative of AOP (AOPdp/dt) were detected in the HO-1 Tg group compared to the NTg values. In HO-1 Tg hearts treated with 50 μmol/kg of tin protoporphyrin IX (SnPPIX), an HO enzyme inhibitor, abolished the post-ischemic cardiac recovery. HO-1 related carbon monoxide (CO) production was detected in NTg, HO-1 Tg and HO-1 Tg + SnPPIX treated groups, and a substantial increase in CO production was observed in the HO-1 Tg hearts subjected to ischemia/reperfusion. Moreover, in ischemia/reperfusion-induced tissue Na⁺ and Ca²⁺ gains were reduced in HO-1 Tg group in comparison with the NTg and HO-1 Tg + SnPPIX treated groups; furthermore K⁺ loss was reduced in the HO-1 Tg group. The infarct size was markedly reduced from its NTg control value of 37 ± 4% to 20 ± 6% (P < 0.05) in the HO-1 Tg group, and was increased to 47 ± 5% (P < 0.05) in the HO-1 knockout (KO) hearts. Parallel to the infarct size reduction, the incidence of total and sustained ventricular fibrillation were also reduced from their NTg control values of 92% and 83% to 25% (P < 0.05) and 8% (P < 0.05) in the HO-1 Tg group, and were increased to 100% and 100% in HO-1 KO^−/− hearts. Immunohistochemical staining of HO-1 was intensified in HO-1 Tg compared to the NTg myocardium. Thus, the HO-1 Tg mouse model suggests a valuable therapeutic approach in the treatment of ischemic myocardium.     

Time-dependent loss of Mac-1 from infiltrating neutrophils in the reperfused myocardium

Numerous studies have shown that polymorphonuclear neutrophils (PMNs) infiltrate the myocardium immediately after reperfusion of infarcted tissue. Studies with mAbs in vivo and cellular studies in vitro suggest that PMN-induced injury of the cardiac myocyte involve Mac-1 adhesion to myocyte ICAM-1. In this study we demonstrate that PMNs that have infiltrated the ischemic area begin to lose Mac-1 within the first 3 h. By the fifth hour of reperfusion, minimal CD11b staining is seen on PMNs using immunostaining, whereas CD11a remained unchanged. Immunoreactivity of postreperfusion cardiac lymph with R15.7 (anti-CD18) or MY904 (anti-CD11b) was positive in all animals but not for CD11a (R7.1), indicating a specific loss of Mac-1. Immunoprecipitation with either R15.7 or MY904 resulted in identical peptides (a doublet at 190 kDa and a band at 80 kDa), suggesting that both alpha and beta subunits of Mac-1 heterodimer were released. Immunoprecipitation of control PMN lysates revealed bands of 198 kDa and 91 kDa slightly greater than those from the released Mac-1. An in vitro model of homotypic aggregation showed a similar loss of Mac-1 from PMNs; immunoprecipitates of the supernatant demonstrated peptide bands identical with those found in postischemic cardiac lymph. The appearance of soluble Mac-1 in vitro was prevented by anti-CD18 mAb, R15.7, and also by protease inhibition by PMSF. Thus, in vivo and in vitro, activated PMNs lose Mac-1 in a process that may be dependent upon adhesion and subsequent proteolysis.     

The use of proteome similarity for the qualitative and quantitative profiling of reperfused myocardium

An LC-MS-based approach is presented for the identification and quantification of proteins from unsequenced organisms. The method relies on the preservation of homology across species and the similarity in detection characteristics of proteomes in general. Species related proteomes share similarity that progresses from the amino acid frequency distribution to the complete amino sequence of matured proteins. Moreover, the comparative analysis between theoretical and experimental proteome distributions can be used as a measure for the correctness of detection and identification obtained through LC-MS-based schemes. Presented are means to the identification and quantification of rabbit myocardium proteins, immediately after inducing cardiac arrest, using a data-independent LC-MS acquisition strategy. The employed method of acquisition affords accurate mass information on both the precursor and associated product ions, whilst preserving and recording the intensities of the ions. The latter facilitates label-free quantification. The experimental ion density observations obtained for the rabbit sub proteome were found to share great similarity with five other mammalian samples, including human heart, human breast tissue, human plasma, rat liver and a mouse cell line. Redundant, species-homologues peptide identifications from other mammalian organisms were used for initial protein identification, which were complemented with peptide identifications of translated gene sequences. The feasibility and accuracy of label-free quantification of the identified peptides and proteins utilizing above mentioned strategy is demonstrated for selected cardiac rabbit proteins.     

IL-10 is induced in the reperfused myocardium and may modulate the reaction to injury

Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory response leading to TNF-alpha release, IL-6 induction, and subsequent neutrophil-mediated cytotoxic injury. Because inflammation is also an important factor in cardiac repair, we hypothesized the presence of components of the inflammatory reaction with a possible role in suppressing acute injury. Thus, we investigated the role of IL-10, an anti-inflammatory cytokine capable of modulating extracellular matrix biosynthesis, following an experimental canine myocardial infarction. Using our canine model of myocardial ischemia and reperfusion, we demonstrated significant up-regulation of IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10 expression was first detected at 5 h and peaked following 96-120 h of reperfusion. In contrast, IL-4 and IL-13, also associated with suppression of acute inflammation and macrophage deactivation, were not expressed. In the ischemic canine heart, CD5-positive lymphocytes were the predominant source of IL-10 in the myocardial infarct. In the absence of reperfusion, no significant induction of IL-10 mRNA was noted. In addition, IL-12, a Th1-related cytokine associated with macrophage activation, was not detected in the ischemic myocardium. In vitro experiments demonstrated late postischemic cardiac-lymph-induced tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in isolated canine mononuclear cells. This effect was inhibited when the incubation contained a neutralizing Ab to IL-10. Our findings suggest that lymphocytes infiltrating the ischemic and reperfused myocardium express IL-10 and may have a significant role in healing by modulating mononuclear cell phenotype and inducing TIMP-1 expression.     

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。     

Role of epoxyeicosatrienoic acids in protecting the myocardium following ischemia/reperfusion injury

Cardiomyocyte injury following ischemia-reperfusion can lead to cell death and result in cardiac dysfunction. A wide range of cardioprotective factors have been studied to date, but only recently has the cardioprotective role of fatty acids, specifically arachidonic acid (AA), been investigated. This fatty acid can be found in the membranes of cells in an inactive state and can be released by phospholipases in response to several stimuli, such as ischemia. The metabolism of AA involves the cycloxygenase (COX) and lipoxygenase (LOX) pathways, as well as the less well characterized cytochrome P450 (CYP) monooxygenase pathway. Current research suggests important differences with respect to the cardiovascular actions of specific CYP mediated arachidonic acid metabolites. For example, CYP mediated hydroxylation of AA produces 20-hydroxyeicosatetraenoic acid (20-HETE) which has detrimental effects in the heart during ischemia, pro-inflammatory effects during reperfusion and potent vasoconstrictor effects in the coronary circulation. Conversely, epoxidation of AA by CYP enzymes generates 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) that have been shown to reduce ischemia-reperfusion injury, have potent anti-inflammatory effects within the vasculature, and are potent vasodilators in the coronary circulation. This review aims to provide an overview of current data on the role of these CYP pathways in the heart with an emphasis on their involvement as mediators of ischemia-reperfusion injury. A better understanding of these relationships will facilitate identification of novel targets for the prevention and/or treatment of ischemic heart disease, a major worldwide public health problem.     

Selenium status as determinant of connexin-43 dephosphorylation in ex vivo ischemic/reperfused rat myocardium.

Recent studies have demonstrated that electrical uncoupling at gap junctions during ischemia is associated with cardiac Connexin-43 (Cx43) dephosphorylation. Whether oxidative stress is involved in this phenomenon still remains unclear. In the present study, we examined the influence of selenium intake on reperfusion-induced Cx43 dephosphorylation. Male Wistar rats were fed a diet containing either 0.05 mg/kg (Low-Se, n = 13) or 1.5 mg/kg (High-Se, n = 11) selenium for 8 weeks. At the end of this diet, hearts were isolated and subjected to 10 min regional ischemia followed by 10 min reperfusion. The level of dephosphorylated Cx43 was determined in tissue samples from ischemic/reperfused and non-ischemic regions of the hearts. At the end of the experiemental diet, the activity of the antioxidant enzyme glutathione peroxidase (GSH-Px) was increased in high-Se hearts compared with low-Se hearts (+ 13%; p < 0.05). After ischemia/reperfusion, in low-Se hearts, Cx43 dephosphorylation appeared significantly increased in the left ventricle compared to the non-ischemic right ventricle (+ 149%; p < 0.05). The high-Se diet significantly reduced Cx43 dephosphorylation in the left ventricle (p < 0.05 vs. low-Se diet). In conclusion, our results suggest that oxidative stress may be involved in Cx43 dephosphorylation during myocardial ischemia/reperfusion, thereby contributing to arrhythmogenesis.     

Norepinephrine of the rat myocardium during repeated ischemia

Character of tissue changes as well as their reversibility could vary depending on the duration of myocardial ischemia. Long (over 30 min) ischemia leads to a massive release of myocardial interstitial norepinephrine. We tried to investigate changes in the myocardial sympathetic system produced by a relatively long episode of ischemia-repeperfusion. Myocardial norepinephrine has been collected by means of microdialysis probe during repeated occlusions of the left descending coronary artery. It was shown that long episode of occulusion-reperfusion resulted in suppression of massive norepinephrine release in response to second (test) occlusion. The features of norepinephrine release during successive occlusions make it possible to associate this process with the reversibility of the ischemic tissue damages.     

Extent of damage in ischemic,nonreperfused, and reperfused myocardium of anesthetized rats

To investigate the localization of the earliest damage in ischemic and ischemic-reperfused myocardium, anesthetized rats were subjected to coronary occlusion for 15, 30, 45, or 90 min. One-half of the animals in each group had no reperfusion, whereas the other half was reperfused for 14 min. With the use of histological methods, preferentially in the periphery of the area at risk, localized zones were detected that lacked the hypoxia-specific increase in NADH fluorescence. The extent of these areas displaying injured tissue was found to be significantly smaller in the ischemic-nonreperfused hearts than in the ischemic-reperfused organs (15-min ischemia: 0.22 +/- 0.12% vs. 43.0 +/- 5.0%; 30-min ischemia: 5.7 +/- 2.7% vs. 64.6 +/- 2.9%; 45-min ischemia: 5.6 +/- 1.2% vs. 66.0 +/- 7.5%; 90-min ischemia: 39.3 +/- 5.5% vs. 86.7 +/- 1.8% of the area at risk). The results point to a localized initiation of the damage close to the surrounding oxygen-supplied tissue during ischemia and an expansion of this injury by intercellular actions into yet-intact areas upon reperfusion.     

Reduction of infarct size by orally administered des-aspartate-angiotensin I in the ischemic reperfused rat heart

Occlusion of the left main coronary artery for 45 min caused sizable infarct scaring of the left ventricular wall in the rat heart at 14 days post-reperfusion. Daily oral administration of des-aspartate-angiotensin I (DAA-I) for 14 days attenuated the area of the infarct scar and transmurality. The attenuation was dose-dependent and biphasic; maximum effective dose was 1524 nmol/kg, and doses higher than this were progressively inactive. The exact mechanism of the biphasic attenuation is not known, and receptor down-regulation by internalization, which has been implicated in a similar biphasic nature for the anticardiac hypertrophic action of DAA-I, could be a likely cause. Indomethacin (101 μmol/kg, i.p.), administered sequentially after the daily oral dose of DAA-I (1524 nmol/kg), completely inhibited the attenuation at 14 days post-reperfusion, indicating that prostaglandins may be involved in transducing the attenuation. The present findings support earlier indications that DAA-I exerts protective actions in cardiovascular pathologies in which angiotensin II is implicated. It is suggested that DAA-I exerts the cardioprotective action by acting on the same indomethacin-sensitive angiotensin AT₁ receptor. Although similar array of protective actions are also seen with another endogenous angiotensin, angiotensin-(1–7), the present findings demonstrate for the first time the ability of an endogenous angiotensin to reduce the infarct size of an ischemic-reperfusion injured rat heart.     

川芎嗪联用L-精氨酸对缺血/再灌注损伤兔心肌线粒体功能的影响

探讨川芎嗪联用L-精氨酸对心肌缺血／再灌注损伤（MI／RI）时心肌细胞线粒体功能的影响。方法：选用日本大耳白兔50只,随机分为正常对照组（A组）、心肌缺血／再灌注组（B组）、心肌缺血／再灌注＋川芎嗪治疗组（C组）、心肌缺血／再灌注＋L-精氨酸治疗组（D组）和心肌缺血／再灌注＋川芎嗪＋L-精氨酸治疗组（E组）。观察心肌线粒体呼吸功能、Ca^2＋浓度（[Ca^2＋]m）、丙二醛浓度（MDA）、超氧化物歧化酶活性（SOD）和心肌组织三磷酸腺苷（ATP）、能荷（EC）的变化。结果：C、D、E组与B组比较,线粒体呼吸控制率（RCR）、Ⅲ态呼吸速率（ST3）、SOD明显升高,Ⅳ态呼吸速率（ST4）、[Ca^2＋]m、MDA显著降低,心肌组织ATP、EC均明显增高;且与A组比较,E组上述指标均无明显差异。结论：川芎嗪联用L-精氨酸可通过降低氧自由基水平和减轻钙超载,而改善缺血／再灌注损伤心肌的线粒体功能。     

心肌缺血／再灌注损伤后血清和心肌组织Leptin水平的变能

目的：观察大鼠心肌缺血／再灌注损伤对血清和心肌组织瘦素（Leptin）表达的影响,探讨Leptin在心肌缺血／再灌注损伤中的作用。方法：建立大鼠心肌缺血／再灌注模型,检测血清乳酸脱氢酶（LDH）和Leptin浓度,并用HE染色和免疫组织化学观察心肌组织病理学及Lepfin表达水平。结果：缺血组、再灌注组血清LDH水平显著升高（P〈0．05）,表明该模型制作成功,造成心肌局部一定程度的损伤。缺血组血清Leptin含量（6．34±2．49）ng／ml显著低于对照组（7．50±2．93ng／ml,P〈0．05）;再灌注后Leptin水平缓慢恢复,于再灌注2h时Leptin达到（8．32±1．74）ng／ml,恢复到损伤前水平（8．38±2．56）ng／ml,且随再灌注时间延长有升高趋势。免疫纽化显示与假手术纽心肌Leptin蛋白表达水平相比,其他四组均有显著降低（P〈0．01）,按缺血45min后再灌注1h组、缺血45min后再灌注3h组、单纯缺血45min组、缺血45min后再灌注2h组依次递减。结论：Leptin在心肌缺血／再灌注损伤后早期45min血中有明显减少,心肌组织中也明显表达下降。心肌组织病理损伤与Leptin的改变可能有一定的关系。     

Blocking the MyD88-dependent pathway protects the myocardium from ischemia/reperfusion injury in rat hearts

We examined whether blocking the MyD88 mediated pathway could protect myocardium from ischemia/reperfusion (I/R) injury by transfecting Ad5-dnMyD88 into the myocardium of rats (n=8) 3 days before the hearts were subjected to ischemia (45min) and reperfusion (4h). Ad5-GFP served as control (n=8). One group of rats was (n=8) subjected to I/R without transfection. Transfection of Ad5-dnMyD88 significantly reduced infarct size by 53.6% compared with the I/R group (15.1+/-3.02 vs 32.5+/-2.59) while transfection of Ad5-GFP did not affect I/R induced myocardial injury (35.4+/-2.59 vs 32.5+/-2.59). Transfection of Ad5-dnMyD88 significantly inhibited I/R-enhanced NFkappaB activity by 50% and increased the levels of phospho-Akt by 35.6% and BCL-2 by 81%, respectively. Cardiac myocyte apoptosis after I/R was significantly reduced by 59% in the Ad5-dnMyD88 group. The results demonstrate that both inhibition of the NFkappaB activation pathway and activation of the Akt signaling pathway may be responsible for the protective effect of transfection of dominant negative MyD88.