Preconditioning in Globally Ischemic Isolated Rat Hearts: Effect on Function and Metabolic Indices of Myocardial Damage

We assessed the effects of ischemic preconditioning on heart recovery and metabolic indices of damage following global ischemia and reperfusion, in relationship to post-ischemic lactate release. Three groups of Langendorff rat hearts were studied: (1) A control group of 40 min global ischemia and 45 min reperfusion; (2) preconditioning by 5 min global ischemia and 15 min reperfusion prior to sustained ischemia and reperfusion; (3) Preconditioning by three episodes of brief ischemia–reperfusion prior to sustained ischemia. Repetitive episodes of brief ischemia–reperfusion were associated with increased reactive hyperemia, decreased release of purines and prostaglandin 6-keto F_1α, lower tissue glycogen but no change in lactate washout. After 40 min ischemia, release of lactate was 173±17, 196±6 and 149±9μmol/g in groups 1, 2 and 3, respectively (P<0.01, group 2vgroup 3). Preconditioning had no effect on ischemic arrest but had divergent effects on the development and the magnitude of ischemic contracture: delay and attenuation in group 2 but enhanced onset in group 3. Preconditioning provided a dose-dependent protection from the increase in left ventricular end-diastolic pressure, reduced the reperfusion release of purine metabolites and of creatine kinase, but neither improved systolic function nor prevented arrhythmia. 6-Keto F_1αproduction was 87±13, 132±19 and 241±35 pmol/g in groups 1, 2, 3, respectively (P<0.01 group 1vgroup 3). We conclude that when subjected to prolonged global ischemia, preconditioned isolated rat hearts develop less post-ischemic contracture, lose less purine nucleosides and creatine kinase activity. In addition, preconditioning leads to increased production of prostacyclin. Differences among preconditioning protocols in lactate production seem to be related to the ischemic contracture development, but may not play an ultimate role in attenuation of myocardial damage or improvement of postischemic recovery.     

病理状态下心肌预适应的研究进展

研究表明,在衰老、心肌肥厚、糖尿病和动脉粥样硬化等病理状态下,心肌预适应和正常状态下预适应的作用存在一定差异,但由于受到不同的种属和实验模型,不同的预适应模式或方案以及对疾病病程等方面的影响,预适应对心脏作用的研究结果很不一致。为此进一步加强在病理状态下预适应作用及其机制的探讨,特别是阐明预适应的有效性、适应证、合理选择预适应方法和强度以及联合治疗本身疾病的药物是否影响预适应的效果等问题,将有助于预适应逐步迈向临床应用。     

Pharmacological Preconditioning in Type 2 Diabetic Rat Hearts: The Roles of Mitochondrial ATP-Sensitive Potassium Channels and the Phosphatidylinositol 3-Kinase-Akt Pathway

Purpose  

The authors examined whether olprinone, a phosphodiesterase type 3 inhibitor, or isoflurane, a volatile anesthetic, could protect the heart against myocardial infarction in type 2 diabetic rats and whether the underlying mechanisms involve protein kinase C (PKC), mitochondrial ATP-sensitive potassium (m-K_ATP) channels, or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway.     

钙预适应对膨胀离体大鼠心室所致心律失常的影响

为了证实钙预适应对膨胀离体大鼠心室所诱发的心律失常具有抑制作用 ,采用Langendorff方法灌流离体大鼠心脏。用正常灌流液灌流平衡后 ,离体心被随机分为 4组 :牵张对照组 ,钙预适应组 ,钙预适应 +维拉帕米组和钙预适应 +格列本脲组。维拉帕米和格列本脲在灌流液中的浓度分别为 1μmol/L和 10 μmol/L ,对钙预适应进行干预后及时用正常灌流液将他们从离体心洗脱。将可充灌液体的乳胶球囊通过左房及二尖瓣置于左室 ,通过向乳胶球囊注射液体对左室进行膨胀 ,记录膨胀前和膨胀过程中左室心电图和左室压力、冠脉流量和心率 ,观察心电图ST段和T波的变化 ,并计算对照组和各实验组由膨胀诱发心律失常的发生率和持续时间。结果 :通过乳胶球囊膨胀左室 ,使左室舒张末压增加相同的情况下 ,对照组心律失常总发生率达 10 0 % ,持续时间为 2 .5 6± 0 .4 6s ;钙预适应组较对照组心律失常总发生率明显降低 (40 % ) ,持续时间缩短到 1.6 7± 0 .6 1s(P均 <0 .0 5 ) ;用维拉帕米对钙预适应进行干预后 ,心律失常总发生率较钙预适应组增高 (90 % ) ,持续时间延长 (2 .5 0± 0 .4 6s) ;钙预适应 +格列本脲组心律失常发生情况和钙预适应组相似。各组离体心在膨胀前后冠脉流量和心率、心电图ST段和T波均未见明显变化。结论 :?     

不同缺血预处理对心肌细胞的影响

目的：明确缺血预处理的刺激量与其心肌细胞保护效果的关系。方法：以Ｗｉｓｔａｒ大鼠为模型，开胸安置缺血再灌注装置。将４８只大鼠分为４组，每组各１２只：Ａ组持续阻断冠状动脉１小时，再灌注２小时；Ｂ组缺血５分钟，再灌注５分钟，阻断冠状动脉１小时，再灌注２小时；Ｃ组缺血５分钟，再灌注５分钟，重复３次，阻断冠状动脉１小时，再灌注２小时；Ｄ组缺血１０分钟，再灌注５分钟，阻断冠状动脉１小时，再灌注２小时。检测血浆肌酸激酶浓度与心脏切片氯化硝基四氮唑蓝染色，判断心肌梗死范围；磁带记录心电，分析心律失常。结果：缺血预处理对缺血心肌有明显的保护作用；增加刺激量（缺血预处理的时间与次数），没有增加保护效果。结论：心肌细胞的缺血预处理保护可能依靠阈值触发，无论刺激强弱，一旦启动则是充分完全地发挥保护作用。而超过阈值的强刺激可能有害无益     

Loss of Pacing-induced Preconditioning in Rat Hearts: Role of Nitric Oxide and Cholesterol-enriched Diet

We examined whether the inhibition of nitric oxide (NO) synthesis by N^G-nitro- -arginine ( NNA) abolished pacing-induced preconditioning, and if prolonged exposure to cholesterol-enriched diet led to the loss of preconditioning due to decreased cardiac NO formation. Therefore, Wistar rats fed 2% cholesterol-enriched diet or standard diet for 24 weeks were treated with a single dose of 1 mg/kg NNA or its solvent at the end of the week 24, respectively. Isolated hearts from all groups were subjected to either preconditioning induced by three consecutive periods of pacing at 600 beats/min for 5 min, with 5-min interpacing periods, or time-matched non-preconditioning perfusion, followed by a 10-min coronary occlusion, respectively. In the control group, coronary occlusion after a non-preconditioning protocol decreased aortic flow (AF) from 45.4±2.4 to 15.6±1.5 ml/min, and resulted in a lactate dehydrogenase (LDH) release of 219±55 mU/min/g, however, preconditioning attenuated the consequences of coronary occlusion [AF: 27.3±1.7 ml/min (P<0.05); LDH: 44±14 mU/min/g (P<0.05)]. Preconditioning did not confer protection in the NNA-treated (AF: 17.4±1.5 ml/min; LDH: 151±21 mU/min/g), and/or in the high-cholesterol-fed groups (AF: 15.7±1.2 ml/min; LDH: 168±22 mU/min/g). Preconditioning was preserved however, when hearts were treated with NNA after the preconditioning protocol [AF: 29.6±2.2 ml/min (P<0.05); LDH: 48±17 mU/min/g (P<0.05)]. Both NNA treatment and cholesterol-enriched diet markedly decreased cardiac NO content assayed by electron spin resonance spectroscopy. We conclude that NO may be involved in the triggering mechanism of pacing-induced preconditioning, the protective effect of which is blocked by sustained exposure to dietary cholesterol, possibly by influencing cardiac metabolism of NO.     

缺血后处理对大鼠急性心肌缺血再灌注损伤的影响

目的观察在体条件下缺血后处理对大鼠心肌缺血再灌注损伤的作用及可能的途径。方法建立大鼠在体缺血再灌注模型,将30只大鼠随机分为假手术组、缺血再灌注组、缺血后处理组、缺血预适应组。于再灌注末测定心肌酶,超氧化物歧化酶(SOD)及丙二醛(MDA)的含量,并测定心肌组织梗死面积。结果与缺血再灌注组相比,缺血后处理组与缺血预适应组心肌梗死面积明显减小,血浆肌钙蛋白I及MDA的含量均降低(P<0.05),血浆SOD活性升高(P<0.05)。结论缺血后处理可减轻心肌缺血再灌注损伤,具有心肌保护效应。     

芍药苷预处理对大鼠离体缺血再灌注损伤心脏的保护作用

目的 观察芍药苷预处理对大鼠离体缺血再灌注损伤心脏的保护作用,探讨其可能机制.方法 建立Langendorff离体心脏灌注模型.随机将SD雄性大鼠分为6组,即空白对照(Con)组、单纯缺血再灌注(I/R)组、缺血预处理(IPC)组、不同浓度(15 mg/L,30 mg/L,60 mg/L)芍药苷预处理组(PF1PF3组).Chart5软件分析缺血前,再灌注后20 min、40 min心功能参数,包括:左心室收缩压(LVSP)、左心室内压变化最大速率(dp/dtmax)、心率(HR)、冠脉流出量(CF);收集灌注末心脏标本,制备心肌匀浆,检测超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)、肌酸激酶(CK)值;电镜观察再灌注末心肌细胞超微结构并拍照.结果 IPC、PF预处理组在心功能恢复及心肌酶指标活力方面均优于I/R组(P<0.01);心肌超微结构损伤减轻.结论 芍药苷预处理对大鼠离体心脏能产生保护作用.     

Ischemic Preconditioning in Cardiac Surgery

Murry and colleagues in 1986 described a paradoxical phenomenon that exposure of the heart to repeated brief ischemic episodes delayed the severity of myocardial infarction of a following prolonged myocardial ischemia and termed it myocardial ischemic preconditioning (IP). From then on, IP had been extensively studied in various kinds of experimental models as well as in humans. Besides delaying myocardial infarction, IP has been proved to be a potent endogenous factor in preserving high energy phosphates, suppressing arrhythmias and improving postischemic functional recovery. Delaying myocardial ultrastructure damage, reducing lactate, reducing the utilization of myocardial glycogen and protecting coronary endothelium after IP has also been found. Different experimental models and conditions resembling cardiac surgery have also been investigated. The mechanism of IP is not yet clear, however. The conclusions derived from animal studies could not totally stand for the situation in open heart surgery. IP was found effective in preserving high energy phosphate, improving heart performance and decreasing cardiac troponin T release during open heart surgery. Controversial exists because in same reports there were no effects of IP. The difference in IP protocol, myocardial protective method and study object might be the reason of the controversial results. Several clinical factors may affect IP effects during clinical practice. The purpose of the present study was to review the recent literature on IP, especially the IP mechanism, IP phenomenon in cardiac surgery and the possible influential factors, to summarize whether IP might prefer additional protection of current protective strategy in cardiac surgery.     

急性心肌梗死大鼠心脏一氧化氮合酶表达的改变

目的 通过建立大鼠心肌梗死模型，观察急性心肌梗死对大鼠心脏内皮型一氧化氮合酶mRNA和诱导型一氧化氮合酶蛋白表达的影响。方法48只健康成年SD大鼠（体重200～250g）随机分为假手术组和缺血组，取1、2、8和24h四个不同时间点观察。采用开胸结扎冠状动脉左前降支建立心肌缺血模型，逆转录聚合酶链反应检测大鼠心肌梗死后1、2及24h三个时段缺血心肌内皮型一氧化氮合酶mRNA的表达；免疫组织化学染色检测冠状动脉结扎后8h缺血心肌诱导型一氧化氮合酶蛋白的表达。结果冠状动脉结扎后2h，缺血组大鼠缺血心肌组织内皮型一氧化氮合酶mRNA表达下降（P〈0．05），并持续至结扎后24h；结扎后24h组内皮型一氧化氮mRNA的表达与结扎后2h组相比无显著性差异（P〉0．05）。冠状动脉结扎后8h，梗死区存活心肌组织细胞诱导型一氧化氮合酶蛋白大量表达，而假手术组未见诱导型一氧化氮合酶蛋白表达。结论正常大鼠心肌组织有内皮型一氧化氮合酶基因表达，无诱导型一氧化氮合酶蛋白表达。在心肌梗死早期缺血心肌内皮型一氧化氮合酶mRNA表达减少。心肌急性缺血刺激早期诱导大鼠缺血心肌组织诱导型一氧化氮合酶蛋白大量表达。     

Sodium Thiosulfate Preconditioning Ameliorates Ischemia/Reperfusion Injury in Rat Hearts Via Reduction of Oxidative Stress and Apoptosis

Purpose

Sodium thiosulfate (STS) has of late been proven efficacious in models of urolithiasis and vascular calcification. However, its cardiovascular effects on ischemia reperfusion injury (IR) have not been revealed. Being an antioxidant and calcium chelator, it is assumed to play a vital role in IR as ROS production and calcium overload are major perpetrators of IR injury.

Methods

The cardioprotective effect of STS was evaluated in vitro using H9C2 cardiomyocytes and in vivo using both isolated rat heart and intact left anterior descending artery (LAD) occlusion models of ischemia reperfusion injury. Finally, in silico tools were utilized to establish its possible mode of action. Myocardial injury markers and expression of apoptotic proteins were studied along with myocardial histopathology.

Results

STS of 1 mM recovered H9C2 cells from glucose oxidase/catalase-induced apoptosis. The isolated rat heart treated with STS prior to IR injury improved its hemodynamics and reduced the infarct size to 9%. This was supported by the absence of derangement of cardiac fibers from H&E stained section of LAD-occluded rats. Plasma troponin levels decreased by 15% compared to IR and the myocardium showed diminished apoptotic proteins. An in silico docking analysis revealed higher binding affinity of STS for caspase-3 with a binding energy of ? 60.523 kcal/mol for the complex.

Conclusion

The effectiveness of STS as a cardioprotective agent is attributed to the reduction of apoptosis by binding to the active site of caspase-3 in silico, which was substantiated by the reduced expression of caspase-3 and poly ADP ribose polymerase levels.

     

Signal Transduction in Ischemic Preconditioning:

Ischemic preconditioning is a phenomenon whereby exposure of the myocardium to a brief episode of ischemia and reperfusion markedly reduces tissue necrosis induced by a subsequent prolonged ischemia. Therefore, it is hoped that elucidation of the mechanism of preconditioning will yield therapeutic strategies capable of reducing myocardial infarction. In the rabbit, the brief period of preconditioning ischemia and reperfusion releases adenosine, bradykinin, opioids, and oxygen radicals that summate to induce the translocation and activation of protein kinase C (PKC). PKC appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in preconditioned hearts. Current evidence indicates that PKC activates a tyrosine kinase that leads to the activation of p38 mitogen-activated protein (MAP) kinase or JNK, or possibly both. The stimulation of these stress-activated protein kinases ultimately induces the opening of mitochondrial K(ATP) channels that may be the final mediator of protection by ischemic preconditioning.     

优化能量代谢治疗缺血性心脏病

随着缺血性心脏病心肌血运重建治疗 [药物溶栓、经皮腔内冠状动脉成形术 (PTCA)、冠状动脉旁路移植术等 ]时代的到来 ,心肌缺血再灌注 (ischemi a reperfusion ,I/R)损伤越来越成为临床上一常见现象。因此对于缺血性心脏病的治疗也就包括了两个方面 :当心肌缺血时 ,采取措施延迟细胞坏死并尽快恢复病变区心肌血供 ;缺血心肌恢复血供后 ,防治再灌注损伤。经研究发现 ,缺血再灌注心肌能量代谢的某些变化不利于当时状况下心肌细胞的存活或功能恢复。通过药物调节代谢 ,为病变心肌创造一个适宜的能量代谢环境来保护心肌细胞 ,已成为治疗缺血…     

Bradykinin Protects Against Infarction but Does not Mediate Ischemic Preconditioning in the Isolated Rat Heart

The aim of the study was to test if pre-ischemic treatment with bradykinin can protect against infarction in an isolated rat heart model of regional ischemia and reperfusion, and if any such protection is dependent upon activation of protein kinase C (PKC) or mediated through the nitric oxide (NO) pathway. We also investigated if bradykinin B₂receptor activation, alone or in combination with activation of adenosine receptors andα-adrenoceptors, are involved in the infarct size reducing effect of ischemic preconditioning. Buffer-perfused rat hearts were subjected to 30 min regional ischemia and 120 min reperfusion. Risk zone was determined by fluorescent particles and infarct size by tetrazolium staining. Treatment with bradykinin (0.5μmol/l) prior to ischemia significantly reduced infarct size in percentage of risk zone compared to control experiments (infarct size: 9.6±1.3%v41.8±3.6%,P<0.001). An inhibitor of NO synthesis, NOARG (100μmol/l), did not interfere with the bradykinin induced protection (infarct size: 13.3±2.0%), while chelerythrine (2μmol/l), an inhibitor of protein kinase C, reversed the effect of bradykinin (infarct size: 30.0±2.8%). NOARG did not influence infarct size in the control group (infarct size: 40.1±3.2%). Ischemic preconditioning with three cycles of 5 min global ischemia+5 min reperfusion offered protection similar to bradykinin (infarct size: 8.4±2.0%). The bradykinin antagonist HOE 140 (1μmol/l) reversed the effect of bradykinin (infarct size: 42.5±3.1%), but did not interfere with ischemic preconditioning (infarct size: 7.7±1.6%). Similarily, combined blockade ofα-adrenergic, adenosine and bradykininB₂receptors with p-benzamine (10μmol/l), SPT (100μmol/l) and HOE 140 did not interfere with ischemic preconditioning (infarct size: 7.8±1.1%). Thus, bradykinin can protect against infarction via protein kinase C, but independently of NO. A role for bradykinin in mediating ischemic preconditioning against infarction could not be demonstrated.     

菟丝子黄酮对缺血再灌注大鼠心肌细胞凋亡的影响

目的观察菟丝子黄酮对缺血再灌注大鼠心肌细胞凋亡及相关基因表达的影响。方法结扎大鼠冠状动脉左前降支30 min,再灌注2 h建立心肌缺血再灌注模型。将50只SD大鼠随机分为假手术组、缺血再灌注组及菟丝子黄酮低、高剂量组和消心痛组。再灌注结束后检测血清肌酸激酶和乳酸脱氢酶,DNA末端标记法计算凋亡指数,免疫组织化学法和Western Blotting法检测心肌Bcl-2和Bax蛋白表达,计算Bcl-2/Bax值。结果与假手术组比较,缺血再灌注组心肌酶肌酸激酶、乳酸脱氢酶和凋亡指数显著增高(P<0.01),Bcl-2和Bax蛋白表达增强(P<0.01);与缺血再灌注组比较,菟丝子黄酮低、高剂量组及消心痛组能降低血清肌酸激酶、乳酸脱氢酶含量和凋亡指数(P<0.01),增加Bcl-2而减少Bax的表达(P<0.01)。结论菟丝子黄酮能够抑制缺血再灌注损伤大鼠的心肌细胞凋亡,与消心痛效果相近。     

内源性降钙素基因相关肽在整体大鼠心肌缺血预适应中的作用

为确定预适应缺血时释放的内源性降钙素基因相关肽是否在整体大鼠心肌缺血预适应中起重要作用,分别用放射免疫法测定缺血预适应、降钙素基因相关肽多克隆抗体及对照组的血浆降钙素基因相关肽浓度。并用硝基四唑氮兰染色判定梗死区面积,以坏死区占危险区的百分数表示。结果表明,缺血预适应组第一次和第三次缺血末的血浆降钙素基因相关肽浓度均较对照组显著增高（Ｐ〈０．０１）,以后者增加更为明显。缺血预适应能明显抑制缺血／现     

Antiapoptosis and Mitochondrial Effect of Pioglitazone Preconditioning in the Ischemic/reperfused Heart of Rat

Purpose  Pioglitazone, used clinically in the treatment of type 2 diabetes mellitus, has been implicated as a regulator of cellular inflammatory and ischemic responses. The present study examined whether pioglitazone could inhibit cadiocyte apoptosis and reduce mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of the rat. Furthermore, we investigated whether the protective effect of pioglitazone was related to opening of the mitochondrial_ATP-sensitive potassium channels. Methods  Adult male Sprague–Dawley rats were subjected to 30 min of ischemia followed by 4 h of reperfusion. At 24 h before ischemia, rats were randomized to receive 0.9% saline, 5-hydroxydecanoate (5-HD, 10 mg kg⁻¹, i.v.) plus pioglitazone (3 mg kg⁻¹, i.v.) or pioglitazone (3 mg kg⁻¹, i.v.). One group served as sham control. We investigated mitochondrial structure, apoptosis rate and Bcl-2, Bax and Caspase-3 proteins by immunohistochemistry staining. RT-PCR was used to determine the expression of P38MAPKmRNA and JNKmRNA. Western blotting was used to measure the expression of P38MAPK, JNK and NFκB P65. A second group of rats were randomly divided into sham-operated, ischemia/reperfusion (I/R), pioglitazone treatment, 5-HD + pioglitazone and 5-HD groups and the size of myocardial infarction was determined. Primary cultured cardiomyocytes of neonatal Sprague–Dawley rats were divided into control, hypoxia reoxygenation, different concentrations of pioglitazone and 5-HD + pioglitazone groups. JC-1 staining flowcytometry was used to examine mitochondrial membrane potential (ΔΨm). Results  Pioglitazone decreased mitochondrial ultrastructural damage compared to I/R, and reduced infarct size from 34.93 ± 5.55% (I/R) to 20.24 ± 3.93% (P < 0.05). Compared with the I/R group, the apoptosis rate and positive cell index (PCI) of Bax and Caspase-3 proteins in the pioglitazone group were significantly decreased (P < 0.05), while the PCI of Bcl-2 protein was increased (P < 0.05). There was no significant difference between the I/R and 5-HD + pioglitazone groups. Compared with the sham-operated group, the expression of P38MAPK mRNA, JNK mRNA and protein of P38MAPK, JNK and NFκB P65 in I/R was increased (P < 0.05). Pioglitazone did inhibit the increase in expressions vs I/R (P < 0.05). The rate of loss ΔΨm cells in the pioglitazone group was significantly lower than in the hypoxia reoxygenation group, while the addition of 5-HD inhibited the effect of pioglitazone. Conclusion  Pioglitazone inhibited cadiocyte apoptosis and reduced mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of rat. These protective effects of pioglitazone may be related to opening mitochondrial_ATP-sensitive potassium channels.     

细胞骨架在原代培养乳鼠窦房结细胞模拟缺血预适应中的作用研究

观察细胞骨架在乳鼠窦房结细胞模拟缺血预适应(IP)中的作用。取培养 2d的乳鼠窦房结细胞,随机分为①对照组;②模拟缺血 /再灌注(I/R)组;③模拟IP组;④phalloidin(微丝聚合剂 ) +I/R组;⑤cytochalasinD(微丝解聚剂) +IP组:⑥Taxol(微管聚合剂 ) +I/R组;⑦colchicine(微管解聚剂 ) +IP组。以FITC phalloidin及SABC cy3试剂盒分别标记F actin及β tubulin,用激光共聚焦显微镜检测各组窦房结细胞荧光强度改变。结果:①phalloi din预处理能显著增强再灌注后窦房结细胞F actin及β tubulin的荧光强度,并维持其形态、结构的相对完整性,模拟IP效应。②cytochalasinD及colchicine均能阻断模拟IP的细胞骨架保护作用,显著降低窦房结细胞F actin及β tubulin的荧光强度,导致细胞骨架的解体。③Taxol未能对I/R窦房结细胞提供IP样保护作用。结论:维持微丝结构的相对完整性可以减轻窦房结细胞I/R损伤,模拟IP效应;维持细胞骨架的相对完整性是IP产生的重要前提。