Cardiac ischemia and reperfusion in spontaneously diabetic rats with and without application of EGb 761: I. cardiomyocytes

Diabetic cardiomyopathy is known to result in increased mortality after ischemic events. Permanently increased oxidative stress with formation of oxygen-free radicals plays a key role in the development of specific heart muscle disease. Associated lesions include structural alterations to cardiomyocytes. Antioxidative treatment in addition to the usual insulin substitution would seem sensible in preventing or delaying long-term diabetic complications and protecting the myocardium against acute ischemic events. We investigated the effects of radical scavenger Ginkgo biloba extract EGb 761 against diabetes-induced damage to cardiomyocytes and additional ischemia/reperfusion injury in spontaneously diabetic BioBreeding/Ottawa Karlsburg (BB/OK) rats, as a model of diabetic myocardium infarction. Morphological and morphometric parameters of heart muscles were analyzed by light and electron-microscopic techniques. We used immunohistochemistry to evaluate parameters of oxidative stress (superoxide dismutase [SOD]) and inducible nitric oxide synthase (iNOS) protein expression. Our results indicated that A) Diabetic myocardium appears more vulnerable to ischemia/reperfusion damage concerning ultrastructure of cardiomyocytes (sarcomeres, vacuoles, mitochondria), expression of antioxidative enzymes (CuZnSOD, MnSOD), and iNOS than normal myocardium; B) Pre-treatment of diabetic myocardium with EGb and additional ischemia/reperfusion leads to a relative improvement in myocardial ultrastructure compared to unprotected myocardium. In summary, EGb appears to be promising as an adjuvant therapeutic drug in diabetics with respect to ischemic myocardium injury. It may contribute to the prevention of late diabetic complications in diabetic cardiomyopathy.     

Involvement of adrenergic pathways in activation of catalase by myocardial ischemia-reperfusion

In situ rabbit hearts were subjected to 15 min of regional myocardial ischemia, and at various time points of reperfusion, antioxidant enzyme activity and mRNA expression were measured in ischemic and nonischemic myocardium. Catalase activity increased significantly in both ischemic and nonischemic myocardium, peaking at 1 h after reperfusion and then gradually returning to the control level. Northern blot analysis showed enhanced expression of catalase mRNA in both areas. There were no changes in redox status, because glutathione levels were not altered by ischemia-reperfusion (I/R). We also tested whether catalase activation in the heart results from signaling pathways that might influence not only the heart but also other organs. We found that catalase activity in the brain was increased after myocardial I/R and ischemic stress to the intestine was equipotent to myocardial I/R in catalase activation. We next sought to elucidate the possible involvement of the adrenergic system in catalase stimulation induced by ischemic stimuli. After pretreatment with the alpha-adrenergic receptor antagonist prazosin, I/R failed to increase catalase activity in the heart and brain. Intravenous norepinephrine increased catalase activity in the heart, brain, and liver. This study shows that brief I/R activates a signaling mechanism to induce catalase activation in multiple organs and the alpha-adrenergic system is involved as an intermediate pathway in this signal transmission.     

迷走神经和乙酰胆碱对缺血心肌保护作用的研究新进展

缺血性心脏病是危害人类健康的主要疾病之一。新近研究发现,心肌缺血与迷走神经活性降低及交感神经活性升高密切相关。本文从缺血性心脏病时心脏迷走神经调控的改变、迷走神经及其递质乙酰胆碱对缺血心肌的保护作用和其在缺血预适应、缺血后适应中可能的信号转导途径等方面,对迷走神经及其递质保护缺血心肌的作用机制研究的新进展予以综述,将有助于深入理解缺血性心脏病的发病机制及防治措施,为该疾病的防治开辟新思路。     

Healing the diabetic heart: does myocardial preconditioning work?

Diabetes mellitus-associated ischemic heart disease is a major public burden in industrialized countries. Reperfusion to a previously ischemic myocardium is obligatory to reinstate its function prior to irreversible damage. However, reperfusion is considered ‘a double-edged sword’ as reperfusion per se could augment myocardial ischemic damage, known as myocardial ischemia-reperfusion (I/R) injury. The brief and repeated cycles of I/R given before a sustained ischemia and reperfusion are represented as ischemic preconditioning, which protects the heart from lethal I/R injury. Few studies have demonstrated preconditioning-mediated cardioprotection in the diabetic heart. In contrast, considerable number of studies suggests that myocardial defensive effects of preconditioning are abolished in the presence of chronic diabetes mellitus that raised questions over preconditioning effects in the diabetic heart. It is evidenced that chronic diabetes mellitus-associated deficit in survival pathways, impaired function of mito-K_ATP channels, MPTP opening and high oxidative stress play key roles in paradoxically suppressed cardioprotective effects of preconditioning in the diabetic heart. These controversial results open up a new area of research to identify potential mechanisms influencing disparities on preconditioning effects in diabetic hearts. In this review, we discussed first the discrepancies on the modulatory role of diabetes mellitus in I/R-induced myocardial injury. Following this, we addressed whether preconditioning could protect the diabetic heart against I/R-induced myocardial injury. Moreover, potential mechanisms pertaining to the attenuated cardioprotective effects of preconditioning in the diabetic heart have been delineated. These are important to be understood for better exploitation of preconditioning strategies in limiting I/R-induced myocardial injury in the diabetic heart.     

Preconditioning the hyperlipidemic myocardium: fact or fantasy?

Ischemic heart disease is a leading cause of death worldwide. Myocardial ischemia results in reduced coronary flow, followed by diminished oxygen and nutrient supply to the heart. Reperfusion to an ischemic myocardium often augments the ischemic damage, known as ischemia-reperfusion (I/R) injury. Number of studies demonstrated that the hyperlipidemic myocardium is rather sensitive and more vulnerable to I/R-induced myocardial injury. Repeated brief ischemia and reperfusion cycles, termed as ischemic preconditioning, given before a sustained ischemia is known to reduce myocardial damage occur as a result of I/R. A plethora of evidence supports the fact that preconditioning is one of the promising interventional strategies having an ability to limit I/R-induced myocardial injury. Despite this fact, the preconditioning-mediated cardioprotection is blunted in chronic hyperlipidemic condition. This suggests that preconditioning is moderately a ‘healthy heart protective phenomenon’. The mechanisms by which chronic hyperlipidemia abrogates cardioprotective effects of preconditioning are uncertain and are not completely understood. The impaired opening of mitochondrial-K_ATP channels, eNOS uncoupling and excessive generation of superoxides in the hyperlipidemic myocardium could play a role in attenuating preconditioning-mediated myocardial protection against I/R injury. Moreover, hyperlipidemia-induced loss of cardioprotective effect of preconditioning is associated with redistribution of both sarcolemmal and mitochondrial Connexin 43. We addressed, in this review, the potential mechanisms involved in hyperlipidemia-induced impairment of myocardial preconditioning. Additionally, novel pharmacologic interventions to attenuate hyperlipidemia-associated exaggerated I/R-induced myocardial injury have been discussed.     

Electrocardiography as a tool for validating myocardial ischemia-reperfusion procedures in mice

This paper evaluates the modifications induced by ischemia and ischemia-reperfusion in mice after permanent or transient, respectively, ligation of the left coronary artery and establishes a correlation among the extent of ischemia, electrocardiograph features, and infarct size. The left coronary artery was ligated 1 mm distal from the tip of the left auricle. Histologic analysis revealed that 30-min ischemia (n = 9) led to infarction involving 9.7% ± 0.5% of the left ventricle, whereas 1-h ischemia (n = 9) resulted in transmural infarction of 16.1% ± 4.6% of the left ventricle. In contrast, 24-h ischemia (n = 8) and permanent ischemia (n = 8) induced similarly sized infarcts (33% ± 2% and 31.8% ± 0.7%, respectively), suggesting ineffective reperfusion after 24-h ischemia. Electrocardiography revealed that ligation of the left coronary artery led to ST height elevation (204 compared with 14 μV) and QTc prolongation (136 compared with 76 ms). Both parameters rapidly normalized on reperfusion, demonstrating that electrocardiography was important for validating correct ligation and reperfusion. In addition, electrocardiography predicted the severity of the myocardial damage induced by ischemia. Our results show that electrocardiographic changes present after 30-min ischemia were reversed on reperfusion; however, prolonged ischemia induced pathologic electrocardiographic patterns that remained even after reperfusion. The mouse model of myocardial ischemia-reperfusion can be improved by using electrocardiography to validate ligation and reperfusion during surgery and to predict the severity of infarction.     

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.     

Ischemia episode detection in ECG using kernel density estimation, support vector machine and feature selection

ABSTRACT: BACKGROUND: Myocardial ischemia can be developed into more serious diseases. Early Detection of the ischemic syndrome inelectrocardiogram (ECG) more accurately and automatically can prevent it from developing into a catastrophicdisease. To this end, we propose a new method, which employs wavelets and simple feature selection. METHODS: For training and testing, the European ST-T database is used, which is comprised of 367 ischemic ST episodes in90 records. We first remove baseline wandering, and detect time positions of QRS complexes by a method basedon the discrete wavelet transform. Next, for each heart beat, we extract three features which can be used fordifferentiating ST episodes from normal: 1) the area between QRS offset and T-peak points, 2) the normalizedand signed sum from QRS offset to effective zero voltage point, and 3) the slope from QRS onset to offset point.We average the feature values for successive five beats to reduce effects of outliers. Finally we apply classifiersto those features. RESULTS: We evaluated the algorithm by kernel density estimation (KDE) and support vector machine (SVM) methods.Sensitivity and specificity for KDE were 0.939 and 0.912, respectively. The KDE classifier detects 349 ischemicST episodes out of total 367 ST episodes. Sensitivity and specificity of SVM were 0.941 and 0.923, respectively.The SVM classifier detects 355 ischemic ST episodes. CONCLUSIONS: We proposed a new method for detecting ischemia in ECG. It contains signal processing techniques of removingbaseline wandering and detecting time positions of QRS complexes by discrete wavelet transform, and featureextraction from morphology of ECG waveforms explicitly. It was shown that the number of selected featureswere sufficient to discriminate ischemic ST episodes from the normal ones. We also showed how the proposedKDE classifier can automatically select kernel bandwidths, meaning that the algorithm does not require anynumerical values of the parameters to be supplied in advance. In the case of the SVM classifier, one has to selecta single parameter.     

Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of reperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG® in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 90 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated short-term ischemia and reperfusion which also reduced the ischemic reperfusion injury as evidenced by better recovery of left ventricular performance in the preconditioned myocardium. The results of this study indicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by myocardial adaptation to ischemia.     

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.     

Perfusion studies of steady flow in poroelastic myocardium tissue

The behaviour of the heart has always elicited interest and particularly the study of its myocardium, as 5–10% of the blood pumped by the heart is passed through the coronary arteries to the myocardium itself. An in-depth investigation of the myocardium behaviour is useful. The present work aims to investigate how myocardium perfusion is influenced by myocardial stress and diseased states, and in general by LV pumping abnormalities. LV myocardial perfusion can then serve as a possible index of the capacity of the LV to respond to its work demand, and thus of the risk of heart failure. The poroelastic analysis of the myocardium based on finite element method (FEM) for regional perfusion through a rectangular element with various physiological ranges of loading conditions was studied.     

Perfusion studies of steady flow in poroelastic myocardium tissue

The behaviour of the heart has always elicited interest and particularly the study of its myocardium, as 5-10% of the blood pumped by the heart is passed through the coronary arteries to the myocardium itself. An in-depth investigation of the myocardium behaviour is useful. The present work aims to investigate how myocardium perfusion is influenced by myocardial stress and diseased states, and in general by LV pumping abnormalities. LV myocardial perfusion can then serve as a possible index of the capacity of the LV to respond to its work demand, and thus of the risk of heart failure. The poroelastic analysis of the myocardium based on finite element method (FEM) for regional perfusion through a rectangular element with various physiological ranges of loading conditions was studied.     

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     

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.     

动态心电图对无症状性心肌缺血的诊断价值

目的:研究动态心电图对无症状性心肌缺血的临床诊断价值。方法:收集我院2014年6月-2015年6月我院接受诊治的冠心病(CAD)患者120例作为研究对象,采用动态心电图仪检测记录24h心电图信息,将动态心电图检测为心肌缺血的患者分为A组(无症状心肌缺血)、B组(有症状心肌缺血),比较两组患者基本信息、心肌缺血发作阵次、ST段下降幅度、ST段下降持续时间、心肌缺血阈变异性、心率及心率失常发生率。结果:120例CAD患者中有95例患者出现心肌缺血,其中A组66例(占69.47%),B组29例(占30.53%),差异显著(P0.05)。A组ST段阵次改变明显高于B组,A组ST段下降幅度、ST段下降持续时间、心肌缺血阈变异性明显低于B组(P0.05)。A、B两组患者心肌缺血发作表现出昼夜节律,在时间段6:00-12:00最高,在时间段0:00-6:00最低(P0.05)。A组患者平均心率显著低于B组患者,心律失常发生率显著高于B组(P0.05)。结论:动态心电图能够准确的诊断出无临床症状心肌缺血,使患者得到及时的治疗,值得临床推广应用。     

New Indices for Evaluating the Functional State in Patients with Angina Pectoris

Traditional indices used to evaluate the functional state in patients with ischemic heart disease (IHD) by testing under conditions of exercise take into account changes in the heart rate (HR), arterial pressure, and the ST segment only during exercise and, for the most part, take into account do not information about the recovery period. The authors show on the basis of the analyses of the regression of ST and HR in patients with angina pectoris that the traditional indices are more effective during exercise. They suggest new standardized nondimensional indices to evaluate the state of patients with myocardial ischemia, i.e., the standardized duration and amplitude of fast recovery (SDFR and SAFR) of ST depression. Most likely, SAFR reflects the share of cardiomyocytes in the metastable state in the total number of the cells affected by short-term ischemia, and SDFR may be an index of the time of the change in the metastable state. Comparative study of the standardized indices of myocardial ischemia showed that the rate–pressure product, SDFR, and SAFR are independent values and may be recommended for evaluating the functional state in patients with IHD.     

Treatment of chronical myocardial ischemia by adenovirus-mediated hepatocyte growth factor gene transfer in minipigs

Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno- virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab- lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran- domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.     

The hypothetical role of potassium conductance on the genesis of R-wave amplitude increase during ischemia in the isolated rat heart.

The effect of increased potassium conductance on the genesis of R-wave amplitude increase during acute myocardial ischemia has been studied in the isolated perfused rat heart by simultaneously recording the R-wave amplitude of epicardial electrograms (VEE), heart rate (HR), coronary flow rate (CFR), left ventricular diastolic pressure (LVDP), and left ventricular systolic pressure (LVSP). The experiments were performed during basal and partial or total ischemic conditions at spontaneous or fixed HR. In some experiments, potassium conductance was increased by means of high-calcium (8 mM) or acetylcholine chloride (10(-6) M) perfusion. In the control experiments, partial ischemic perfusion produced an increase in VEE and LVDP and a decrease in HR, CFR, and LVSP; total ischemic perfusion exaggerated these variations. High-calcium perfusion provoked an increase in VEE and LVDP and a decrease in HR, CFR, and LVSP during basal conditions (p less than 0.01 vs. control experiment); these modifications increased progressively during partial ischemic perfusion (p less than 0.01 vs. control experiment) and during total ischemic perfusion (p less than 0.01 vs. control experiment). Perfusion with acetylcholine chloride produced variations similar to those observed in high-calcium solution except that LVDP under basal conditions remained unchanged from control. When the HR was maintained at a constant value by means of atrial pacing the results were similar to those observed in the unpaced hearts. In conclusion, in the isolated perfused rat heart, increasing potassium conductance may influence the genesis of R-wave amplitude increasing during acute myocardial ischemia.