缺血预处理限制线粒体钙超载保护再灌注大鼠心肌

目的研究缺血预处理(IP)对缺血大鼠心肌的保护作用。方法将Wistar大鼠随机分为对照组(CON组)、缺血预处理组(IP组)、5-羟葵酸拮抗IP组(5HD+IP组),每组8只。建立Langendorff灌注模型,平衡灌注20min,全心缺血40 min,再灌注30 min,观察比较各组血流动力学变化;再灌注末取心肌并制备线粒体,电镜观察比较形态学改变;分离、测定线粒体游离钙浓度。结果IP组与对照组相比,能明显改善缺血再灌注大鼠心脏收缩功能,左室发展压(LVDP)有显著差异(P<0.01);缺血末、再灌注末线粒体游离钙浓度显著低于对照组(P<0.01);IP组明显改善心肌、线粒体显微结构。5HD+IP组与IP组比较,再灌注各时间点大鼠心脏收缩功能指标LVDP有显著性差异(P<0.01),缺血末、再灌注末线粒体游离钙浓度显著高于IP组(P<0.01);5HD+IP组心肌细胞、线粒体形态的保存明显比IP组差。结论①IP能改善缺血再灌注大鼠心肌的收缩功能,减少线粒体钙超载,保护心肌细胞和线粒体形态和结构的完整,产生心肌保护作用;②选择性线粒体钾通道拮抗剂5-羟葵酸能翻转缺血预处理的心肌保护作用;③缺血预处理的心肌保护机制可能是通过激活线粒体ATP敏感性钾通道,限制心肌线粒体钙超载,维护线粒体整体形态和功能的完整,产生心肌保护作用。     

肾脏缺血预处理对麻醉家兔心脏保护的神经机制(英文)

目的　探讨肾神经在肾脏缺血预处理 (RIP)心肌保护中的作用。方法　在麻醉家兔心肌缺血 /再灌注 (MIR)模型上 ,观察MIR和RIP对家兔血流动力学、心肌耗氧量、心外膜电图和心肌梗死范围的影响。结果　在MIR过程中 ,血流动力学指标和心肌耗氧量均明显持续性降低 ;心外膜电图ST段在缺血期明显升高 ,再灌注过程中恢复到基础对照值。单纯MIR时的心肌梗塞范围占缺血心肌的 (55.80± 1.2 5) % ,RIP后心肌梗塞范围为 (3 6.51± 2 .8) % ,较单纯MIR显著减少 (P <0 .0 1)。肾神经切断后对MIR所致的心肌梗死范围无明显影响 ,但可取消RIP对心肌的保护效应。结论　RIP具有心肌保护作用 ,肾短暂缺血 -再灌注所诱发的肾神经传入活动可能参与此心肌保护效应     

Melatonin ameliorates calcium homeostasis in myocardial and ischemia-reperfusion injury in chronically hypoxic rats

Chronic hypoxia (CH) leads to the deterioration of myocardial functions with impaired calcium handling in the sarcoplasmic reticulum (SR), which may be mediated by oxidative stress. We hypothesized that administration of antioxidant melatonin would protect against cardiac and ischemia-reperfusion (I/R) injury by ameliorating SR calcium handling. Adult Sprague-Dawley rats that had received a daily injection of melatonin or vehicle were exposed to 10% oxygen for 4 wk. The heart of each rat was then dissected and perfused using a Langendorff apparatus. The ratio of heart-to-body weight, ventricular hypertrophy and hematocrit were increased in the hypoxic rats compared with the normoxic controls. Malondialdehyde levels were also increased in the heart of hypoxic rats and were lowered by the treatment of melatonin. The hearts were subjected to left coronary artery ischemia (30 min) followed by 120-min reperfusion. Lactate dehydrogenase leakage before ischemia, during I/R and infarct size of the isolated perfused hearts were significantly elevated in the vehicle-treated hypoxic rats but not in the melatonin-treated rats. Spectroflurometric studies showed that resting calcium levels and I/R-induced calcium overload in the cardiomyocytes were more significantly altered in the hypoxic rats than the normoxic controls. Also, the hypoxic group had decreased levels of the SR calcium content and reduced amplitude and decay time of electrically induced calcium transients, indicating impaired contractility and SR calcium re-uptake. Moreover, there were reductions in protein expression of calcium handling proteins, markedly shown at the level of SR-Ca(2+) ATPase (SERCA) in the heart of hypoxic rats. Melatonin treatment significantly mitigated the calcium handling in the hypoxic rats by preserving SERCA expression. The results suggest that melatonin is cardioprotective against CH-induced myocardial injury by improving calcium handling in the SR of cardiomyocytes via an antioxidant mechanism.     

Cardioprotection of PLGA/gelatine cardiac patches functionalised with adenosine in a large animal model of ischaemia and reperfusion injury: A feasibility study

The protection from ischaemia‐reperfusion‐associated myocardial infarction worsening remains a big challenge. We produced a bioartificial 3D cardiac patch with cardioinductive properties on stem cells. Its multilayer structure was functionalised with clinically relevant doses of adenosine. We report here the first study on the potential of these cardiac patches in the controlled delivery of adenosine into the in vivo ischaemic‐reperfused pig heart. A Fourier transform infrared chemical imaging approach allowed us to perform a characterisation, complementary to the histological and biochemical analyses on myocardial samples after in vivo patch implantation, increasing the number of investigations and results on the restricted number of pigs (n = 4) employed in this feasibility step. In vitro tests suggested that adenosine was completely released by a functionalised patch, a data that was confirmed in vivo after 24 hr from patch implantation. Moreover, the adenosine‐loaded patch enabled a targeted delivery of the drug to the ischaemic‐reperfused area of the heart, as highlighted by the activation of the pro‐survival signalling reperfusion injury salvage kinases pathway. At 3 months, though limited to one animal, the used methods provided a picture of a tissue in dynamic conditions, associated to the biosynthesis of new collagen and to a non‐fibrotic outcome of the healing process underway. The synergistic effect between the functionalised 3D cardiac patch and adenosine cardioprotection might represent a promising innovation in the treatment of reperfusion injury. As this is a feasibility study, the clinical implications of our findings will require further in vivo investigation on larger numbers of ischaemic‐reperfused pig hearts.     

超速起搏预适应的延迟保护与热休克蛋白70的表达

目的观察在超速心室起搏（ventricular overdrive pacing,VOP）预适应延迟保护阶段热休克蛋白70（HSP70）的表达水平。方法新西兰兔24只,随机分为3组,单纯结扎组,起搏组,起搏+放线菌素D组。制作超速起搏预适应和缺血/再灌注的动物模型,检测CK和CK-MB的变化,动态描记再灌注时心电图,免疫组织化染色检测HSP70抗原。结果缺血后起搏组心肌酶的水平在再灌注时均低于单纯结扎组和起搏+放线菌素D组（P<0.01）;单纯结扎组中在再灌注过程中共有5只发生心律失常,起搏+放线菌素组也有4只,而起搏组无心律失常发生。起搏组和单纯结扎组之间有显著性差别（P<0.05）。起搏组HSP70的阳性表达的程度明显高于其他两组。结论超速起搏预适应可以模拟缺血预适应,其延迟保护作用可能与HSP70表达增加密切相关。     

Diazoxide acts more as a PKC-epsilon activator, and indirectly activates the mitochondrial K(ATP) channel conferring cardioprotection against hypoxic injury

BACKGROUND AND PURPOSE: Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoK(ATP) channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoK(ATP) channel in diazoxide-induced cardioprotection. EXPERIMENTAL APPROACH: In H9c2 cells and neonatal rat cardiomyocytes, PKC-epsilon activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca(2+) and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay. KEY RESULTS: Diazoxide (100 microM) induced translocation of PKC-epsilon from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-epsilon by either epsilonV1-2 or dominant negative mutant PKC-epsilon (PKC-epsilon KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either epsilonV1-2 or PKC-epsilon KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-epsilon induced by diazoxide. Transfection with wild type PKC-epsilon mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by epsilonV1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca(2+), mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by epsilonV1-2 or 5-HD. CONCLUSIONS AND IMPLICATIONS: Diazoxide induced isoform-specific translocation of PKC-epsilon as an upstream signaling molecule for the mitoK(ATP) channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway.     

Delayed uncoupling contributes to the protective effect of heptanol against ischaemia in the rat isolated heart

1. It is known that infusion of the gap junction uncoupler heptanol, before ischaemia or during reperfusion, limits myocardial infarct size. However, whether this cardiac effect is linked to the effect of heptanol on communication across gap junctions has not been elucidated. The aims of the present study were to examine the effect of heptanol on infarct size, arrhythmias and myocardial tissue resistance and to assess whether changes in electrical coupling correlate with cardiac protection. 2. Rat isolated, perfused hearts were subjected to a 24 min infusion of heptanol (0.05, 0.1, 0.5 or 1.0 mmol/L) followed by 20 min regional ischaemia and 60 min reperfusion, or by 70 min global no-flow ischaemia. The effective refractory period, action potential and conduction velocity were measured in papillary muscles from the right ventricle. Heptanol markedly decreased arrhythmia scores during ischaemia and reperfusion, as well as reducing infarct size to a degree similar to that induced by ischaemic preconditioning. In the prolonged ischaemia model, heptanol delayed the onset of uncoupling, increased time to plateau and decreased the maximal rate of uncoupling during ischaemia. Ischaemic preconditioning had similar effects on these parameters. In papillary muscle, heptanol reduced the conduction velocity of the action potential in a dose-dependent manner, but had no significant effect on resting potential, action potential amplitude, action potential duration, maximal upstroke of depolarization or effective refractory period. 3. These results demonstrate that treatment with the gap junction uncoupler heptanol confers cardioprotection against ischaemia and this effect is related to delayed electrical uncoupling during prolonged ischaemia.     

二甲双胍的心脏保护作用

二甲双胍是临床上广泛应用的治疗2型糖尿病的药物。近年来不断有证据表明二甲双胍具有心脏保护作用,可提高心肌供能,促进一氧化氮的合成,改善胰岛素抵抗、抗炎、抗纤维化,抑制心肌细胞凋亡。同时研究显示二甲双胍的药理作用是通过激活腺苷激活的蛋白激酶实现。腺苷激活的蛋白激酶是一种重要的激酶,在调节糖脂代谢方面发挥着重要作用。     

Post–conditioning reduces infarct size in the isolated rat heart: Role of coronary flow and pressure and the nitric oxide/cGMP pathway

We aimed to assess the role of the nitric oxide (NO)–cGMP pathway in cardioprotection by brief intermittent ischemias at the onset of reperfusion (i.e., post–conditioning (Post–con)). We also evaluated the role of coronary flow and pressure in Post–con. Rat isolated hearts perfused at constant– flow or –pressure underwent 30 min global ischemia and 120 min reperfusion. Post–con obtained with brief ischemias of different duration (modified, MPost–con) was compared with Post–con obtained with ischemias of identical duration (classical, C–Post–con) and with ischemic preconditioning (IP). Infarct size was evaluated using nitro–blue tetrazolium staining and lactate dehydrogenase (LDH) release. In the groups, NO synthase (NOS) or guanylyl–cyclase (GC) was inhibited with LNAME and ODQ, respectively. In the subgroups, the enzyme immunoassay technique was used to quantify cGMP release. In the constant–flow model, M–Post–con and C–Post–con were equally effective, but more effective than IP in reducing infarct size. The cardioprotection by M–Post–con was only blunted by the NOS–inhibitor, but was abolished by the GC–antagonist. Post–ischemic cGMP release was enhanced by MPost–con. In the constant–pressure model IP, M–Post–con and C–Post–con were equally effective in reducing infarct size. Post–con protocols were more effective in the constant–flow than in the constant–pressure model. In all groups, LDH release during reperfusion was proportional to infarct size. In conclusion, Post–con depends upon GC activation, which can be achieved by NOS–dependent and NOS–independent pathways. The benefits of M– and CPost–con are similar. However, protection by Post–con is greater in the constant–flow than in the constant–pressure model.