Asymmetric dimethyl L-arginine (ADMA) is a critical regulator of myocardial reperfusion injury

OBJECTIVE: Endothelial dysfunction by the loss of nitric oxide (NO) is a critical event during reperfusion of ischemic myocardium. Reduced NO availability signals important pathophysiological changes leading to myocardial reperfusion injury. We have recently shown that NO biosynthesis can be disturbed by the endogenous NO synthase (NOS) inhibitor ADMA and that these changes are mediated by an impairment of its metabolism by dimethylarginine dimethylaminohydrolase (DDAH). We therefore analyzed the role of ADMA and its metabolism in the setting of myocardial ischemia and reperfusion. METHODS: C57-bl6 mice underwent myocardial ischemia for exactly 30 min followed by 2, 4, 8, 12, 24, and 72 h of reperfusion achieved by occlusion and re-opening of the left coronary artery. The reperfused left ventricle was subsequently homogenized for measurements of determinants of the NO synthase pathway. Furthermore, the effects and its mechanisms of ADMA on reperfusion injury were analyzed in a genetic mouse model. RESULTS: A significant accumulation of ADMA was found in myocardial tissue when mice were subjected to 30 min of ischemia followed by reperfusion in our in vivo model. The maximum increase of tissue ADMA at 4 h of reperfusion coincided with reductions of NO tissue concentrations and DDAH activity; protein expression of NOS isoforms, however, was not changed. Furthermore, DDAH overexpression in a genetic mouse model as well as treatment with oral L-arginine markedly reduced reperfusion injury by 40-50% at 4 h of reperfusion. The effects of ADMA on reperfusion injury were shown to be mediated by reduced eNOS activity and phosphorylation, expression of adhesion molecules, and leukocyte activity. CONCLUSION: Accumulation of tissue ADMA by impairment of DDAH was found to be a significant determinant of reperfusion injury. Our results indicate that ADMA could be a potential new target for the treatment of myocardial ischemia/reperfusion injury.     

Effect of early coronary artery reperfusion on infarct development in a model of low collateral flow

To characterise collateral blood flow patterns after coronary artery occlusion in the rat and to determine whether tissue can be salvaged by reperfusion in this model, anaesthetised rats were subjected to 20, 30, 40, 60 min of coronary occlusion followed by 24 h of reperfusion or 24 h of permanent occlusion. Relative regional blood flow was measured by radioactive microspheres after 10 min of occlusion in rats undergoing 30 min of occlusion plus reperfusion and in those undergoing 24 h of permanent occlusion. The area at risk was determined by in vivo injection of fluorescent microspheres and necrosis delineated by in vitro tetrazolium staining. Tracings of heart slices were planimetered and the area of necrosis and transmural extent of the infarct measured. Blood flow in the area at risk during occlusion was similar in both the reperfused and permanent occlusion groups. In the 30 min group mean(SEM) subendocardial flow was reduced to 13(5)% of normal and subepicardial flow to 9(3)% and in the permanent occlusion group to 11(2)% and 8(3)% respectively. As delineated by fluorescent microspheres the area at risk was transmural in all rats; however, infarct size expressed as a percentage of the area at risk increased as the duration of occlusion increased. In rats reperfused up to 30 min after occlusion the area of necrosis as a percentage of the area at risk was significantly decreased compared with that in the permanent occlusion group (36.4(9.2)% in rats with 30 min occlusion plus 24 h reperfusion and 78.6(7.4)% in rats with permanent occlusion).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged depletion of ATP and of the adenine nucleotide pool due to delayed resynthesis of adenine nucleotides following reversible myocardial ischemic injury in dogs

After 15 min of severe ischemia induced by circumflex artery occlusion in open-chest dogs, 65% of the ATP and 50% of the total adenine nucleotide (ΣAd) pool is lost from the subendocardial myocardium [12]. Nevertheless, this injury is reversible if the affected tissue is reperfused with coronary arterial blood. In the present experiment, we assessed the effects of various periods of arterial reflow following 15 min of ischemic injury, on resynthesis of ATP and ΣAd. The circumflex artery was occluded for 15 min and reperfused for 20 or 60 min, or 24 or 96 h. Ten seconds prior to excision of the heart, the circumflex artery was reoccluded and the fluorescent dye thioflavine S was injected intravenously in order to identify the ischemic or the reperfused tissue which had been ischemic. The mean ATP after 15 min of ischemia was reduced 62% from 5.42 ± 0.33 to 2.08 ± 0.21 μmol/g; and the total nucleotide content was reduced by 50%. ATP content recovered slightly during the first 20 min of reperfusion but remained markedly depressed for at least 24 h because of the initial depletion of adenine nucleotides and because minimal salvage or de novo synthesis occurred in the injured muscle during this time period. By 4 days, ATP and total adenine nucleotides were still slightly depressed but had recovered to 88% and 91% of control. Electrolyte changes and an increased inulin diffusible space, which are characteristic of irreversibly injured myocardium reperfused for 20 or 60 min, were not observed. Also tissue necrosis was absent in the hearts reperfused for 24 or 96 h. These observations indicate that the marked depression of ATP and adenine nucleotides and the slow recovery of these metabolites occurred in myocardium which nevertheless was reversibly injured in terms of cellular viability.     

The pathophysiology of myocardial stunning: reversibility, accumulation and continuity of the ischemic myocardial damage after reperfusion.

In order to understand the pathophysiology of myocardial stunning, reversibility, accumulation and continuity of ischemic myocardial damage after reperfusion should be studied. Then, to analyze these three factors, myocardial function, metabolism and morphology under ischemia and reperfusion were studied in anesthetized, open-chest dogs. When myocardial ischemia was induced by occlusion of the left anterior descending coronary artery, percentage regional systolic shortening (%SS) of ischemic myocardium sharply decreased and became stable 10 min after occlusion. After reperfusion, ischemic myocardium showed active shortening after within 30-min occlusion, but did not after more than 60-min occlusion. During 90-min of ischemia, extracellular K+ concentration (Ke) steeply increased for first 10 min and was almost stable for next 10 min. Then, Ke straightly increased till 90 min. Metabolic rates, calculated from myocardial tissue CO2 and pH, steeply increased for first 20 min and sharply decreased for next 10 min. After 30 min, these two variables were almost stable, near zero. By electron-microscopy with cytochemistry, distribution of Na/K ATPase to myocardial cell membrane was observed to be almost after 15-min occlusion but distinctly sparse with destruction of cell membrane after 30-min occlusion. Therefore, irreversible myocardial damage appears after about 20-min ischemia and is almost complete after 60 min. Reversibility of damage to ischemic myocardium after reperfusion may mainly occur within 60-min ischemia. Although stunned myocardium in a narrow sense is may appear after reperfusion within less than 20-min of ischemia, stunned myocardium in a broad sense may appear within less than 60-min ischemia. When reversible myocardial ischemia (4- or 15-min occlusion) was repeated after short time intervals (20-min reperfusion), %SS of ischemic myocardium was gradually decreased with each ischemic episode. Active shortening of ischemic myocardium disappeared after more than two episodes of 15-min occlusion. Fluctuation of PCO2, pH and Ke of ischemic myocardium was gradually depressed with each occlusion. Metabolic viability of ischemic myocardium was cumulatively depressed by repeated brief occlusion. Naturally, myocardial damage was more severe after repeated 15-min occlusion than after 4-min occlusion. Accumulation of ischemic myocardial damage may arise as brief ischemia, which only induces reversible damage, is repeated. At last, continuity of ischemic myocardial damage was studied. The effect of 5-min occlusion to %SS of ischemic myocardium was apparently reversed after 90-min reperfusion. Early contractile failure was advanced even after very short duration of ischemia. Thus, myocardial function will be latently damaged.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of hypothermia on induction of heat shock protein (HSP) - 72 in ischemic brain

Intra-ischemic hypothermia has been demonstrated to be protective against ischemic neuronal injury. The present study examined the effect of moderate hypothermia on the expression of heat shock protein (HSP)-72 following transient forebrain ischemia in gerbils by immunohistochemistry. Global forebrain ischemia with concurrent moderate hypothermia (30°C) was induced in gerbils by 10-minute bilateral carotid artery occlusion followed by recirculation periods of 1 hour (h), 6h, 24h, and 48h. Normothermic forebrain ischemic animals with similar recirculation periods were utilized for comparison of the HSP expression. Sham-operated normothermic and hypothermic animals were also included. 72-kDa heat shock protein immunoreactivity was demonstrated in the hippocampus and neocortex of the normothermic ischemic animals following 24h and 48h recirculation similar to that reported previously. However, the immunoreactivity was absent in the brains of the animals subjected to hypothermic ischemia or sham-operation. Only the ependymal cells were immunopositive in all hypothermic brains as was the case with all normothermic brains. The hypothermic ischemic brains showed no significant necrosis in the hippocampus. These findings suggest that the protection of ischemic neuronal necrosis conferred by intra-ischemic hypothermia is not associated with induction of HSP-72 protein and that mechanisms other then HSP-72 protein induction are likely to be responsible for this neuroprotective effect.     

Myocardial temperature reduction attenuates necrosis after prolonged ischemia in rabbits

OBJECTIVE: Previously we observed that a large reduction in infarct size was attained by cooling the risk region of the heart, either before or early after the onset of a 30-min coronary artery occlusion. While this is a standard duration of ischemia used in the rabbit model of infarction, it may not reflect the situation of patients who are reperfused late. The effects of regional hypothermia with a longer duration of ischemia, and when the intervention is applied later, are unknown. This study tests the hypothesis that a local reduction in cardiac temperature protects myocardium during prolonged ischemia (2 h) even if begun well after coronary artery occlusion. METHODS: Anesthetized rabbits received 2 h of coronary artery occlusion and 3 h of reperfusion. Rabbits were randomly assigned to a treated group: topical myocardial cooling starting 30 min after coronary occlusion (n = 14), or control group, no intervention (n = 12). Myocardial temperature in the risk zone, hemodynamics and regional myocardial blood flow were measured. RESULTS: Ischemic zone temperature was similar in both groups at 30 min post occlusion, but the cooling maneuver produced a reduction in temperature in the risk region of the treated group such that myocardial temperature was reduced an average of 10 degrees C between 30 and 60 min of coronary artery occlusion. Myocardial temperature in the control group remained within 0.3 degree C of baseline during coronary artery occlusion and into reperfusion. Core temperatures were similar in both groups. Hemodynamic parameters and collateral blood flow during occlusion were also equivalent in both groups. After 120 min of coronary occlusion, necrosis in the control group comprised 72 +/- 3% of the ischemic risk region. However, in cooled hearts, infarct size, expressed as a fraction of the risk region was significantly lower. Infarct size in this group averaged 59 +/- 3% of the risk region (p < 0.004 vs. controls), and thus cooling resulted in a salvage of approximately 18% of the risk region. CONCLUSION: These results show that reducing myocardial temperature protects ischemic myocardium during a long duration of ischemia even if initiated after coronary artery occlusion.     

Reperfusion alters the relation between blood flow and the remaining myocardial infarction

This study evaluated whether or not reperfusion of ischemic myocardium 2 hours after occlusion alters the basic relation between myocardial blood flow and infarction occurring during permanent occlusion. Awake mongrel dogs chronically instrumented with proximal circumflex coronary occluders were subjected to permanent occlusion (group A, n = 10) or occlusion followed by reperfusion 2 hours later (group B, n = 11). Myocardial blood flow was quantified with radioactive microsphere injections before, 6 hours after occlusion (group A), immediately before release, and 4 hours after reperfusion (group B). Three days later, the dogs were killed, and the heart was sectioned systematically into approximately 80 1-2-g circumferential and transmural samples for radioactive counting and histologic infarct quantification. Epimyocardial and endomyocardial samples from the permanent occlusion group (A) and the reperfused group (B) were separated by infarct range and related to regional myocardial blood flow measurements. In groups A and B, regional myocardial blood flow in endomyocardial and epimyocardial layers were inversely related to the extent of infarction. For given degrees of infarction, myocardial blood flow was significantly higher (greater than twofold) in the reperfused group. Myocardial samples with extensive infarction (51-75%) showed only mild (20-30%) reductions in blood flow when compared with nonischemic regions in the reperfused group. Thus, although early reperfusion may salvage ischemic myocardium, these studies showed that reperfusion causes a new relation between blood flow to the ischemic region and eventual histologic infarct size. When myocardial blood flow is used as an index of myocardial salvage after reperfusion, the basic relation obtained from permanent occlusion studies substantially overestimates the extent of myocardial salvage and underestimates the degree of remaining infarction.     

The time course and characterization of myocardial hemorrhage after coronary reperfusion in the anesthetized dog

We quantitated hemorrhage associated with reperfusion after varying periods of myocardial ischemia and examined the flow characteristics that accompany reperfusion hemorrhage. Anesthetized dogs were reperfused after 2, 6 or 24 hours of circumflex occlusion. A control group underwent coronary occlusion without reperfusion. Radioactive microspheres were injected before and 5 minutes and 24 hours after reperfusion. The papillary muscles were analyzed for hemoglobin content, flow during myocardial ischemia and flow early and 24 hours after reperfusion. Myocardial creatine kinase activity was assayed to determine the severity of myocardial necrosis in the papillary muscles. Hemorrhage into the posterior papillary muscle was dependent upon the duration of coronary artery occlusion. Posterior papillary hemoglobin averaged 14 mg/g in the 2-hour group, 28 mg/g in the 6-hour group and 36 mg/g in the group reperfused 24 hours after occlusion, compared with 8.7 mg/g in the control group. Myocardial hemorrhage was associated with severe depression in myocardial CK and marked depression in flow to the ischemic area (i.e., collateral flow) during the occlusion. Early reflow averaged 112 ml/min/100 g in the 2-hour group, 61 ml/min/100 g in the 6-hour group and only 5.8 ml/min/100 g in the 24-hour group. Therefore, myocardial hemorrhage induced by reperfusion of the acutely ischemic myocardium is associated with severe ischemia during occlusion and severe myocardial necrosis, but does not depend upon the magnitude of early reflow. Myocardial hemorrhage may occur even though initial reflow values are markedly decreased.     

Flavopiridol对大鼠局灶性脑缺血后神经元细胞周期蛋白依赖蛋白激酶-4蛋白表达的影响

目的　观察大鼠局灶性脑缺血后神经元内细胞周期蛋白依赖蛋白激酶 4 (cdk4 )的蛋白表达与神经细胞凋亡的关系以及cdk4阻滞剂———Flavopiridol对其的影响。方法　采用线栓法大鼠大脑中动脉持续栓塞模型 ,应用免疫组织化学和原位末端标记 (TUNEL)染色方法观察缺血组、Flavopiridol治疗组 [根据剂量多少又分为FH(多剂量 )组和FL(少剂量 )组 ]和假手术组神经元阳性细胞染色数量和分布情况。结果　cdk4蛋白和凋亡细胞自缺血后 12h开始表达 ,前者缺血后 4 8h达高峰 ,后者缺血后 72h达高峰 ;FH组和FL组各相应时间点cdk4蛋白表达均明显减少 (P<0 .0 1) ;FL组各相应时间点凋亡细胞明显减少 (P <0 .0 1) ,FH组仅在缺血 4 8h凋亡细胞明显减少。相邻切片可见cdk4蛋白表达和凋亡细胞染色区域基本相同。结论　cdk4的蛋白表达可能诱发缺血神经细胞的凋亡 ,Flavopiri dol通过抑制cdk4的表达而减少缺血后神经细胞的损害 ,但同时Flavopiridol本身也可能引起神经细胞凋亡。     

No-reflow disrupts the expression and distribution of connexin 43 in a swine model

Introduction and objectives

Ischemia and ischemia/reperfusion can dephosphorylate and redistribute Connexin 43 (Cx43). But it is unknown whether no-reflow phenomenon has an effect on the expression and distribution of Cx43 after acute infarction and reperfusion.

Methods

21 open-chest pigs were divided into three groups. Left anterior descending artery (LAD) occlusion for 90 min before 180 min of reperfusion was made in ischemia/reperfusion group. The pigs in ischemia groups were either subjected to LAD ligation for 90 min or for 270 min. No-reflow and risk regions were determined pathologically by dye staining. Cx43 expression was measured by western blotting and quantitative RT-PCR analysis. Cx43 spatial distribution was shown by immunofluorescence examination.

Results

The content of phosphorylated and mRNA of Cx43 were higher in reflow region than in the no-reflow or sustained ischemic region. The distribution of Cx43 was also altered in no-reflow region.

Conclusions

There are some differences in synthesis, expression and distribution of myocardial Cx43 at microvascular level after ischemia/reperfusion. Cx43 is partially rephosphorylated with reperfusion only in the reflow myocardium.     

Xanthine oxidase is not a source of free radicals in the ischemic rabbit heart

The xanthine oxidase pathway has been proposed as a source of oxygen-derived free radicals in ischemic and reperfused myocardium. A spectrophotometric assay was employed to measure the xanthine oxidase activity of rat and rabbit hearts exposed to varying durations of global ischemia. In the rat 24.6 +/- 4.8 mIU/g wet wt of xanthine dehydrogenase + xanthine oxidase activity were detected in both ischemic and normally perfused myocardium. In the non-ischemic state only 6% of this activity was associated with the free radical-producing oxidase form. After 5 min of ischemia however about 25% of the enzyme was in the oxidase form, a value which remained unchanged over the following 25 min. Neither xanthine dehydrogenase nor xanthine oxidase could be detected in the rabbit heart. Failure of allopurinol, an inhibitor of xanthine oxidase, to limit infarct size in a rabbit model of ischemia/reperfusion provides further evidence that this species has insignificant amounts of xanthine oxidase in its heart. Anesthetized rabbits were subjected to coronary artery ligation for 45 min and 3 h of reperfusion. The volume of the zone of underperfusion was assessed with fluorescent microspheres and infarct size was assessed by tetrazolium staining. In control animals 67.5 +/- 3.8% of the zone of underperfusion became necrotic. In rabbits given superoxide dismutase (15000 IU/kg) + catalase (50,000 IU/kg) for 90 min starting 15 min before occlusion, infarct size was only 35.4 +/- 3.3% of the zone of underperfusion. However, in rabbits pretreated with allopurinol (75 mg p.o. 24 h before study + 30 mg/kg 5 min before occlusion) infarct size was 65.8 +/- 8.7%.(ABSTRACT TRUNCATED AT 250 WORDS)     

银杏叶提取物对大鼠心肌缺血再灌注损伤热休克蛋白表达的影响

目的:观察热休克蛋白70(HSP70)在心肌缺血再灌注损伤过程中的表达规律及银杏叶提取物(EGB)干预后的变化.方法:65只SD大鼠随机分为假手术组、模型组和治疗组.试验前30 min,治疗组大鼠腹腔注射银杏叶提取物50 mg/kg,结扎左冠状动脉前降支建立大鼠急性心肌缺血再灌注损伤模型.于再灌注不同时间点(1、3、6、12、24、48 h)取出心脏左前降支支配区的全层心肌组织,用免疫组织化学和Western Blot方法观察心肌HSP70的表达.结果:心肌缺血再灌注损伤时HSP70主要表达在心肌细胞和微血管上,表达水平随再灌注时间延长而增加,24 h时达高峰;与模型组相比,治疗组心肌组织HSP70表达水平明显上调,表达时间明显延长.结论:EGB促进HSP70在心肌表达增强和延长,可能与其提高心肌对缺血再灌注损伤的耐受性有关.     

Heat shock protein induction in rat hearts. A role for improved myocardial salvage after ischemia and reperfusion?

BACKGROUND. To test the hypothesis that the heat shock response is associated with improved myocardial salvage after myocardial ischemia and reperfusion, rats treated with prior whole-body hyperthermia and 24 hours of recovery (n = 26) or 20 minutes of ischemic pretreatment and 8 hours of recovery (n = 24) and control rats (n = 27, n = 24, for hyperthermic and ischemic pretreatment, respectively) were subjected to 35 minutes of left coronary artery (LCA) occlusion and 120 minutes of reperfusion. METHODS AND RESULTS. Although ventricular samples from rats subjected to either hyperthermia (n = 7) or ischemic pretreatment (n = 6) all showed induction of HSP72 (heat shock protein), Western blot analysis revealed significantly greater amounts of HSP72 in samples obtained from rats subjected to hyperthermia compared with those from rats subjected to ischemic pretreatment. Control rats (n = 7) showed no significant presence of myocardial HSP72. After 35 minutes of LCA occlusion and 2 hours of reperfusion, infarct size was significantly reduced in heat-shocked rats compared with controls (8.4 +/- 1.7%, n = 26 versus 15.5 +/- 1.9%, n = 27; p = 0.007; mean +/- SEM; infarct mass/left ventricular mass x 100). There were no significant differences in left ventricular (LV) systolic pressure, heart rate, LV dP/dt, or rate-pressure product between heat-shocked (n = 11) and control (n = 14) rats during the ischemic period. There were no differences in infarct size between ischemically pretreated and control rats subjected to 35 minutes of ischemia and reperfusion (9.7 +/- 2.1%, n = 23 versus 10.0 +/- 2.1, n = 24; p = NS). CONCLUSIONS. In this model of ischemia and reperfusion, prior heat shock was associated with significantly improved myocardial salvage after 35 minutes of LCA occlusion and reperfusion. This improved salvage was correlated with marked HSP72 induction and was independent of the hemodynamic determinants of myocardial oxygen supply and myocardial oxygen demand during the ischemic period. In contrast, mild HSP72 induction by ischemic pretreatment was not associated with improved myocardial salvage after myocardial ischemia and reperfusion. Thus, the absolute levels of HSP72 may be important in conferring protection from ischemic injury in this animal model.     

Protective effects of ginsenosides in myocardial ischemia and reperfusion injury

To verify whether ginsenosides will attenuate the myocardial ischemia and reperfusion injury, the left anterior descending coronary artery (LAD) was snared for 2 hours in 23 dogs and then the ischemic myocardium was reperfused. 45 minutes after ischemia, the animals were randomly divided into a ginsenosides group (n = 11, receiving a slow IV bolus of ginsenosides 10 mg/kg and then a continuous infusion of 80 micrograms/kg/min) and a saline solution group (n = 12 receiving equal amount of glucose in saline). The treatment was started 45 minutes after coronary occlusion and stopped one hour after reperfusion. 24 hours later, the dogs were killed and the extent of myocardial necrosis was determined histologically. The LVEDP, arterial pressure and heart rate were markedly lower in the ginsenosides group. Electrocardiographic findings of myocardial ischemia were significantly improved in the ginsenosides group. 8 controls developed malignant arrhythmia after reperfusion, but none in ginsenosides group. The myocardial ultrastructure can be protected by ginsenosides during the period of ischemia and reperfusion. The infarct size in saline group was 22.7 +/- 3.2% while in the ginsenosides group it was 5.2 +/- 1.3% (P less than 0.05). These results show that ginsenosides can protect the ischemic myocardium and reperfusion injury of myocardium.     

诱导型一氧化氮合酶和解偶联蛋白-2对心肌缺血预适应的作用

目的 探讨诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）和解偶联蛋白-2（uncoupling protein-2,UCP2）对大鼠心肌缺血预适应的保护机制。方法 结扎左冠状动脉复制大鼠心肌缺血再灌注模型。预适应组行3次缺血5min,再灌注10min的预处理,分别于预处理0,6,12,24和48h（分别为0,6,12,24和48h亚组）后行30min缺血及120min再灌注：对照组开胸后不结扎左冠状动脉,电镜观察心肌超微结构,据Rainio评分标准进行心肌超微结构损伤程度的半定量分析。采用Western Blot及比色法检测心肌UCP2活性及iNOS活性。结果 预适应各亚组UCP2活性均增高（P〈0．05）,0h亚组UCP2表达水平最高（P〈0．01）,24小时亚组和48小时亚组心肌iNOS活性显著升高（P〈0．05）。结论 UCP2和iNOS共同参与了大鼠心肌缺血预适应心肌保护作用。     

Inhibition of Rho-kinase protects the heart against ischemia/reperfusion injury

OBJECTIVE: To investigate the role of Rho A and Rho-kinase in acute myocardial ischemia/reperfusion injury and the protective effect of Rho-kinase inhibitor, Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide]. METHODS AND RESULTS: Male CD1 mice were subjected to 30 min of coronary occlusion and 24 h reperfusion. Ischemia/reperfusion upregulated expression of Rho A in ischemic myocardium, and subsequently activated Rho-kinase. Y-27632 significantly inhibited the activation of Rho-kinase following ischemia/reperfusion. Treatment with Y-27632 at 10 and 30 mg/kg oral administration, reduced infarct size by 30.2% and 41.1%, respectively (P<0.01 vs. vehicle). Y-27632 also enhanced post-ischemia cardiac function. Left ventricular systolic pressure, +dP/dt and -dP/dt were significantly improved by 23.5%, 52.3%, and 59.4%, respectively (P<0.01 vs. vehicle). Moreover, Y-27632 reduced ischemia/reperfusion-induced myocardial apoptosis. The apoptotic myocytes in ischemic myocardium after 4 h reperfusion were reduced from 13.1% in vehicle group to 6.4% in Y-27632-treated group (P<0.01). Meanwhile, ischemia/reperfusion-induced downregulation of Bcl-2 in myocardium was remarkably attenuated in the treated animals. Ischemia/reperfusion resulted in remarkable elevation in serum levels of proinflammatory cytokines, interleukin-6 (IL-6), keratinocyte chemoattractant (KC) and granulocyte colony-stimulating factor (G-CSF), which was significantly suppressed by Y-27632. In addition, Y-27632 decreased ischemia/reperfusion-induced accumulation of neutrophils in the heart by 45% (P<0.01). CONCLUSIONS: These results suggest that Rho-kinase plays a pivotal role in myocardial ischemia/reperfusion injury. The cardiac protection provided by treatment with a selective Rho-kinase inhibitor is likely via anti-apoptotic effect and attenuation of ischemia/reperfusion-induced inflammatory responses. The finding of this study suggest a novel therapeutic approach to the treatment of acute myocardial ischemia/reperfusion injury.     

Mitochondrial function, oxygen extraction, epicardial S-T segment changes and tritiated digoxin distribution after reperfusion of ischemic myocardium.

This study examines the effect of 2 hours of reperfusion on transiently ischemic myocardium in pigs. Indexes of myocardial viability measured were mitochondrial function, oxygen extraction, epicardial S-T segment change and distribution of tritiated digoxin. Results were as follows: (1) Mitochondrial function was markedly impaired in the reperfused area after 60 minutes or more of coronary occlusion. The defect would seem to be a block in electron flow near site I, which can be partially bypassed with succinate. (2) An apparent inability of the reperfused myocardium to extract oxygen did not improve with 2 hours of reperfusion. (3) Epicardial S-T segment mapping suggested that necrosis occurred during reperfusion. (4) There was an altered distribution of tritiated digoxin in the reperfused area. The results show that reperfusion for 2 hours did not improve myocardial viability after 60 minutes or more of ischemia.     

Functional and metabolic responses to ischemia in the isolated perfused hypothyroid rat heart.

It has been demonstrated that the V3 cardiac myosin isozyme has a higher efficiency and consumes less oxygen in doing mechanical work than the V1 myosin isozyme. The purpose of the present study is to investigate the functional and metabolic responses to ischemia following reperfusion of the hypothyroid heart, in which ventricular myosin isozyme is shifted from V1 predominance to V3 predominance. The heart was perfused by the working heart method for 15 min, and then global ischemia was induced for 10, 20 and 30 min with pacing at a rate of 320/min until cardiac cessation. The ischemic heart was reperfused for 30 min. The extent of recovery of pressure-rate product after reperfusion, following 20 min of ischemia in the hypothyroid heart, was higher than that in the control heart (p less than 0.01). The level of adenosine triphosphate (ATP) declined more slowly in the hypothyroid heart than in the control heart. The level of ATP and energy charge potential in the hypothyroid heart reperfused after 20 min of ischemia were higher than those in the reperfused control heart (p less than 0.01, p less than 0.05, respectively), though they did not differ from each other in preischemia. There were no significant differences in the levels of tissue lactate between the 2 hearts during ischemia and reperfusion. The recovery rate of the coronary flow of the 2 hearts did not differ from each other significantly. These data suggest that there is probability of the V3 predominant heart recovering from ischemic damage to the metabolism and cardiac function better than the V1 predominant heart because the transformation of myocardium, due to an increase in V3 into a slower but more efficiently working muscle results in conservation of tissue ATP during ischemia.     

Response of isolated perfused heart to ischemia after long-term treatment of spontaneously hypertensive rats with diltiazem

The effects of long-term treatment with diltiazem on the heart in normotensive (WKY) and spontaneously hypertensive rats (SHR) were studied. Diltiazem was added to the drinking fluid (900 mg/liter) and given ad libitum from 19 to 26 weeks of age, whereas tap water was given to the control animals. Although diltiazem did not decrease blood pressure in SHR, it decelerated the increase in their left ventricular weight (p less than 0.01). Hearts were removed and perfused by the working heart technique for 15 min, and then global ischemia was induced for either 10 or 30 min. The ischemic heart was reperfused for 30 min. The extent of recovery of coronary flow after reperfusion, following 30 min of ischemia in the diltiazem-treated SHR, was higher than that in the control SHR (p less than 0.01). The levels of adenosine triphosphate (ATP), creatine phosphate (CrP), and energy charge potential in the SHR heart reperfused after 30 min of ischemia were lower than those in the reperfused WKY heart (p less than 0.01, respectively). Diltiazem improved the restoration of ATP and CrP and prevented the decrease in energy charge potential in SHR after reperfusion following 30 min of ischemia (p less than 0.01, respectively). In conclusion, long-term treatment of SHR with diltiazem may protect the myocardium when myocardial ischemia occurs.     

缺血预适应减少心肌缺血损伤机制的研究

目的　通过对心肌缺血预适应的动物模型观察 ,探讨细胞凋亡在其中的作用 ,以及p5 3,bcl 2 ,Bax基因对其发生进行的调控。方法　采用TUNEL标记技术研究心肌缺血预适应心肌细胞中细胞凋亡现象 ,并采用免疫组化染色技术及原位分子杂交技术研究p5 3,bcl 2及Bax基因的蛋白及mRNA的表达。结果　缺血预适应组 (P)及非缺血预适应组 (NP)非缺血区均未见凋亡细胞 ,但在P组缺血区可见散在的凋亡细胞 ,而在NP组缺血区则多见。P组p5 3蛋白表达显著低于NP组 ,bcl 2蛋白表达在P组显著高于NP组 ,Bax蛋白表达在P组显著低于NP组 ,并且bcl 2 /Bax的比值P组与NP组相比显著升高。P组p5 3基因mRNA表达显著低于NP组 ,bcl 2基因mRNA表达在P组显著高于NP组。结论　心肌缺血预适应对心肌的保护可通过抑制细胞凋亡来实现 ,并且通过bcl 2表达增加 ,p5 3、Bax表达减少对其进行调控。