Protein kinase C-epsilon coimmunoprecipitates with cytochrome oxidase subunit IV and is associated with improved cytochrome-c oxidase activity and cardioprotection

We have utilized an in situ rat coronary ligation model to establish a PKC-epsilon cytochrome oxidase subunit IV (COIV) coimmunoprecipitation in myocardium exposed to ischemic preconditioning (PC). Ischemia-reperfusion (I/R) damage and PC protection were confirmed using tetrazolium-based staining methods and serum levels of cardiac troponin I. Homogenates prepared from the regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell-soluble (S), 600 g low-speed centrifugation (L), Percoll/Optiprep density gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. COIV immunoreactivity and cytochrome-c oxidase activity measurements estimated the percentages of cellular mitochondria in S, L, M, and P fractions to be 0, 55, 29, and 16%, respectively. We observed 18, 3, and 3% of PKC-delta, -epsilon, and -zeta isozymes in the M fraction under basal conditions. Following PC, we observed a 61% increase in PKC-epsilon levels in the RAR M fraction compared with the RNAR M fraction. In RAR mitochondria, we also observed a 2.8-fold increase in PKC-epsilon serine 729 phosphoimmunoreactivity (autophosphorylation), indicating the presence of activated PKC-epsilon in mitochondria following PC. PC administered before prolonged I/R induced a 1.9-fold increase in the coimmunoprecipitation of COIV, with anti-PKC-epsilon antisera and a twofold enhancement of cytochrome-c oxidase activity. Our results suggest that PKC-epsilon may interact with COIV as a component of the cardioprotection in PC. Induction of this interaction may provide a novel therapeutic target for protecting the heart from I/R damage.     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning

We examined whether the mitochondrial ATP-sensitive K channel (K(ATP)) is an effector downstream of protein kinase C-epsilon (PKC-epsilon) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 +/- 6.8% of the left ventricle to 20.3 +/- 3.7%. PC significantly increased PKC-epsilon protein in the particulate fraction from 51 +/- 4% of the total to 60 +/- 4%, whereas no translocation was observed for PKC-delta and PKC-alpha. In mitochondria separated from the other particulate fractions, PC increased the PKC-epsilon level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) blocker, after PC abolished the cardioprotection of PC, whereas PKC-epsilon translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial K(ATP) opener, infused 10 min before ischemia limited infarct size to 5.2 +/- 1.4%, but this agent neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. Together with the results of earlier studies showing mitochondrial K(ATP) opening by PKC, the present results suggest that mitochondrial K(ATP)-mediated cardioprotection occurs subsequent to PKC-epsilon activation by PC.     

Role of endogenous opioid peptides in protection of ischemic preconditioning in rat small intestine

This study investigated the protective effects of ischemic preconditioning on intestinal ischemic injury and the role of endogenous opioid peptides (EOP) in these effects. Ischemia-reperfusion (I/R) induced by 30-min of ischemia and 60-min of reperfusion significantly increased the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) and resulted in serious intestinal edema (wet weight/dry weight). The ischemic preconditioning (PC) elicited by three 8-min occlusion periods interspersed with 10-min reperfusion markedly attenuated intestinal injury caused by ischemia-reperfusion. Pretreatment with morphine (300 microg x kg(-1), i.v.) 10-min before ischemia and reperfusion mimicked the protection produced by PC. Naloxone (3 mg x kg(-1), i.v.) abolished the protection of morphine-induced preconditioning and ischemic preconditioning in rat intestine. However, there were no changes between naloxone alone and control groups. Treatment with naloxone before ischemia-reperfusion had no effect on animals compared with the I/R group. In addition, we also measured the content of endogenous opioid peptides (Leu-enkephalin) in the effluent which was collected before and during preconditioning. It was shown that the release of leu-enkephalin was markedly increased during preconditioning. These results suggested that EOP might play an important role in PC in rat small intestine.     

VEGFR1 (Flt-1+/-) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium

This report demonstrates that mice deficient in Flt-1 failed to establish ischemic preconditioning (PC)-mediated cardioprotection in isolated working buffer-perfused ischemic/reperfused (I/R) hearts compared to wild type (WT) subjected to the same PC protocol. WT and Flt-1+/- mice were divided into four groups: (1) WT I/R, (2) WT + PC, (3) Flt-1+/- I/R, and (4) Flt-1+/- + PC. Group 1 and 3 mice were subjected to 30 min of ischemia followed by 2 h of reperfusion and group 2 and 4 mice were subjected to four episodes of 4-min global ischemia followed by 6 min of reperfusion before ischemia/reperfusion. For both wild-type and Flt-1+/- mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the knockout mice was less compared to the WT mice even in preconditioning. The myocardial infarction and apoptosis were higher in Flt-1+/- compared to wild-type I/R. Flt-1+/- KO mice demonstrated pronounced inhibition of the expression of iNOS, p-AKT & p-eNOS. Significant inhibition of STAT3 & CREB were also observed along with the inhibition of HO-1 mRNA. Results demonstrate that Flt-1+/- mouse hearts are more susceptible to ischemia/reperfusion injury and also document that preconditioning is not as effective as found in WT and therefore suggest the importance of VEGF/Flt-1 signaling in ischemic/reperfused myocardium.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion

Reactive oxygen species (ROS) are believed to be involved in triggering cardiac ischemic preconditioning (IPC). Decreased formation of ROS on reperfusion after prolonged ischemia may in part underlie protection by IPC. In heart models, these contentions have been based either on the effect of ROS scavengers to abrogate IPC-induced preservation or on a measurement of oxidation products on reperfusion. Using spectrophotofluorometry at the left ventricular wall and the fluorescent probe dihydroethidium (DHE), we measured intracellular ROS superoxide (O(2)(-).) continuously in isolated guinea pig heart and tested the effect of IPC and the O(2)(-). scavenger manganese(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) on O(2)(-). formation throughout the phases of preconditioning (PC), 30-min ischemia and 60-min reperfusion (I/R). IPC was evidenced by improved contractile function and reduced infarction; MnTBAP abrogated these effects. Brief PC pulses increased O(2)(-). during the ischemic but not the reperfusion phase. O(2)(-). increased by 35% within 1 min of ischemia, increased further to 95% after 20 min of ischemia, and decreased slowly on reperfusion. In the IPC group, O(2)(-). was not elevated over 35% during index ischemia and was not increased at all on reperfusion; these effects were abrogated by MnTBAP. Our results directly demonstrate how intracellular ROS increase in intact hearts during IPC and I/R and clarify the role of ROS in triggering and mediating IPC.     

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount of mtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interval. Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO₂) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO₂ and efficiency and mitochondrial oxygen tension (mitoPO₂) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ?G_ATP) were determined following peptide treatment. When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reduced MVO₂ and improved energetics (increased PCr) before ischemia, without affecting MVO₂/RPP ratio or mitoPO₂. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO₂ or decreased cardiac energetics before damage occurs.     

吸入一氧化碳对肢体严重缺血/再灌注损伤及其所致休克的影响

目的：观察吸入适量一氧化碳（CO）对肢体严重缺血/再灌注（I/R）损伤及其所致休克的影响。方法：SD大鼠36只,随机分为I/RI、/R吸入CO（RC）及假手术（S）组。用夹闭双侧股动脉及绑扎后肢根部的办法使后肢缺血8 h,之后行再灌注12 h1、0 d,以此建立肢体严重I/R损伤模型。RC组于再灌注前2 h时开始持续吸入含有CO的医用空气6 h（CO的体积分数为0.075%）,S、I/R组呼吸自然空气。观测对比尾动脉压（CAP）、10 d存活率及肢体损伤指标的变化。结果：I/R组再灌注后CAP快速下降,再灌注至8 h时CAP均值降为（5.3259±0.3832）kPa;RC组CAP下降较I/R组速率慢、幅度小,再灌注至8 h时CAP均值为（8.3300±0.4224）kPa。I/R组用于10 d存活率观察的8只大鼠,于再灌注13~20 h间全部死亡,RC组8只中有1只死亡,其余7只再灌注10 d时仍存活。与I/R组比较,RC组肢体的病理学表现明显减轻,肢体湿干重比值、血清乳酸脱氢酶和肌酸激酶活性显著下降（P〈0.05）。结论：吸入适量CO对肢体严重I/R损伤所引起的血压下降有反向调节作用,可有效防止休克的发生,减轻肢体损伤,显著提高动物的存活率。     

Signal transduction mechanisms involved in cardiac preconditioning: Role of Ras-GTPase, Ca2 +/calmodulin-dependent protein kinase II and epidermal growth factor receptor

It is well established that brief episodes of ischemia/reperfusion (I/R) [preconditioning (PC)] protect the myocardium from the damage induced by subsequent more prolonged I/R. However, the signaling pathways activated during PC or I/R are not well characterized. In this study, the role of Ras-GTPase, tyrosine kinases (TKs), epidermal growth factor receptor (EGFR) and Ca^{2 +}/calmodulin-dependent protein kinase II (CaMK II) in mediating PC in a perfused rat heart model was investigated. A 40-min episode of global ischemia in perfused rat hearts produced significantly impaired cardiac function, measured as left ventricular developed pressure (P_max) and left ventricular end-diastolic pressure (LVEDP), and impaired coronary hemodynamics, measured as coronary flow (CF) and coronary vascular resistance (CVR). PC significantly enhanced cardiac recovery after I/R. Combination of PC and FPT III (Ras-GTPase inhibitor FPT III; 232 ng/min for 6 days) treatment did not produce any additive benefits as compared to PC alone. In contrast, PC-induced improvements in cardiac function after I/R were significantly attenuated by pretreatment with genistein (1mg/kg/day for 6 days), a broad-spectrum inhibitor of TKs, or AG1478 (1mg/kg/day for 6 days), a specific inhibitor of EGFR tyrosine kinase or KN-93 (578 ng/min for 6 days), a CaMK II inhibitor, before PC. These observations suggest that PC and FPT III pretreatment may produce cardioprotection via similar mechanisms. Present results also indicate that activation of TKs and specifically activation of EGFR-mediated TKs and CaMK II-mediated regulation of calcium homeostasis are part of the PC mechanisms that improve recovery after I/R. (Mol Cell Biochem 268: 175–183, 2005)     

C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.     

Endurance Exercise Accelerates Myocardial Tissue Oxygenation Recovery and Reduces Ischemia Reperfusion Injury in Mice

Exercise training offers cardioprotection against ischemia and reperfusion (I/R) injury. However, few essential signals have been identified to underscore the protection from injury. In the present study, we hypothesized that exercise-induced acceleration of myocardial tissue oxygenation recovery contributes to this protection. C57BL/6 mice (4 weeks old) were trained on treadmills for 45 min/day at a treading rate of 15 m/min for 8 weeks. At the end of 8-week exercise training, mice underwent 30-min left anterior descending coronary artery occlusion followed by 60-min or 24-h reperfusion. Electron paramagnetic resonance oximetry was performed to measure myocardial tissue oxygenation. Western immunoblotting analyses, gene transfection, and myography were examined. The oximetry study demonstrated that exercise markedly shortened myocardial tissue oxygenation recovery time following reperfusion. Exercise training up-regulated Kir6.1 protein expression (a subunit of ATP-sensitive K⁺ channel on vascular smooth muscle cells, VSMC sarc-K_ATP) and protected the heart from I/R injury. In vivo gene transfer of dominant negative Kir6.1AAA prolonged the recovery time and enlarged infarct size. In addition, transfection of Kir6.1AAA increased the stiffness and reduced the relaxation capacity in the vasculature. Together, our study demonstrated that exercise training up-regulated Kir6.1, improved tissue oxygenation recovery, and protected the heart against I/R injury. This exercise-induced cardioprotective mechanism may provide a potential therapeutic intervention targeting VSMC sarc-K_ATP channels and reperfusion recovery.     

Protective effects of circulating microvesicles derived from ischemic preconditioning on myocardial ischemia/reperfusion injury in rats by inhibiting endoplasmic reticulum stress

Microvesicles (MVs) have been shown to be involved in pathophysiology of ischemic heart diseases. However, the underlying mechanisms are still unclear. Here we investigated the effects of MVs derived from ischemic preconditioning (IPC-MVs) on myocardial ischemic/reperfusion (I/R) injury in rats. Myocardial IPC model was elicited by three cycles of ischemia and reperfusion of the left anterior descending (LAD) coronary artery. IPC-MVs from the peripheral blood of the above animal model were isolated by ultracentrifugation and characterized by flow cytometry and transmission electron microscopy. IPC-MVs were administered intravenously (7 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min ischemia and 120-min reperfusion of LAD in rats. We found that total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<?1 µm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats respectively. Additionally, treatment with IPC-MVs significantly alleviated damage of myocardium, and restored cardiac function of I/R injury rats, as evidenced by increased heart rate, and decreased the elevation of ST-segment. The size of myocardial infarction, lactate dehydrogenase activity, and the number of apoptotic cardiomyocytes were also reduced significantly with IPC-MVs treatment, coincident with the above function amelioration. Moreover, IPC-MVs decreased the activity of caspase 3, and the expression of endoplasmic reticulum stress (ERS) markers, GRP78, CHOP and caspase 12 indicating the involvement of ERS-specific apoptosis in I/R injury, and cardioprotective effects of IPC-MVs. In summary, our study demonstrated a novel mechanism of IPC in which circulating IPC-MVs could protect hearts from I/R injury in rats through attenuation of ERS-induced apoptosis. These findings provide new insight into therapeutic potential of IPC-induced MVs in cardioprotection against I/R injury.     

Mitochondrial Src tyrosine kinase plays a role in the cardioprotective effect of ischemic preconditioning by modulating complex I activity and mitochondrial ROS generation

Abstract

While ischemic preconditioning (IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion (I/R) injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC modulates complex I activity at reperfusion by activating mitochondrial Src tyrosine kinase, and induces cardioprotection against I/R injury. Isolated rat hearts were preconditioned by three cycles of 5-min ischemia and 5-min reperfusion prior to 30-min index ischemia followed by 2 h of reperfusion. Mitochondrial Src phosphorylation (Tyr⁴¹⁶) was dramatically decreased during I/R, implying inactivation of Src tyrosine kinase by I/R. IPC increased mitochondrial Src phosphorylation upon reperfusion and this was inhibited by the selective Src tyrosine kinase inhibitor PP2. IPC's anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity was significantly increased upon reperfusion, an effect that was prevented by IPC in a Src tyrosine kinase-dependent manner. In support, Src and phospho-Src were found in complex I. Furthermore, IPC prevented hypoxia/reoxygenation-induced mitochondrial reactive oxygen species (ROS) generation and cellular injury in rat cardiomyocytes, which was revoked by PP2. Finally, IPC reduced LDH release induced by both hypoxia/reoxygenation and simulated ischemia/reperfusion, an effect that was reversed by PP2 and Src siRNA. These data suggest that mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I and mitochondrial ROS generation, and thereby plays a role in the cardioprotective effect of IPC.     

Catestatin Improves Post-Ischemic Left Ventricular Function and Decreases Ischemia/Reperfusion Injury in Heart

The Chromogranin A (CgA)-derived anti-hypertensive peptide catestatin (CST) antagonizes catecholamine secretion, and is a negative myocardial inotrope acting via a nitric oxide-dependent mechanism. It is not known whether CST contributes to ischemia/reperfusion injury or is a component of a cardioprotective response to limit injury. Here, we tested whether CST by virtue of its negative inotropic activity improves post-ischemic cardiac function and cardiomyocyte survival. Three groups of isolated perfused hearts from adult Wistar rats underwent 30-min ischemia and 120-min reperfusion (I/R, Group 1), or were post-conditioned by brief ischemic episodes (PostC, 5-cycles of 10-s I/R at the beginning of 120-min reperfusion, Group 2), or with exogenous CST (75 nM for 20 min, CST-Post, Group-3) at the onset of reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated with nitroblue-tetrazolium staining. The CST (5 nM) effects were also tested in simulated ischemia/reperfusion experiments on cardiomyocytes isolated from young-adult rats, evaluating cell survival with propidium iodide labeling. Infarct size was 61 ± 6% of risk area in hearts subjected to I/R only. PostC reduced infarct size to 34 ± 5%. Infarct size in CST-Post was 36 ± 3% of risk area (P < 0.05 respect to I/R). CST-Post reduced post-ischemic rise of diastolic LVP, an index of contracture, and significantly improved post-ischemic recovery of developed LVP. In isolated cardiomyocytes, CST increased the cell viability rate by about 65% after simulated ischemia/reperfusion. These results suggest a novel cardioprotective role for CST, which appears mainly due to a direct reduction of post-ischemic myocardial damages and dysfunction, rather than to an involvement of adrenergic terminals and/or endothelium.     

Delta-opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-epsilon-mediated phosphorylation of connexin 43

The aim of this study was to examine the hypothesis that delta-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the delta-opioid receptor activation. A delta-opioid receptor agonist, [D-Ala(2),D-Leu(5)]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-epsilon inhibitor [PKC-epsilon translocation inhibitory peptide (PKC-epsilon-TIP)] but was not affected by a PKC-delta inhibitor (rottlerin). After DADLE infusion, PKC-epsilon, but not PKC-delta, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser(368)) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-epsilon-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-epsilon activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-epsilon plays a crucial role in delta-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic delta-opioid receptor activation.     

SAPKs regulation of ischemic preconditioning

The role of stress-activated protein kinases (SAPKs), c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase, in preconditioning (PC) was examined with the use of isolated rat hearts subjected to four cyclic episodes of 5-min ischemia and 10-min reperfusion followed by 30-min ischemia and 2-h reperfusion (I/R). A group of hearts was preperfused with 100 microM curcumin, a c-Jun and JNK1 inhibitor, or 5 microM SB 203580, a p38 MAP kinase inhibitor. Another group of hearts was preperfused with 20 microM anisomycin, a stimulator for both JNK and p38 MAP kinases. I/R increased the protein levels of JNK1, c-Jun, and p38 MAP kinase. PC also enhanced the induction of these kinases, but subsequent I/R-mediated increase was blocked by PC. Curcumin blocked I/R- and PC-mediated increase in JNK1 and c-Jun protein levels, whereas it had no effects on p38 MAP kinase. SB 203580, on the other hand, was equally effective in reducing the p38 MAP kinase activation but exerted no effects on JNK1 and c-Jun induction. I/R-mediated increased myocardial infarction was reduced by any of the following compounds: anisomycin, curcumin, and SB 203580. The cardioprotective effects of PC were abolished by either curcumin or SB 203580. The results demonstrate that PC is mediated by a signal-transduction pathway involving both JNK1 and p38 MAP kinase. Activation of SAPKs, although transient, is obligatory for PC.     

吸入一氧化碳抗肢体缺血/再灌注损伤的实验研究

目的：观察吸入适量一氧化碳（CO）对大鼠肢体缺血/再灌注（I/R）损伤的防治作用。方法：SD大鼠44只,随机分为假手术（S）、I/R、I/R吸入CO（RC）组;通过夹闭股动脉4h、再开放48h,复制肢体I/R损伤模型;RC组行再灌注时,使动物吸入含有CO的医用空气（CO的体积分数为0.05%）,其余两组呼吸正常空气;对比观测缺血肢体大体及骨骼肌组织病理学、缺血肢体湿干重比值（W/D）的变化,流式细胞仪检测肌组织中Bax、Bcl-2的表达水平及细胞凋亡百分比,全自动生化分析仪检测血清乳酸脱氢酶（LDH）和肌酸激酶（CK）的变化。结果：与I/R组比较,RC组动物W/D、血清LDH及CK含量、肌组织中Bax表达水平及细胞凋亡百分比均显著降低,肌组织Bcl-2表达水平显著升高,缺血肢体大体观及肌组织病理学明显改善。结论：吸入适量浓度的外源性CO对肢体I/R损伤有防治作用。     

Preconditioning attenuates ischemia-reperfusion-induced remodeling of Na+-K+-ATPase in hearts

The aim of this study was to determine whether changes in protein content and/or gene expression of Na+-K+-ATPase subunits underlie its decreased enzyme activity during ischemia and reperfusion. We measured protein and mRNA subunit levels in isolated rat hearts subjected to 30 min of ischemia and 30 min of reperfusion (I/R). The effect of ischemic preconditioning (IP), induced by three cycles of ischemia and reperfusion (10 min each), was also assessed on the molecular changes in Na+-K+-ATPase subunit composition due to I/R. I/R reduced the protein levels of the alpha2-, alpha3-, beta1-, and beta2-isoforms by 71%, 85%, 27%, and 65%, respectively, whereas the alpha1-isoform was decreased by <15%. A similar reduction in mRNA levels also occurred for the isoforms of Na+-K+-ATPase. IP attenuated the reduction in protein levels of Na+-K+-ATPase alpha2-, alpha3-, and beta2-isoforms induced by I/R, without affecting the alpha1- and beta1-isoforms. Furthermore, IP prevented the reduction in mRNA levels of Na+-K+-ATPase alpha2-, alpha3-, and beta1-isoforms following I/R. Similar alterations in protein contents and mRNA levels for the Na+/Ca2+ exchanger were seen due to I/R as well as IP. These findings indicate that remodeling of Na+-K+-ATPase may occur because of I/R injury, and this may partly explain the reduction in enzyme activity in ischemic heart disease. Furthermore, IP may produce beneficial effects by attenuating the remodeling of Na+-K+-ATPase and changes in Na+/Ca2+ exchanger in hearts after I/R.