In vivo heat shock preconditioning mitigates calcium overload during ischaemia/reperfusion in the isolated, perfused rat heart

Heat shock (HS) pretreatment of the heart is effective in mitigating the deleterious effects of ischaemia/reperfusion. The main objective of this study was to determine whether the beneficial effect of HS is associated with the preservation of intracellular Ca²⁺ handling in the ischaemic/reperfused, isolated rat heart. Twenty-four hours after raising body core temperature to 42 °C for 15 min, rat hearts were perfused according to Langendorff and subjected to 30 min ischaemia followed by 20 min reperfusion. Cyclic changes of cytoplasmic calcium ion [Ca²⁺_i] levels were measured by surface fluorometry using Indo-1 AM. Reperfused HS hearts showed improved recovery of contractile function compared with control hearts: end-diastolic pressure: 45±11 vs. 64±22 mm Hg; developed pressure: 72±12 vs. 41±20 mm Hg; maximum rate of pressure increase (+dP/dt_max): 1,513±305 vs. 938±500 mm Hg/s; maximum rate of pressure decrease (–dP/dt_max): –1,354±304 vs. –806±403 mm Hg/s. HS hearts displayed a significantly lower end-diastolic cytosolic [Ca²⁺] ([Ca²⁺]_i) after reinstallation of flow. The dynamic parameters of the Ca²⁺_i transients, i.e. the maximum rate of increase/decrease (±dCa²⁺_i/dt_max) and amplitude, did not differ between reperfused control and HS hearts. The novel finding of this study is that improved performance of the HS-preconditioned heart after an ischaemic insult is associated with a reduced end-diastolic Ca²⁺_i load, and most likely, preserved Ca²⁺ sensitivity of the myocardial contractile machinery.     

非创伤性预处理在离体心脏抗再灌注损伤中作用机制的探讨

目的:观察非创伤性肢体缺血预处理对大鼠离体再灌注心肌是否有保护作用。方法:实验采用体重(250±30)gSD雄性大鼠25只随机分成3组,在Langendorff装置上对大鼠离体心脏进行灌流。对照组(C,n=8):在灌注全程均用富氧K-H液(充以95%O₂+5%CO₂),在恒压(8.33kPa)、恒温(37℃)条件下灌注;缺氧/复氧组(A,n=8):预灌15min后,灌注心脏先全心缺血缺氧15min,随后15min复氧再灌注(37℃);非创伤性肢体缺血预处理组(N-WIP,n=9):先将大鼠双后肢捆绑5min,松开5min,反复4次后,随后的方法同R组。在相应时点分别测定冠脉流出液和心肌匀浆中超氧化物歧化酶(SOD)活性,Ca²⁺-Mg²⁺-ATP酶活性和丙二醛(MDA)含量,同时记录心肌细胞的单相动作电位(MAP)和心肌收缩张力曲线。结果:非创伤性肢体缺血预处理能使再灌注心律失常发生率显著低于A组;心肌组织中MDA含量显著低于A组,心肌组织中SOD活性显著高于A组,心肌细胞的膜电位、Ca²⁺-Mg²⁺-ATP酶活性及肌张力较稳定。结论:非创伤性肢体缺血预处理对大鼠离体再灌注心肌有明显的保护作用,可能是通过增强心肌的抗氧化能力、稳定心肌Ca²⁺-Mg²⁺-ATP酶活性和膜相结构等途径,提高心肌细胞对再灌注损伤的抵抗力。     

血红素氧化酶-1在离体大鼠心肌缺血预处理中的作用

目的：观察缺血预处理对离体大鼠心肌缺血再灌注后心功能、乳酸脱氢酶（LDH）活性和丙二醛（MDA）含量及血红素氧化酶-1活性的影响。方法：应用Langendorff离体大鼠等容收缩灌流模型行离体大鼠心脏灌流。缺血预处理方案为停灌5 min后再灌5 min反复3次,持续缺血再灌方案为停灌40 min后再灌20 min,监测正常对照组、缺血再灌组（IR）和缺血预处理组（IPC）心功能指标的同时,测定冠脉流出液LDH活性、心肌MDA含量和HO-1活性变化。结果：IPC组再灌20 min时的心功能恢复率显著高于IR组（P<0.01）,心肌血红素氧化酶-1活性也明显高于IR组（P<0.05）,而LDH活性及MDA含量显著少于IR组（均为P<0.01）。结论：血红素氧化酶-1活性增高可能与缺血预处理对大鼠缺血再灌心肌的保护作用有关。     

Influence of selective 5-HT agonists and myocardial preconditioning on ischaemia/reperfusion induced changes in isolated heart of the rat

Inflammation Research -     

Remote vs. local ischaemic preconditioning in the rat heart: infarct limitation,suppression of ischaemic arrhythmia and the role of reactive oxygen species

The unmet clinical need for myocardial salvage during ischaemia–reperfusion injury requires the development of new techniques for myocardial protection. In this study the protective effect of different local ischaemic preconditioning (LIPC) and remote ischaemic preconditioning (RIPC) protocols was compared in the rat model of myocardial ischaemia–reperfusion, using infarct size and ischaemic tachyarrhythmias as end‐points. In addition, the hypothesis that there is involvement of reactive oxygen species (ROS) in the protective signalling by RIPC was tested, again in comparison with LIPC. The animals were subjected to 30‐min coronary occlusion and 90‐min reperfusion. RIPC protocol included either transient infrarenal aortic occlusion (for 5, 15 and 30 min followed by 15‐min reperfusion) or 15‐min mesenteric artery occlusion with 15‐min reperfusion. Ventricular tachyarrhythmias during test ischaemia were quantified according to Lambeth Conventions. It was found that the infarct‐limiting effect of RIPC critically depends on the duration of a single episode of remote ischaemia, which fails to protect the heart from infarction when it is too short or, instead, too prolonged. It was also shown that RIPC is ineffective in reducing the incidence and severity of ischaemia‐induced ventricular tachyarrhythmias. According to our data, the infarct‐limiting effect of LIPC could be partially eliminated by the administration of ROS scavenger N‐2‐mercaptopropionylglycine (90 mg/kg), whereas the same effect of RIPC seems to be independent of ROS signalling.     

Cyclooxygenase-2 mediates the delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes

We previously reported that hydrogen sulfide (H₂S) preconditioning (SP) produces cardioprotection in isolated rat cardiomyocytes. The present study was designed to determine the involvement of cyclooxygenase-2 (COX-2) in the SP-induced delayed cardioprotection. Isolated cardiac myocytes were treated with NaHS (100 μM, a H₂S donor) for 30 min and then cultured for 20 h followed by ischemia/reperfusion insults. SP significantly increased cell viability, percentage of rod-shaped cells, and myocyte contractility after 10 min of reperfusion. Given 30 min before and during lethal ischemia, two selective COX-2 inhibitors, NS-398 and celebrex, abrogated SP-induced cardioprotective effects. Moreover, SP upregulated the expression of COX-2 and increased PGE₂ production in the cardiac myocytes. These effects were significantly attenuated by glibenclamide, an ATP-sensitive K⁺ channel (K_ATP) blocker, and chelerythrine, a selective protein kinase C (PKC) inhibitor, suggesting that activation of both K_ATP and PKC is required for the stimulation of COX-2. Additionally, NG-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, failed to regulate COX-2 protein expression but inhibited SP-enhanced COX-2 activity and PGE₂ production. In conclusion, we provided the first evidence that SP may produce delayed cardioprotection via K_ATP/PKC dependent induction of COX-2 expression and via nitric oxide-induced COX-2 activation.     

Compartmentation of glycolysis and glycogenolysis in the perfused rat heart

Developing methods that can detect compartmentation of metabolic pathways in intact tissues may be important for understanding energy demand and supply. In this study, we investigated compartmentation of glycolysis and glycogenolysis in the isolated perfused rat heart using (13)C NMR isotopomer analysis. Rat hearts previously depleted of myocardial glycogen were perfused with 5.5 mm [U-(13)C]glucose plus 50 mU/mL insulin until newly synthesized glycogen recovered to new steady-state levels ( approximately 60% of pre-depleted values). After a short wash-out period, the perfusate glucose was then switched to [1-(13)C]glucose, and glycolysis and glycogenolysis were stimulated by addition of glucagon (1 microg/ml). A (13)C NMR multiplet analysis of the methyl resonance of lactate provided an estimate of pyruvate derived from glucose vs glycogen while a multiplet analysis of the C4 resonance of glutamate provided an estimate of acetyl-CoA derived from glycolytic pyruvate vs glycogenolytic pyruvate. These two indices were not equivalent and their difference was further magnified in the presence of insulin during the stimulation phase. These combined observations are consistent with functional compartmentation of glycolytic and glycogenolytic enzymes that allows pyruvate generated by these two processes to be distinguished at the level of lactate and acetyl-CoA.     

大鼠脑缺血预适应对脑线粒体功能的影响

观察缺血预适应对大鼠大脑中动脉梗塞的保护作用 ,以及对大鼠脑线粒体功能的影响。观察大鼠双侧颈总动脉缺血预适应后 ,短暂缺血作用对脑梗塞面积、神经症状评分、倾斜板停留时间的影响 ,并测定线粒体复合酶活性、膜肿胀、膜电位、膜流动性的变化。与大脑中动脉梗塞 (MCAO)组比较 ,预适应可以显著降低大脑梗塞面积 (P <0 0 0 1) ;明显改善神经症状评分 ;延长在倾斜板上停留时间 (P <0 0 0 1) ;并可增强线粒体复合酶Ⅰ、Ⅲ、Ⅳ活性 ;线粒体膜肿胀降低 (P <0 0 0 1) ,流动性好于MCAO组 (P <0 0 1) ;膜电位无显著性变化。结果表明 ,缺血预适应对脑有保护作用 ;线粒体的膜受到了保护     

The role of endogenous angiotensin II in ischaemia,reperfusion and preconditioning of the isolated rat heart

We examined the possibility that endogenous angiotensin II (AII) is involved in the regulation of the cardiac Na(+)/H(+) exchanger (NHE1) during ischaemia, reperfusion and preconditioning. Mechanical function and intracellular sodium ([Na(+)](i)) were studied in isolated, perfused rat hearts. To test whether AII production might underlie the increased activity of NHE1 on reperfusion, we applied the AII receptor antagonist losartan during ischaemia and reperfusion. Losartan significantly improved mechanical performance on reperfusion and reduced the peak [Na(+)](i) on reperfusion. It has been proposed that preconditioning inhibits the activity of NHE1 in early reperfusion. To test whether this might be because of impaired action of AII on NHE1 we applied AII throughout ischaemia and reperfusion in preconditioned hearts. AII abolished the improved mechanical recovery caused by preconditioning and the peak [Na(+)](i) on reperfusion was similar to that after ischaemia alone. Addition of the NHE1 antagonist cariporide or losartan simultaneously with AII, reversed the deleterious effects of AII on the preconditioned heart. These studies suggest that AII contributes to the activation of NHE1 in early reperfusion and that part of the beneficial effect of preconditioning may be attributed to the abolition of AII-induced activation of NHE1.     

NO参与缺血预处理对大鼠骨骼肌缺血再灌注损伤的保护作用

目的研究缺血预处理(IPC)对大鼠骨骼肌缺血再灌注损伤的保护作用及一氧化氮(NO)是否参与此保护作用。方法采用Wistar雌性大鼠,在左后肢跟部止血,同时使用血流监测仪测股四头肌的血流量,调整止血带的松紧程度,使血流量控制在上止血带前的30%。30只大鼠随机分成3组,分别为缺血再灌注组(n=10)、缺血预处理组(n=10)、缺血预处理+NAME组(n=10)。应用尼克酰胺腺嘌呤二核苷酸黄递酶2+2+(NADH)染色,检测股四头肌各型肌纤维横截面积,钙-腺苷三磷酸酶(Ca-ATPase)染色,观察细胞膜CaATPase分布。透射电镜观测肌细胞的超微结构变化。结果缺血再灌注组出现明显肌纤维破裂溶解,许多白细胞浸润,电镜下线粒体中含有大量空泡变性、肌纤维断裂、溶解和Z线排列整齐膜脂质过氧化物的增加。与缺血再灌注组相比,缺血预处理组显示了轻微的损害,正常的纤维和血管变形少,股四头肌各型肌纤维横2+截面积降低,细胞膜Ca-ATPase数量增加。缺血预处理+NAME组与缺血再灌注组相比没有明显改变。结论缺血预处理对大鼠骨骼肌缺血再灌注损伤有明显保护作用,NO参与这一保护作用。     

Fatty acids are important for the Frank–Starling mechanism and Gregg effect but not for catecholamine response in isolated rat hearts

In some pathophysiological conditions myocardial metabolism can switch from mainly long chain fatty acid (LCFA) oxidation to mainly glucose oxidation. Whether the predominant fatty acid or glucose oxidation affects cardiac performance has not been defined. In a buffer perfused isovolumetrically contracting rat heart, oxidation of endogenous pool LCFA was avoided by inhibiting carnitine‐palmitoyl‐transferase I (CPT‐I) with oxfenicine (2 mm ). In order to restore fatty acid oxidation, hexanoate (1 mm ), which bypasses CPT‐I inhibition, was added to the perfusate. Three groups of hearts were subjected to either an increase in left ventricular volume (VV, +25%) or an increase in coronary flow (CF, +50%), or inotropic stimulation with isoproterenol (10^?8 and 10^?6 m ). The increase in VV (the Frank–Starling mechanism) increased rate–pressure product (RPP) by 21 ± 2% under control conditions, but only by 6 ± 2% during oxfenicine‐induced CPT‐I inhibition. The contractile response to changes in VV recovered after the addition of hexanoate. Similar results were obtained in hearts, in which an increase in CF was elicited (the Gregg phenomenon). Isoproterenol caused a similar increase in contractility regardless of the presence of oxfenicine or hexanoate. In all groups, a commensurate increase in oxygen consumption accompanied the increase in contractility. The fatty acid oxidation is necessary for an adequate contractile response of the isolated heart to increased pre‐load or flow, whereas the inotropic response to adrenergic β‐receptor stimulation is insensitive to changes in substrate availability.     

Effect of hypobaric hypoxia on the development of long-term posttetanic potentiation in slices of rat olfactory cortex: Correction with hypoxic preconditioning

We studied the effect of in vivo hypobaric hypoxia on the development (after 3 h) of in vitro long-term posttetanic potentiation in Wistar rats. Severe hypoxia suppressed induction of posttetanic potentiation in slices of the olfactory cortex. Preconditioning exposure (moderate hypobaric hypoxia) prevented inhibition of posttetanic potentiation induced by severe hypoxia. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 487–489, November, 2006     

Characterization of contractions in mechanically and enzymatically isolated myocardial cells from the rat heart ventricles

Single spontaneously beating myocardial cells were prepared by mechanical or enzymatical isolation. Using a TV camera and videotape recorder, beating parameters were characterized in different buffer solutions in regard to temperature and integrity of sarcolemma, which was tested by ability of cells to exclude a vital stain, Evans blue. The mechanically disaggregated cells were totally permeable to this stain, but about 60% of the enzymatically isolated myocytes were able to exclude the stain. The myocytes prepared in the presence of calcium were most tolerant to physiological concentration of calcium. Contractions were mainly of phasic type where the velocity of contraction wave was very slow (50–330 μm/s). The velocity was not correlated to the beat rate. Temperature dependence of the velocity was positively correlated with the integrity of sarcolemma. Beat rate was inversely related to the integrity of sarcolemma being slowest in calcium tolerant enzymatically isolated cells. Beat rate of calcium sensitive myocytes was higher but unstable and the temperature dependence of the rate was steeply reduced and even reversed during the incubation. Some calcium tolerant myocytes generated occasionally an electric type of contraction which came in bursts and was characterized by synchronous sarcomere shortening without any contraction wave.     

Apamin,a selective blocker of SKCa channels,inhibits posthypoxic hyperexcitability but does not affect rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro

The aim of this study was to investigate the effects of apamin, a selective blocker of SK_Ca channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. Application of apamin (50 nM) to the hippocampal slices during hypoxic episodes significantly abolished posthypoxic hyperexcitability induced by brief hypoxic episodes. However, in contrast to our previous results with iberiotoxin, a selective blocker of BK_Ca channels, apamin significantly enhanced the depressive effect of brief hypoxia on the PS amplitude during hypoxic episode and did not abolish the rapid hypoxic preconditioning in CA1 pyramidal neurons. Present results indicate that SK_Ca channels, along with previously implicated BK_Ca channels, play an important role in the development of posthypoxic hyperexcitability induced by brief hypoxic episodes in CA1 pyramidal neurons. However, SK_Ca channels, in contrast to the BK_Ca channels, are not involved in the rapid hypoxic preconditioning in CA1 hippocampal region in vitro.     

Effects of high oxygen tension on the metabolism of vasoactive hormones in isolated perfused rat lungs

The effect of exposure of rats to high concentrations of oxygen (90–95%, normobaric) on the activation of angiotensin I to angiotensin II and on the inactivation of bradykinin, prostaglandin E₂ (PGE₂) and 5-hydroxytryptamine (serotonin) in the pulmonary circulation of isolated perfused rat lungs was investigated. After 36 h exposure, PGE₂ survival in the pulmonary circulation increased and reached 3 times the control value after 48 h exposure. A decrease in the conversion of angiotensin I to angiotensin II was seen after 48 h exposure. No decrease in the inactivation of 5-hydroxytryptamine was seen until after 60 h exposure. At this time bradykinin inactivation was also decreased. The decrease in metabolism of angiotensin I and PGE₂ following 48 h exposure to oxygen was reversed by a subsequent exposure to room air for 12 h. In these experiments, therefore, the earliest sign of oxygen toxicity was a decrease in PGE₂ metabolism, a reaction associated with cells other than endothelial cells.     

Bromocriptine and lisuride stimulate the accumulation of cyclic AMP in intact slices but not in homogenates of rat neostriatum.

The effects of bromocriptine and lisuride on cyclic AMP concentrations in homogenates and in intact slices of rat neostriatum were investigated. Significant increases in cyclic AMP concentration were found after a 10-min exposure to bromocriptine and lisuride in striatal intact slices. On the contrary, as previously found, the two dopaminergic ergot derivatives did not stimulate dopamine-senstiive adenylate cyclase present in striatal homogenates. The stimulatory effects observed only in intact tissues were blocked by the specific dopamine receptor blocking agent fluphenazine. It is tempting to conclude that dopaminergic ergot derivatives have a site of action different from that stimulated by classic dopamine agonists in tissue homogenates.     

Alteration of cardiac energy state during development of hypertension in rats of the Lyon strain: a 31 P-NMR study on the isolated rat heart

The effect of different chronic blood pressure levels on cardiac energy metabolism was studied by ³¹P-NMR spectroscopy in perfused hearts from the Lyon strains of hypertensive (LH), normotensive (LN) and hypotensive (LL) rats at the ages of 12 and 21 weeks. The in vivo assessment of haemodynamic parameters measured at 21 weeks in anaesthetized rats with an ultraminiature catheter pressure transducer confirmed that left ventricular systolic pressure and mean aortic pressure were significantly greater (+ 25 %) in LH rats than in LN and LL rats. In the LL animals, left ventricular systolic pressure was slightly reduced (–10%) and cardiac contractility (estimated by LV dP/dt_max) showed a 24% decreased compared to normotensive animals. The energy state of the cardiomyocytes was characterized at different work levels of isolated rat hearts, by determining the concentration of the free phosphorylated compounds at each work level. Changes in workload were induced by varying the calcium concentration in the perfusion fluid. Increasing extracellular calcium concentration resulted in a similar increase in left ventricular developed pressure (LVDP) in all groups studied. Intracellular pH was not influenced by either the age of the animals or the level of cardiac work, in the three groups of animals. ATP content of the LN and LL rats remained constant during the whole perfusion period while the 12 week-old LH rats showed a decreased ATP content with increasing cardiac work. In the older LH rats, ATP content was decreased at the highest work level (corresponding to 2 mM calcium). In response to the increase in work, phosphocreatine (PCr) content diminished and inorganic phosphate (Pi) content increased in both LN, LH and LL animals. PCr degradation and Pi accumulation were higher in the LH rats and less in LL rats compared to the LN. These changes were more important in the younger than in the older hypertensive animals. The relationship between LVDP and [Pi]/[PCr] indicates that oxidative metabolism is maximally activated in the young hypertensive rats and suggests that this maximal activation represents an adaptative phase to the increase in blood pressure. Since the difference between the metabolic pattern of the 21 week-old LH rats and age-matched LN rats was less pronounced, it is likely that a compensatory stage has been reached at that age.