The role of inhibition of NO formation in the metabolic recovery of ischemic rat heart by apelin-12

The effects of apelin-12, a 12 amino acid peptide (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A-12), on recovery of energy metabolism and cardiac function have been studied in isolated working rat hearts perfused with Krebs buffer (KB) containing 11 mM glucose and subjected to global ischemia and reperfusion. Infusion of 140 μM A-12 before ischemia enhanced myocardial ATP, the total pool of adenine nucleotides (ΣAN = ATP+ADP+AMP) and the energy charge of cardiomyocytes ((ATP + 0.5ADP)/ΣAN) at the end of reperfusion compared with control (KB infusion) and decreased lactate content and lactate/pyruvate ratio in the reperfused myocardium up to the initial values. This was accompanied by improved recovery of coronary flow and cardiac function. Co-administration of A-12 and 100 μM L-NAME (an inhibitor of NO synthases) significantly attenuated the A-12 effects on metabolic and functional recovery of reperfused hearts. These results indicate involvement of NO in mechanisms of cardioprotection that are tightly associated with recovery of energy metabolism in the postischemic heart.     

Protection of the reperfused heart by L-propionylcarnitine.

The effects of L-propionylcarnitine on mechanical function, creatine phosphate and ATP content, and lactate dehydrogenase leakage were studied in isolated perfused rat hearts exposed to global no-flow ischemia for 30 min followed by reperfusion for 20 min. Five and 10 mM L-propionylcarnitine resulted in a 100% recovery of left ventricular-developed pressure, whereas the recovery was only 40% in the hearts perfused without this agent. Ischemia-reperfusion caused a 85% loss of creatine phosphate and a 77% loss of ATP, which was prevented by 10 mM L-propionylcarnitine. Five millimolar L-propionylcarnitine protected the heart from the loss of creatine phosphate but not from the loss of ATP. Ten millimolar L-propionylcarnitine failed to improve the postischemic left ventricular-developed pressure, when it was added to the perfusate only after ischemia. L-propionylcarnitine alleviated the decrease of coronary flow in the reperfused hearts. Lactate dehydrogenase leakage was aggravated in the beginning of the reperfusion period by 10 mM L-propionylcarnitine. This adverse effect was, however, transient. L-Propionylcarnitine provides protection for the postischemic reperfused heart in a dose-dependent manner. The optimal time for administration is before the ischemic insult. High doses of this compound may perturb cell membrane integrity. Moreover, the present data point to an intracellular, metabolic, and perhaps anaplerotic mechanism of action of L-propionylcarnitine in cardiac ischemia-reperfusion injury.     

The effect of myosin ATPase inhibition on metabolic and functional recovery of isolated rat heart after global ischemia

The effect of myosin ATPase inhibitor, 2,3-butanedione monoxime (BDM) used in the range of concentrations 1.25–10.0 mM), on recovery of functions of isolated rat heart subjected to normothermic (37 °C) total ischemia for 35 min has been investigated. BDM perfusion was performed at a flow rate of 4 ml/min during 5 min before ischemia (BDM-I) or before 25-min reperfusion (BDM-R). Control hearts were perfused with Krebs solution at the same flow rate. The highest functional recovery of heart and coronary vessels was observed during infusion of 2.5 mM BDM before ischemia. At the end of reperfusion ATP and phosphocreatine (PCr) content in hearts of this group was significantly higher whereas the level of lactate was two times lower than in control; total creatine content (ΣCr) did not differ from the initial level. Similar but less pronounced changes in the improvement of aerobic metabolism and maintenance of ΣCr after reperfusion were also observed in the case of infusion of 2.5 mM BDM before reperfusion. They were consistent with reduced recovery of functions of heart and coronary flow compared with these parameters observed in the BDM-I group. 2.5 mM BDM caused almost 2-fold decrease in release of cardiac lactate dehydrogenase into myocardial perfusate in the BDM-I and BDM-R groups (compared with control); this suggests lower damage of cell membranes. These results suggest that improvement of energy supply of postischemic cardiomyocytes may be a key factor determining cardioprotector effectiveness of short-term administration of BDM before ischemia.     

Allopurinol-Enhanced Postischemic Recovery in the Isolated Pat Heart Involves Repletion of High-Energy Phosphates

The effects of allopurinol (AP) on functional and metabolic recovery of the isolated rat heart after global ischemia were studied. Hearts were subjected to aerobic perfusion (30 min), cardioplegic infusion (5 min), normothermic ischemia (37 min), and reperfusion (50 min) which was started with secondary cardioplegic infusion (10 min). AP was injected into rats (44 mg/kg body wt ip 2 h before heart excision) and added to cardioplegic solution (2 mM) prior and after ischemia. AP treatment significantly improved postischemic recovery of the function and reduced the leakage of lactate dehydrogenase from reperfused hearts. These beneficial effects were accompanied by a better preservation of tissue content of ATP, the total adenine nucleotides, phosphocreatine, and the total creatine at the end of reperfusion. Inhibition of xanthine oxidase by AP substantially decreased pre- and postischemic release of xanthine and uric acid and increased postischemic release of hypoxanthine into the coronary effluent. Despite this, AP treated hearts did not exhibit a reduction in hydroxyl radical adduct formation in the effluents at reperfusion assessed by the spin-trap measurements. The results suggest that AP may protect the heart from ischemia/reperfusion injury due to enhanced energy provision rather than by prevention of oxygen-derived free radical formation.     

Effect of various reperfusion regimens on the recovery of myocardial contractile function after total ischemia

Isolated guinea pig hearts were subjected to 25-min total ischemia at 37 degrees C followed by 30-min reperfusion. The product of the left ventricular isovolumic systolic pressure and heart rate representing the cardiac work index was restored to 33 +/- 5% of initial value and diastolic pressure (DP) remained substantially elevated by 47 +/- 9 mm Hg if reperfusion was resumed with initial rate 10 ml/min. The gradual restoration of perfusion rate initiating from 2 or 4 ml/min was performed in other series, and was associated with slower but higher recovery of cardiac work and lower DP by the end of reperfusion. The similar result was observed when reperfusion was resumed with initial rate but a modified solution was used for first 5 min. In which Ca++ content was reduced while K+ and Mg++ elevated. In this case final recovery of cardiac work was 59 +/- 2% and DP completely returned to initial level. It is suggested that optimal reperfusion mode should be associated with slower work recovery.     

Metabolic protection of the reperfused canine endocardium by the thromboxane synthetase inhibitor, dazoxiben

This study investigated whether dazoxiben, a thromboxane synthesis inhibitor, could reverse regional contractile dysfunction and protect against adenine nucleotide loss in the "stunned myocardium". Hearts from anesthetized dogs were "stunned" by 15 min of left anterior descending coronary artery occlusion followed by 3 hr of reperfusion. Left ventricular segment shortening (%SS) and regional myocardial blood flow (RMBF) were measured by sonomicrometry and the radioactive microsphere technique, respectively. Local coronary venous blood was withdrawn and thromboxane A2 and prostacyclin measured by radioimmunoassay. Transmural biopsies from the reperfused and nonischemic areas were taken at 3 hr following reperfusion for tissue metabolite analysis. During ischemia, %SS, RMBF and area at risk were decreased to similar levels in both control and dazoxiben-treated hearts indicating equivalent degrees of flow deprivation. During reperfusion, %SS recovered only partially and was not significantly improved by dazoxiben. Dazoxiben augmented peak prostacyclin production (123 +/- 31% vs. 292 +/- 49% of preocclusion values) following reperfusion, while it completely blocked thromboxane A2 production. Dazoxiben attenuated the decline in endocardial ATP (69 +/- 5% vs. 92 +/- 9% normalized to the nonischemic zone) and total adenine nucleotides. The results indicate that dazoxiben may elicit a cardioprotective effect on energy metabolism in the reperfused heart, but this is dissociated from any improvement in regional contractile function.     

Energy metabolism and mechanical recovery after cardioplegia in moderately hypertrophiedrats

It is well established that severe hypertrophy induces metabolic and structural changes in the heart which result in enhanced susceptibility to ischemic damage during cardioplegic arrest while much less is known about the effect of cardioplegic arrest on moderately hypertrophied hearts. The aim of this study was to elucidate the differences in myocardial high energy phosphate metabolism and in functional recovery after cardioplegic arrest and ischemia in mildly hypertrophied hearts, before any metabolic alterations could be shown under baseline conditions.Cardiac hypertrophy was induced in rats by constriction of the abdominal aorta resulting in 20% increase in heart weight/body weight ratio (hypertrophy group) while sham operated animals served as control. In both groups, isolated hearts were perfused under normoxic conditions for 40 min followed by infusion of St.Thomas' Hospital No. 1 cardioplegia and 90 min ischemia at 25øC with infusions of cardioplegia every 30 min. The changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were followed by³¹ P nuclear magnetic resonance (NMR) spectroscopy. Systolic and diastolic function was assessed with an intraventricular balloon before and after ischemia.Baseline concentrations of PCr, ATP and Pi as well as coronary flow and cardiac function were not different between the two groups. However, after cardioplegic arrest PCr concentration increased to 61.8 ± 4.9 mol/g dry wt in the control group and to 46.3 ± 2.8 mol/g in hypertrophied hearts. Subsequently PCr, pH and ATP decreased gradually, concomitant with an accumulation of Pi in both groups. PCr was transiently restored during each infusion of cardioplegic solution while Pi decreased. PCr decreased faster after cardioplegic infusions in hypertrophied hearts. The most significant difference was observed during reperfusion: PCr recovered to its pre-ischemic levels within 2 min following restoration of coronary flow in the control group while similar recovery was observed after 4 min in the hypertrophied hearts. A greater deterioration of diastolic function was observed in hypertrophied hearts.Moderate hypertrophy, despite absence of metabolic changes under baseline conditions could lead to enhanced functional deterioration after cardioplegic arrest and ischemia. Impaired energy metabolism resulting in accelerated high energy phosphate depletion during ischemia and delayed recovery of energy equilibrium after cardioplegic arrest observed in hypertrophied hearts could be one of the underlying mechanisms.     

Ischemic preconditioning and superoxide dismutase protect against endothelial dysfunction and endothelium glycocalyx disruption in the postischemic guinea-pig hearts

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.     

Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release

We investigated the contribution of dexamethasone treatment on the recovery of postischemic cardiac function and the development of reperfusion-induced arrhythmias in ischemic/reperfused isolated rat hearts. Rats were treated with 2 mg/kg of intraperitoneal injection of dexamethasone, and 24 hours later, hearts were isolated according to the 'working' mode, perfused, and subjected to 30 min global ischemia followed by 120 min reperfusion. Cardiac function including heart rate, coronary flow, aortic flow, and left ventricular developed pressure were recorded. After 60 min and 120 min reperfusion, 2 mg/kg of dexamethasone significantly improved the postischemic recovery of aortic flow and left ventricular developed pressure from their control values of 10.7 +/- 0.3 ml/min and 10.5 +/- 0.3 kPa to 22.2 +/- 0.3 ml/min (p < 0.05) and 14.3 +/- 0.5 kPa (p < 0.05), 19.3 +/- 0.3 ml/min (p < 0.05) and 12.3 +/- 0.5 kPa (p < 0.05), respectively. Heart rate and coronary flow did not show a significant change in postischemic recovery after 60 or 120 min reperfusion. In rats treated with 0.5 mg/kg of actinomycin D injected i.v., one hour before the dexamethasone injection, suppressed the dexamethasone-induced cardiac protection. Electrocardiograms were monitored to determine the incidence of reperfusion-induced ventricular fibrillation. Dexamethasone pretreatment significantly reduces the occurrence of ventricular fibrillation. Cytochrome c release was also observed in the cytoplasm. The results suggest that the inhibition of cytochrome c release is involved in the dexamethasone-induced cardiac protection.     

Evaluation of the role of xanthine oxidase in myocardial reperfusion injury

The free radical-generating enzyme xanthine oxidase has been hypothesized to be a central mechanism of the injury which occurs in postischemic tissues; however, its importance remains controversial. Much attention has focused on the role of this enzyme in myocardial reperfusion injury. While xanthine oxidase has been observed in ischemic tissue homogenates, the presence and importance of radical generation by the enzyme in intact tissues are unknown. Therefore, we performed electron paramagnetic resonance, nuclear magnetic resonance and hemodynamic studies to measure the presence and significance of xanthine oxidase-mediated free radical generation in the isolated rat heart. When isolated perfused rat hearts were reperfused after 30 min of global ischemia, myocardial function and coronary flow were significantly improved in the presence of the definitive xanthine oxidase blocker oxypurinol. Free radical concentrations measured by spin-trapping with 5,5'-dimethyl-1-pyrroline-N-oxide were significantly decreased by oxypurinol and the energetic state of the heart was improved as reflected by an increased recovery of phosphocreatine and a higher phosphocreatine/Pi ratio. ATP recovery, however, was not altered, indicating that the improved functional and metabolic state of the heart was not due to ATP salvage. Spectrophotometric assays for the enzyme showed an increase in the amount of xanthine oxidase relative to dehydrogenase following ischemia, and a total available xanthine oxidase pool in the rat heart of approximately 150 milliunits/g of protein. Thus, xanthine oxidase is a significant source of the oxidative injury which occurs upon reperfusion of the ischemic rat heart.     

一氧化氮和线粒体ATP敏感性钾通道介导血管紧张素转换酶抑制剂加强阈下预处理的作用

实验采用离体大鼠心脏Langendorff灌流模型,观察含巯基（卡托普利）和不含巯基（培哚普利拉）的两种血管紧张素转换酶抑制剂（angiotensin-converting enzyme inhibitors,ACEI）对抗心肌缺血的作用,并探讨一氧化氮（nitric oxide,NO）和线粒体ATP敏感性钾通道（mimchondrial ATP-sensitive potassium channel,mitoKATP channel）是否参与ACEI的心肌保护作用。结果表明：（1）给予大鼠心脏2min全心停灌和10min复灌作为闽下缺血预处理（subthreshold preconditioning,sPC）、卡托普利或培哚普利拉单独使用,均不能改善长时间缺血复灌（缺血30min＋复灌120min）引起的心肌损伤。（2）当两种ACEI分别和sPC联合使用时,与sPC组相比,缺血心脏在长时间缺血后的复灌期问左室舒张末压（left ventricular end-diastolic pressure,LVEDP）明显降低,左宦发展压（left ventricular developed pressure,LVDP）和冠脉流量明显增高,乳酸脱氢酶（lactate dehydrogenase,LDH）的释放量和心肌梗死面积明显低于sPC组。（3）利用NOS抑制剂L-NAME和mitoKATP通道的抑制剂5-HD灌流10min后,可明显抑制卡托普利／培哚普利拉和sPC联合使用引起的LVEDP降低,并使LVDP和冠脉流量降低,LDH的释放量和心肌梗死面积明显增高（P〈0．05）。（4）sPC、卡托普利或培哚普利拉单独使用,心脏NO的产生增加。ACEI和sPC联合使用,与三者单独使用相比NO的浓度亦明显增高（P〈0．05）。结果提示：含与不含巯基的ACEI与闽下缺血预处理联合使用均可使大鼠心脏功能明显改善,其心肌保护作用的机制可能通过NO途径,并和mitoKATP通道的激活有关。     

Relationship between oxidative phosphorylation and adenine nucleotide translocase activity of two populations of cardiac mitochondria and mechanical recovery of ischemic hearts following reperfusion

The possible relationship of the atractyloside-sensitive adenine nucleotide translocase activity, oxidative phosphorylation, and the recovery of ventricular contractility following reperfusion of the ischemic isolated rat heart was studied. Five minutes of total global ischemia without reperfusion produced a significant depression in adenine nucleotide translocase in subsarcolemmal mitochondria (SLM), whereas a minimum of 10 min ischemia was required to observe a significant depression in interfibrillar mitochondria (IFM). Increasing durations of ischemia resulted in a progressively larger depression in translocase activity, with a maximum depression of approximately 75% seen in both populations following 20 min ischemia. In contrast, oxidative phosphorylation was totally unaffected in either mitochondrial population following up to 20 min of ischemia. We assessed whether translocase activity or oxidative phosphorylation were related to contractile recovery in hearts reperfused following various durations of ischemia. In SLM, translocase activity was further depressed following reperfusion compared with pre-reperfusion ischemic values, whereas with IFM only reperfusion following 5 min ischemia produced a further depression in translocase values. Oxidative phosphorylation rates of SLM and IFM were significantly depressed following reperfusion of ischemic hearts, although SLM exhibited a generally higher sensitivity in this regard. In reperfused hearts, an overall significant relationship was found between oxidative phosphorylation rate and adenine translocase activity as well as between translocase activity and post-reperfusion contractile recovery. These data show that ischemia can produce a significant depression in translocase activity in the absence of any change in oxidative phosphorylation. The results also suggest that the depression in mitochondrial ADP/ATP translocase and subsequent inhibition of oxidative phosphorylation in the reperfused heart may represent one of the important contributory mechanisms involved in cardiac failure and injury during acute ischemia and reperfusion.     

Effects of epinephrine on underperfusion-reperfusion injuries in diabetic and non-diabetic rat hearts

The sympathetic nervous systems may bear relevance to the increased incidence of heart failure in diabetes (DM). In our isolated rat hearts perfused at constant low flow rate, norepinephrine dose-dependently enhanced diabetic myocardial damage, particularly during underperfusion. The purpose of this investigation is to examine the effects of epinephrine on the ischemic injury and on the reperfusion injury in DM and non-DM rat hearts, and to clarify whether the cardiac states during underperfusion at constant low pressure are similar to those at constant low flow rate. Isolated streptozotocin-induced 6-week DM and non-DM rat hearts with a balloon in the left ventricle (LV) were paced and normal perfused at 75 cm H₂O with normoxic Krebs-Henseleit solution. Then the hearts were underperfused at 35 cm H₂O, a constant low pressure with below one-third of the pre-ischemic coronary perfusion flow (CPF) level. Four min after the start of underperfusion, the perfusate was changed to that containing epinephrine 10^–6 M. After 45 min underperfusion with or without epinephrine, all of the hearts were reperfused without epinephrine at 75 cm H₂O for 45 min. To detect changes in LV stiffness, the isometric tension along the longitudinal direction of the whole heart and the LV isovolumic pressure were monitored simultaneously. In DM hearts, the underperfusion alone caused a slight increase in LV stiffness, and all the changes recovered to the pre-ischemic levels during reperfusion. Epinephrine during underperfusion accelerated the start of increase in LV stiffness and the decrease in CPF. During reperfusion the changes recovered partly to the control levels. In non-DM hearts, epinephrine during underperfusion caused only a slight increase in LV stiffness though a similar low CPF to DM hearts. However, the reperfusion caused a marked increase in LV stiffness and a lower recovery of CPF. Epinephrine at constant low pressure, as well as norepinephrine at constant low flow rate, enhanced the ischemic injury, particularly in DM hearts, while aggravated the reperfusion injury in non-DM hearts.     

二氮嗪在长时程心脏低温保存中的作用

延长心脏的体外有效保存时间对临床心脏移植具有重要意义。本文旨在研究线粒体ATP敏感性钾通道开放剂二氮嗪(diazoxide,DE)在离体大鼠心脏长时程低温保存中的作用。SD大鼠随机分成5组,包括对照组(单纯Celsior保存液),DE组(Celsior液中含15、30或45μmol／L的DE)和DE 5-HD组[Celsior液中含30μmol／L的DE和100μmol／L的5-羟基葵酸盐(5-hydroxydecanoate,5-HD)]。利用Langendorff离体鼠心灌注法,观察心脏在4℃条件下保存10h后,复灌期血流动力学恢复、冠脉流出液中心肌酶漏出量及心肌水含量变化,并做心肌超微结构检查。结果显示：与对照组比较,DE处理后,复灌期的左心室舒张末期压力明显降低,心率、左心室发展压、左心室压力变化率、冠脉流出量等的恢复率在多个复灌时间点上优于对照组,且能显著减少复灌过程中心肌酶(乳酸脱氢酶、磷酸肌酸激酶及谷草转氨酶)的漏出量,降低心肌水含量;其中30和45μmol／LDE组的保护作用优于15μmol／LDE组;电镜结果显示DE对长时程低温保存心脏的超微结构有较好的保护作用。DE的上述作用可被线粒体ATP敏感性钾通道的特异性阻断剂5-HD所取消。以上结果提示：DE可通过激活线粒体ATP敏感性钾通道显著改善离体大鼠心脏长时程低温保存效果。     

The effect of dichloroacetate on the isolated no flow arrested rat heart

Ischemic dysfunction, including contracture, has been attributed to lack of ATP, although previous work has not been consistent with this concept. We describe here a model of no flow ischemic arrest, characterized by depressed levels of mechanical function upon reperfusion and high energy phosphate stores within normal limits. The decreased mechanical function bears an inverse relationship to myocardial lactate levels after twenty-minutes of reperfusion in the absence or presence of dichloroacetic acid (DCA). Post-ischemic non-DCA treated hearts attained peak work of only 25% of that of controls, while those treated with DCA following ischemia performed almost as well as controls. ATP and CP levels remained high in both DCA treated and non-DCA treated hearts. Lactate levels were high in hearts immediately following ischemia, but were reduced to control levels in post-ischemic hearts perfused with DCA within twenty minutes, whereas those not treated with DCA had lactate levels two to three times that of controls within the same time period. Pyruvate dehydrogenase (PDH) activity was reduced in non-DCA treated post ischemic hearts after twenty minutes reperfusion but was elevated above controls in hearts reperfused with DCA. The data indicates that DCA increases mechanical performance of the isolated post-ischemic rat heart and the proposed mechanism for this increase is the oxidative removal of lactate resulting from an increase in PDH activity.     

Effects of genetic deletion of angiotensin-(1-7) receptor Mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart

In this study we investigated the role of Mas on cardiac function during ischemia/reperfusion in isolated perfused mouse heart. Following a stabilization period of 30 min, hearts from WT and Mas KO mice were subjected to global ischemia. After 20 min of ischemia, the flow was restarted and the hearts were reperfused for 30 min. An additional group of WT mice was perfused with solution containing the Ang-(1-7) receptor Mas antagonist A-779. Isolated heart of Mas KO and WT treated with A-779 presented an increase in the perfusion pressure in the baseline period. This difference increased with 5 min of reperfusion reaching similar values to baseline period at the end of the reperfusion. Isolated hearts of Mas KO and WT treated with A-779 also presented a decreased systolic tension, +/-dT/dt, and HR. Upon global ischemia WT hearts showed a significant decrease in systolic tension and an increase in diastolic tension. During reperfusion an increase in systolic and diastolic tension was observed in WT mice. Deletion or blockade of Mas markedly attenuated these changes in isolated hearts. These results indicate that Mas plays an important role in cardiac function during ischemia/reperfusion which is in keeping with the cardiac and coronary effects previously described for Ang-(1-7).     

Microdialysis study in vivo of the release of adenine nucleotide degradation products into intercellular space of canine myocardium during regional ischemia and reperfusion

Intercellular concentrations of adenine nucleotide degradation products (ANDP)--adenosine inosine and hypoxanthine--in ischemic and control regions of the canine myocardium were measured by microdialysis technique during 20- and 40-min coronary artery occlusion and reperfusion. In hearts that fibrillated on reperfusion during the ischemic 40-min period catabolism of adenine nucleotides was more intensive, which could be the min cause of the reperfusion ventricular fibrillation. Reperfusion ventricular fibrillation was accompanied by an increase in the intercellular ANDP level in the control region, that indicated the development of the total myocardial ischemia. During the initial period of reperfusion after 20-min, a sharp increase in the interstitial ANDP level was observed in the ischemic region as compared with the end of the ischemia which could be explained as a result of demasking of reperfusion damage in such a case. The 40-min reperfusion induced slow reduction of the intercellular ANDP level in the ischemic region, while the regional blood flow already 5 min after the reperfusion did not differ from the blood flow in the control region. It is supposed that a slow washout of ANDP could be caused by the "no-reflow" phenomenon.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

Acetaminophen in the post-ischemia reperfused myocardium

Acetaminophen was administered acutely at the onset of reperfusion after 20 min of low-flow, global myocardial ischemia in isolated, perfused guinea pig hearts (Langendorff) to evaluate its influence in the postischemia, reperfused myocardium. Similarly prepared hearts were treated with vehicle or with uric acid (another phenol for comparison). Functionally, acetaminophen-treated hearts (0.35 mM) achieved significantly greater recovery during reperfusion. For example, left ventricular developed pressures at 40 min reperfusion were 38 +/- 3, 27 +/- 3, and 20 +/- 2 in the presence of acetaminophen (P < 0.05, relative to the other two groups), vehicle, and uric acid, respectively. Coronary perfusion pressures and calculated coronary vascular resistances, in the acetaminophen-treated hearts, were significantly lower at the same time (e.g., coronary perfusion pressures in the three groups, respectively, were 40 +/- 2 [P < 0.05], 51 +/- 3, and 65 +/- 12 mm Hg). Under baseline, control conditions, creatine kinase ranged from 12-15 units/liter in the three groups. It increased to 35-40 units/liter (P < 0.05) during ischemia but was significantly reduced by acetaminophen during reperfusion (e.g., 5.3 +/- 0.8 units/liter at 40 min). Oxidant-mediated chemiluminescence in all three treatment groups during baseline conditions and ischemia was similar (i.e., approximately 1.5-2.0 min for peak luminescence to reach its half maximal value). It took significantly more time during reperfusion for the oxidation of luminol in the presence of acetaminophen (>20 min, P < 0.05) than in its absence (3-8 min in uric acid- and vehicle-treated hearts). These results suggest that administration of acetaminophen (0.35 mM), at the onset of reperfusion, provides anti-oxidant-mediated cardioprotection in the postischemia, reperfused myocardium.     

Lack of both oxygen and glucose contributes to I/R-induced changes in cardiac SR function

Although ischemia-reperfusion(I/R) has been shown to depress cardiac performance and sarcoplasmicreticulum (SR) function, the mechanisms underlying these alterationsare poorly understood. Because lack of oxygen and substrate deprivationare known to occur during the ischemic phase, we examined theeffects of reperfusion on cardiac performance and SR function in heartssubjected to hypoxia and substrate lack. For this purpose, isolated rathearts were perfused with hypoxic and/or glucose-free medium for 30 min and then reperfused with normal medium for 1 h; the SR vesicles were isolated for studying the Ca²⁺-transport activities.Reperfusion with normal medium of hearts deprived of oxygen or glucoseshowed no changes in cardiac performance and SR function. However,reperfusion of hearts perfused with hypoxic glucose-free medium showed~45% decrease in cardiac contractile activities as well as 23 and64% reduction in SR Ca²⁺-uptake andCa²⁺-release activities, respectively, without any changein the level of SR Ca²⁺-cycling proteins. Depressed SRfunction in these hearts was associated with a reduction inCa²⁺/calmodulin-dependent protein kinase (CaMK)phosphorylation of the SR Ca²⁺-cycling proteins and 34%decrease in SR CaMK activity. These changes in cardiac performance, SRfunction, and SR CaMK activity in the hypoxic, glucose-deprived,reperfused hearts were similar to those observed in hearts subjected to30 min of global ischemia and 60 min of reperfusion. Theresults therefore suggest that the lack of both oxygen and substrateduring the ischemic phase may contribute to the I/R-inducedalterations in cardiac performance and SR function. Furthermore, theseabnormalities were associated with reduced SR CaMK activity.