Improved myocardial performance induced by clofibrate during reperfusion after acute myocardial infarction

The increase of cellular fatty acids appears to be one of the causes of the myocardial injury during ischemia and reperfusion. This study was designed to examine whether a hypolipidemic drug such as clofibrate can reduce the myocardial injury during ischemia and reperfusion. Clofibrate was fed to experimental pigs for 9 days. Isolated in situ hearts from both experimental and control pigs were subjected to 60 min of regional ischemia induced by occluding the left anterior descending coronary artery, followed by 60 min of global ischemia by hypothermic cardioplegic arrest and 60 min of reperfusion. The clofibrate feeding resulted in the better cardiac performance as judged by increased coronary blood flow, improved left ventricular function, and reduced myocardial injury as judged by creatine kinase release. Although the clofibrate-fed animals contained higher levels of thiobarbituric reactive materials, the free fatty acid levels of plasma and myocardium were much lower compared with control animals. The clofibrate feeding was also associated with increased peroxisomal catalase and beta-oxidation of fatty acids. These results suggest that decreased levels of free fatty acids in the plasma and the myocardium and increased catalase activity induced by antilipolytic therapy appear to provide beneficial effects to the myocardium during ischemia and reperfusion.     

Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger

A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.     

SMT对大鼠在体心脏缺血-再灌注损伤超微结构的保护作用

目的：研究ＳＭＴ对心脏缺血－再灌注损伤（ＩＲＩ）心肌超微结构的影响。方法：ＳＤ大鼠１８只,体重３２０￣３８０ｇ,随机分为三组：①缺血－再灌注组（ＩＲ）：夹闭冠状动脉左前降支６０ｍｉｎ,松夹２０ｍｉｎ。②缺血－再灌注＋ＳＭＴ组（ＳＭＴ）：再灌注前５ｍｉｎ,股静脉注射ｉＮＯＳ抑制剂Ｓ－ｍｅｔｈｙｌｉｓｏｔｈｉｏｕｒｅａ ｓｕｌｆａｔｅ（ＳＭＴ ５ｍｇ／ｋｇ ｗ）,余同ＩＲ组;③对照组（Ｃ）：暴露心脏后     

Limited area of coronary-occlusion myocardial infarct in rats undergoing antioxidant therapy

The synthetic antioxidant dibunol, (ionol. 2,6-ditret-butyl-4-methylphenol) produces the limitation of the zone of the coronaro-occlusion myocardial infarction in rats by 15.8 and 24.2% on day 7 during daily oral administration in doses of 80 and 120 mg/kg, respectively. In the doses used, dibunol reduces the activity of glutathione peroxidase but does not change the activity of glutathione-S-transferase and superoxide dismutase in the infarction zone of the myocardium. It is concluded that free radical products play an important role in ischemic and infarction damage to the myocardium.     

Combined use of hyperbaric oxygenation and antioxidants in the therapy of experimental myocardial infarct

In rabbits with experimental myocardial infarction, the combined use of hyperbaric oxygenation and ionol greatly potentiated the effect of the antioxidant on contractile function of the left ventricle, the time course of antioxidant lipid activity and superoxide dismutase activity of the myocardium.     

Role of monoaminoxidase in the intensification of peroxidation of lipids in mitochondria during experimental myocardial necrosis

The relationship between lipid peroxidation and rat heart mitochondrial monoamine oxidase activity was studied in experimental myocardial necrosis induced by adrenaline injection. It has been established that both the intensity of peroxidation and the activity of monoamine oxidase in mitochondria from adrenaline-injured rat myocardium were essentially increased. The preliminary administration of antioxidants (vitamin E and ionol) was shown to decrease both the intensity of lipid peroxidation and the activity of monoamine oxidase. It is suggested that intensification of lipid peroxidation which is considered to be the main pathogenic factor in ischemic myocardial injury depends on mitochondrial monoamine oxidase activity. Protective effects of antioxidants are realized by the action on two subsequent chains during the formation of active oxygen forms and destruction of lipid peroxidation products.     

Effect of ischemic preconditioning on free radical centers of the isolated rat heart during ischemia and early reperfusion

The effect of ischemic preconditioning on the free-radical state of isolated rat myocardium fixed by rapid freezing at the 25th min of normothermic total ischemia and the 3rd min of reperfusion was studied by the EPR method. It was shown that EPR spectra registered at -40 degrees C consist of two free-radical signals: of the semireduced forms of ubiquinone and flavine coynzymes. It was found that during ischemia and at the beginning of reperfusion, the preconditioning results in a narrowing of the spectra (as compared with control) due to an increase in the narrow ubisemiquinone EPR signal portion, and a decrease in the total concentration of free-radical centers: by 16% in the case of ischemia, and 23% in the case of reperfusion. It was concluded that in both cases the changes were due to a decrease in the concentration of myocardial flavosemiquinones as a result of ischemic preconditioning. We registered the microvawe power saturation curves for these two stages, which corresponded to control and ischemic preconditioning. In the case of ischemia these dependences had similar shapes; however, in the case of reperfusion they differ from each other due to changes in the relative intensities of the EPR signals from ubisemiquinone and flavosemiquinones in the integral myocardial free-radical spectra.     

Creatine kinase of heart mitochondria. The progressive loss of enzyme activity during in vivo ischemia and its correlation to depressed myocardial function

It is now appreciated that mitochondrial creatine kinase (CKm) may play an important role in heart high-energy phosphate metabolism and that this isozyme is solubilized in vitro by dilute solutions of Pi. Since an increase in cellular Pi is known to occur with even brief periods of myocardial ischemia, we investigated the relationship between CKm activity and myocardial performance in rabbit hearts subjected to total global ischemia. CKm activity is expressed as a ratio to mitochondrial malate dehydrogenase (MDHm), a stable marker enzyme. A significant decline in this ratio was observed after only 10 min of ischemia, a time prior to changes in total homogenate creatine kinase activity. After 60 min of ischemia, the CKm/MDHm ratio was depressed by more than 70%. Since there was no restoration of activity following 30 min of reperfusion, we correlated changes in enzyme activity to contractile dysfunction following variable periods of total ischemia. The data showed a close correlation between the decline in the CKm/MDHm ratio and the reduction in performance, measured as left ventricular developed pressure. No correlation was observed between State 3 respiratory rates and performance. Using KCl arrest at 27 degrees C or hyperthermic ischemia at 40 degrees C, the CKm/MDHm ratio consistently correlated to the degree of postischemic functional depression, independent of the duration of ischemia. Isoenzyme electrophoresis failed to detect soluble CKm activity in the postischemic supernatant. Therefore, CKm activity appears to be altered rapidly and irreversibly by ischemia. The implications of these observations on the integration of myocardial high-energy phosphate metabolism are discussed.     

High mobility box 1 mediates neutrophil recruitment in myocardial ischemia-reperfusion injury through toll like receptor 4-related pathway

This study aimed to explore the role of high mobility box 1 (HMGB1) and its receptor toll like receptor 4 (TLR4) on neutrophils in myocardial ischemia reperfusion (I/R) injury. We constructed TLR4-mutant (C3H/HeJ) and control (C3H/HeN) mouse models of myocardial I/R injury and subjected the mice to 30min of ischemia and 6h of reperfusion. Light microscope was used to observe structural changes in the myocardium. HMGB1 levels were measured using quantitative real-time PCR and immunohistochemistry. Neutrophil accumulation, TNF-a expression and IL-8 levels were analyzed via myeloperoxidase (MPO) biochemical studies, quantitative real-time PCR and ELISA, respectively. The results demonstrated that fewer neutrophils infiltrated in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency markedly decreased the ischemic injury caused by ischemia/reperfusion, and inhibited the expression of HMGB1, TNF-a, and IL-8, all of which were up-regulated by ischemia/reperfusion. These findings suggest that HMGB1 plays a central role in recruiting neutrophils during myocardial I/R leading to worsened myocardial I/R injury. This recruitment mechanism is possibly due to its inflammatory and chemokine functions based on the TLR4-dependent pathway.     

Effects of nicotine on thromboxane/prostacyclin balance in myocardial ischemia

It has been proven that nicotine contributes to cardiovascular diseases, although its precise mechanism of action is still unclear. The purpose of this study is to find how nicotine may complicate myocardial ischemia by affecting the thromboxane/prostacyclin (TXA(2)/PGI(2)) balance. We used four groups (n=7 each) of isolated and perfused rabbit hearts according to Langendorff method: (i) control group; (ii) group submitted to 1 microM nicotine perfusion during 60 min; (iii) group submitted to a regional ischemia by ligation of the left descending coronary artery during 60 min and (iv) group submitted to nicotine perfusion during ischemia. Levels of TXB(2) and 6-keto PGF(1alpha), the stable metabolites of TXA(2) and PGI(2) were then determined in the microsomes of the hearts by radioimmunoassay. The results showed that (1) a TXA(2) synthetase activity is present in the myocardium, and this activity, as well as that of PGI(2) synthetase, is decreased by a 60min ischemia; (2) TXA(2) and PGI(2) activities are not affected by nicotine in the normal myocardium and (3) nicotine infusion during ischemia contributes to the increase of TXA(2)/PGI(2) ratio further by decreasing PGI(2). Therefore, these results provide one explanation on how nicotine might worsen myocardial ischemia.     

Evaluation of structuro-functional heterogeneity of isolated mitochondria from the normal and the ischemic myocardium

The structural and functional heterogeneity of mitochondria isolated from intact and ischemic (after 60 min exposure at 37 degrees C) rabbit myocardium was evaluated. In the presence of cytochrome c. a relatively high (260 +/- 26 ng at O/min . mg of protein) rate of rotenone-sensitive NADH oxidation was observed, which was increased in ischemia. Cytochrome c stimulated the increase of NADH oxidation in mitochondria of normal and ischemic myocardium by the factors of 3.5 and 3.4, respectively. Succinate oxidation in the presence of bromthymol blue in normal and ischemic myocardium mitochondria was activated by cytochrome c 3.3- and 2.9-fold, respectively. The percentage of mitochondria with both structurally damaged membranes was 15% and 25% in normal and ischemic myocardium preparations, respectively. In the absence of ADP, cytochrome c contributed to the increase of the succinate oxidase activity in ischemic mitochondria; that in the 3rd state was inhibited in ischemia and normalized by cytochrome c. A principle was proposed for estimating the percentage of mitochondria with damaged outer membranes, the indices being equal to 34% in control and to 56% in ischemic myocardium. Evidence was obtained suggesting that this mitochondrial fraction was characterized by lowered coupling and absence of rotenone-sensitive NADH: oxidase activity. The percentage of intact mitochondria, in which succinate oxidation is inhibited by bromthymol blue and does not need exogenous cytochrome c, is 51% in control and 19% in ischemic myocardium mitochondria.     

Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.     

Influence of temperature and cyanide on electrical and mechanical activities of isoproterenol-damaged frog heart

The electrical and mechanical activities of myocardial strips from Rana pipiens treated with isoproterenol (ISO) were studied during cyanide hypoxia at different bath temperatures (12, 25 and 35 degrees C). In normal myocardium at 12 degrees C, the action potential duration (APD) was almost unchanged but the isometric force (P) was reduced to about 60% after 30 min in 3 mmol/l NaCN. At 25 degrees C, APD and P decreased to about 80 and 60%, respectively, after exposure to cyanide for 30 min. At 35 degrees C, a fast decrease of APD (to about 30%) and P (to about 10%) was seen within 30 min. In all cases, washout of cyanide interactions was possible. Large effects occurred when ISO-damaged myocardium was exposed to cyanide. During the initial 30 min of CN-treatment, APD and P were significantly reduced in the whole temperature range between 12 and 35 degrees C, when compared with controls. When the cyanide exposure times were long enough, all preparations developed contracture. Increase of the temperature and/or ISO-pretreatment shortened the time-course for resting tension increase. The effects of cyanide on APD and resting tension (RT) were strongly correlated and presumably a result of a cyanide-induced rise of the intracellular free calcium concentration (Ca2+i).     

Cholesteryl Esters Accumulate in the Heart in a Porcine Model of Ischemia and Reperfusion

Myocardial ischemia is associated with intracellular accumulation of lipids and increased depots of myocardial lipids are linked to decreased heart function. Despite investigations in cell culture and animal models, there is little data available on where in the heart the lipids accumulate after myocardial ischemia and which lipid species that accumulate. The aim of this study was to investigate derangements of lipid metabolism that are associated with myocardial ischemia in a porcine model of ischemia and reperfusion. The large pig heart enables the separation of the infarct area with irreversible injury from the area at risk with reversible injury and the unaffected control area. The surviving myocardium bordering the infarct is exposed to mild ischemia and is stressed, but remains viable. We found that cholesteryl esters accumulated in the infarct area as well as in the bordering myocardium. In addition, we found that expression of the low density lipoprotein receptor (LDLr) and the low density lipoprotein receptor-related protein 1 (LRP1) was up-regulated, suggesting that choleteryl ester uptake is mediated via these receptors. Furthermore, we found increased ceramide accumulation, inflammation and endoplasmatic reticulum (ER) stress in the infarcted area of the pig heart. In addition, we found increased levels of inflammation and ER stress in the myocardium bordering the infarct area. Our results indicate that lipid accumulation in the heart is one of the metabolic derangements remaining after ischemia, even in the myocardium bordering the infarct area. Normalizing lipid levels in the myocardium after ischemia would likely improve myocardial function and should therefore be considered as a target for treatment.     

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。     

Effect of endovascular cooling on myocardial temperature, infarct size, and cardiac output in human-sized pigs

Mild hypothermia reduces myocardial infarct size in small animals; however, the extent of myocardial protection in large animals with greater thermal mass remains unknown. We evaluated the effects of mild endovascular cooling on myocardial temperature, infarct size, and cardiac output in 60- to 80-kg isoflurane-anesthetized pigs. We occluded the left anterior descending coronary artery for 60 min, followed by reperfusion for 3 h. An endovascular heat-exchange catheter was used to either lower core body temperature to 34 degrees C (n = 11) or maintain temperature at 38 degrees C (n = 11). Additional studies assessed myocardial viability and microvascular perfusion with (99m)Tc-sestamibi autoradiography. Endovascular cooling reduced infarct size compared with normothermia (9 +/- 6% vs. 45 +/- 8% of the area at risk; P < 0.001), whereas the area at risk was comparable (19 +/- 3% vs. 20 +/- 7%; P = 0.65). Salvaged myocardium showed normal sestamibi uptake, confirming intact microvascular flow and myocyte viability. Cardiac output was maintained in hypothermic hearts because of an increase in stroke volume, despite a decrease in heart rate. Mild endovascular cooling to 34 degrees C lowers myocardial temperature sufficiently in human-sized hearts to cause a substantial cardioprotective effect, preserve microvascular flow, and maintain cardiac output.     

心宁片对糖尿病合并心肌缺血再灌注损伤的保护作用和机制研究

目的:研究心宁片对糖尿病合并心肌缺血再灌注损伤的保护作用及其作用机制。方法:腹腔注射STZ加高脂高糖饲料喂养诱导二型糖尿病小鼠模型,随机分为假手术组、模型组及心宁片高、中、低剂量组(心宁片10、20和30 mg·kg~(-1)),每组10只。在此基础上,制作心肌缺血再灌注模型。测定小鼠体内血糖和血脂水平,测定缺血再灌注后心肌酶(LDH和CK-MB)、心梗面积以及AMPK磷酸化水平。结果:心宁片能够有效的控制糖尿病小鼠体内血糖和血脂水平,减轻胰岛素抵抗情况。心宁片能够减轻缺血再灌注引起的心肌梗死,降低LDH和CK-MB水平,减少MDA水平。同时,还发现心宁片能够促进AMPK蛋白磷酸化。采用AMPK特异性抑制剂Compound C抑制AMPK后,LDH水平显著升高,心宁片的心肌保护作用减弱。结论:心宁片能够保护糖尿病合并缺血再灌注损伤,其机制可能是通过AMPK信号通路。     

Translocation of S100A1(1) calcium binding protein during heart surgery

Myocardial ischemia during cardiopulmonary bypass terminated by reperfusion generally leads to different degrees of damage of the cardiomyocytes induced by transient cytosolic Ca(2+) overload. Recently, much attention has been paid to the role of heart-specific Ca(2+)-binding proteins in the pathogenesis of myocardial ischemia-reperfusion injury. S100A1 is a heart-specific EF-hand Ca(2+)-binding protein that is directly involved in a variety of Ca(2+)-mediated functions in myocytes. The aim of our study was to investigate the localization and translocation of S100A1 in the human heart under normal (baseline) conditions and after prolonged ischemia and reperfusion of the myocardium. Our data suggest that S100A1 is directly involved in the transient perioperative myocardial damage caused by ischemia during open heart surgery in humans. Given its role in the contractile function of muscle cells, this S100 protein could be an important "intracellular link" in ischemia-reperfusion injury of the heart.     

An electrophysiological study of the anti-arrhythmia effect of antioxidant ionol]

The paper describes the study of anti-arrhythmia effects of ionol. In isolated rabbit papillary muscle, ionol (a) had no effect on the depolarization-induced automaticity; (b) did not influence early afterdepolarizations: (c) delayed the onset of post-depolarizations initiated by Ca-overload and therefore inhibited the ectopic focal activity in myocardium. In isolated left auricles of rabbit, ionol suppressed the shortening of the excitation wavelength induced with adrenaline and thus protected the heart of reentry and consequent rhythm disturbances.