Remote preconditioning reduces ischemic injury in the explanted heart by a KATP channel-dependent mechanism

Local and remote ischemic preconditioning (IPC) reduce ischemia-reperfusion (I/R) injury and preserve cardiac function. In this study, we tested the hypothesis that remote preconditioning is memorized by the explanted heart and yields protection from subsequent I/R injury and that the underlying mechanism involves sarcolemmal and mitochondrial ATP-sensitive K(+) (K(ATP)) channels. Male Wistar rats (300-350 g) were randomized to a control (n = 10), a remote IPC (n = 10), and a local IPC group (n = 10). Remote IPC was induced by four cycles of 5 min of limb ischemia, followed by 5 min of reperfusion. Local IPC was induced by four cycles of 2 min of regional myocardial ischemia, followed by 3 min of reperfusion. The heart was excised within 5 min after the final cycle of preconditioning, mounted in a perfused Langendorff preparation for 40 min of stabilization, and subjected to 45 min of sustained ischemia by occluding the left coronary artery and 120 min of reperfusion. I/R injury was assessed as infarct size by triphenyltetrazolium staining. The influence of sarcolemmal and mitochondrial K(ATP) channels on remote preconditioning was assessed by the addition of glibenclamide (10 microM, a nonselective K(ATP) blocker), 5-hydroxydecanoic acid (5-HD; 100 microM, a mitochondrial K(ATP) blocker), and HMR-1098 (30 microM, a sarcolemmal K(ATP) blocker) to the Langendorff preparation before I/R. The role of mitochondrial K(ATP) channels as an effector mechanism for memorizing remote preconditioning was further studied by the effect of the specific mitochondrial K(ATP) activator diaxozide (10 mg/kg) on myocardial infarct size. Remote preconditioning reduced I/R injury in the explanted heart (0.17 +/- 0.03 vs. 0.39 +/- 0.05, P < 0.05) and improved left ventricular function during reperfusion compared with control (P < 0.05). Similar effects were obtained with diazoxide. Remote preconditioning was abolished by the addition of 5-HD and glibenclamide but not by HMR-1098. In conclusion, the protective effect of remote preconditioning is memorized in the explanted heart by a mechanism that involves mitochondrial K(ATP) channels.     

Warm ischemic preconditioning improves mitochondrial redox balance during and after mild hypothermic ischemia in guinea pig isolated hearts

Ischemic preconditioning (IPC) induces distinctive changes in mitochondrial bioenergetics during warm (37 degrees C) ischemia and improves function and tissue viability on reperfusion. We examined whether IPC before 2 h of hypothermic (27 degrees C) ischemia affords additive cardioprotection and improves mitochondrial redox balance assessed by mitochondrial NADH and flavin adenine dinucleotide (FAD) autofluorescence in intact hearts. A mediating role of ATP-sensitive K(+) (K(ATP)) channel opening was investigated. NADH and FAD fluorescence was measured in the left ventricular wall of guinea pig isolated hearts assigned to five groups of eight animals each: hypothermia alone, hypothermia with ischemia, IPC with cold ischemia, 5-hydroxydecanoic acid (5-HD) alone, and 5-HD with IPC and cold ischemia. IPC consisted of two 5-min periods of warm global ischemia spaced 5 min apart and 15 min of reperfusion before 2 h of ischemia at 27 degrees C and 2 h of warm reperfusion. The K(ATP) channel inhibitor 5-HD was perfused from 5 min before until 5 min after IPC. IPC before 2 h of ischemia at 27 degrees C led to better recovery of function and less tissue damage on reperfusion than did 27 degrees C ischemia alone. These improvements were preceded by attenuated increases in NADH and decreases in FAD during cold ischemia and the reverse changes during warm reperfusion. 5-HD blocked each of these changes induced by IPC. This study indicates that IPC induces additive cardioprotection with mild hypothermic ischemia by improving mitochondrial bioenergetics during and after ischemia. Because effects of IPC on subsequent changes in NADH and FAD were inhibited by 5-HD, this suggests that mitochondrial K(ATP) channel opening plays a substantial role in improving mitochondrial bioenergetics throughout mild hypothermic ischemia and reperfusion.     

Ischemic preconditioning in rats: role of mitochondrial K(ATP) channel in preservation of mitochondrial function

We examined the role of the sarcolemmal and mitochondrial K(ATP) channels in a rat model of ischemic preconditioning (IPC). Infarct size was expressed as a percentage of the area at risk (IS/AAR). IPC significantly reduced infarct size (7 +/- 1%) versus control (56 +/- 1%). The sarcolemmal K(ATP) channel-selective antagonist HMR-1098 administered before IPC did not significantly attenuate cardioprotection. However, pretreatment with the mitochondrial K(ATP) channel-selective antagonist 5-hydroxydecanoic acid (5-HD) 5 min before IPC partially abolished cardioprotection (40 +/- 1%). Diazoxide (10 mg/kg iv) also reduced IS/AAR (36.2 +/- 4.8%), but this effect was abolished by 5-HD. As an index of mitochondrial bioenergetic function, the rate of ATP synthesis in the AAR was examined. Untreated animals synthesized ATP at 2.12 +/- 0.30 micromol x min(-1) x mg mitochondrial protein(-1). Rats subjected to ischemia-reperfusion synthesized ATP at 0.67 +/- 0.06 micromol x min(-1) x mg mitochondrial protein(-1). IPC significantly increased ATP synthesis to 1.86 +/- 0.23 micromol x min(-1) x mg mitochondrial protein(-1). However, when 5-HD was administered before IPC, the preservation of ATP synthesis was attenuated (1.18 +/- 0.15 micromol x min(-1) x mg mitochondrial protein(-1)). These data are consistent with the notion that inhibition of mitochondrial K(ATP) channels attenuates IPC by reducing IPC-induced protection of mitochondrial function.     

Preconditioning limits mitochondrial Ca(2+) during ischemia in rat hearts: role of K(ATP) channels

Prolonged myocardial ischemia results in an increase in intracellular calcium concentration ([Ca(2+)]i), which is thought to play a critical role in ischemia-reperfusion injury. Ischemic preconditioning (PC) improves myocardial function during ischemia-reperfusion, a process that may involve opening mitochondrial ATP-sensitive potassium (K(ATP)) channels. Because pharmacological limitation of mitochondrial calcium concentration ([Ca(2+)]m) overload during ischemia-reperfusion has been shown to improve myocardial function, we hypothesized that PC would reduce [Ca(2+)]m during ischemia-reperfusion and that this effect was mediated by opening mitochondrial K(ATP) channels. Isolated rat hearts were subjected to 25 min of global ischemia and 30 min of reperfusion with or without PC in the presence of mitochondrial K(ATP) channel opening (diazoxide, 100 microM) and blockade [5-hydroxydecanoic acid (5-HD), 100 microM]. Contracture during ischemia (end-diastolic pressure) and functional recovery on reperfusion (developed pressure) were assessed. Total [Ca(2+)]i and [Ca(2+)]m were measured using indo 1 fluorescence. Both PC and diazoxide limited the increase in end-diastolic pressure and resulted in greater functional recovery after 30 min of reperfusion, functional effects that were partially or completely abolished by 5-HD. PC and diazoxide also significantly limited the increase in [Ca(2+)]m during ischemia-reperfusion. In addition, PC lowered [Ca(2+)]i during reperfusion, whereas diazoxide paradoxically resulted in increased [Ca(2+)]i during reperfusion. There was an inverse linear relationship between [Ca(2+)]m and developed pressure during reperfusion. PC limits the ischemia-induced increase in mitochondrial, but not total, [Ca(2+)]i, an effect mediated by opening mitochondrial K(ATP) channels. These data suggest that the lowering of mitochondrial calcium overload is a mechanism of cardioprotection in PC.     

Differential role of K(ATP) channels in late preconditioning against myocardial stunning and infarction in rabbits

The role of ATP-sensitive potassium (K(ATP)) channels in the late phase of ischemic preconditioning (PC) remains unclear. Furthermore, it is unknown whether K(ATP) channels serve as end effectors both for late PC against infarction and against stunning. Thus, in phase I of this study, conscious rabbits underwent a 30-min coronary occlusion (O) followed by 72 h of reperfusion (R) with or without ischemic PC (6 4-min O/4-min R cycles) 24 h earlier. Late PC reduced infarct size approximately 46% versus controls. The K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD), given 5 min before the 30-min O, abrogated the infarct-sparing effect of late PC but did not alter infarct size in non-PC rabbits. In phase II, rabbits underwent six 4-min O/4-min R cycles for 3 consecutive days (days 1, 2, and 3). In controls, the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 46% on day 2 and 54% on day 3 compared with day 1, indicating a late PC effect against myocardial stunning. Neither 5-HD nor glibenclamide, given on day 2, abrogated late PC. The K(ATP) channel opener diazoxide, given on day 1, attenuated stunning, and this effect was completely blocked by 5-HD. Thus the same dose of 5-HD that blocked the antistunning effect of diazoxide failed to block the antistunning effects of late PC. Furthermore, when diazoxide was administered in PC rabbits on day 2, myocardial stunning was further attenuated, indicating that diazoxide and late PC have additive anti-stunning effects. We conclude that K(ATP) channels play an essential role in late PC against infarction but not in late PC against stunning, revealing an important pathogenetic difference between these two forms of cardioprotection.     

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.     

Sarcolemmal KATP channel triggers delayed ischemic preconditioning in rats

Previous work from our laboratory has shown that the sarcolemmal K(ATP) channel (sK(ATP)) is required as a trigger for delayed cardioprotection upon exogenous opioid administration. We also established that the mitochondrial K(ATP) (mK(ATP)) channel is not required for triggering delayed delta-opioid-induced infarct size reduction. Because mechanistic differences have been found among delta-opioids and that due to ischemic preconditioning (IPC), we determined whether the triggering mechanism of delayed IPC-induced infarct size reduction involves either the sK(ATP) or mK(ATP). Male Sprague-Dawley rats received either sham surgery or IPC (3- to 5-min cycles of ischemia and reperfusion) 24 h before being subjected to 30 min of ischemia and 2 h of reperfusion. Infarct size was determined and expressed as a percentage of the area at risk, with significance compared with sham reported at P 相似文献   

Myocardial preconditioning against ischemia-reperfusion injury is abolished in Zucker obese rats with insulin resistance

Insulin resistance (IR) precedes the onset of Type 2 diabetes, but its impact on preconditioning against myocardial ischemia-reperfusion injury is unexplored. We examined the effects of diazoxide and ischemic preconditioning (IPC; 5-min ischemia and 5-min reperfusion) on ischemia (30 min)-reperfusion (240 min) injury in young IR Zucker obese (ZO) and lean (ZL) rats. ZO hearts developed larger infarcts than ZL hearts (infarct size: 57.3 +/- 3% in ZO vs. 39.2 +/- 3.2% in ZL; P < 0.05) and also failed to respond to cardioprotection by IPC or diazoxide (47.2 +/- 4.3% and 52.5 +/- 5.8%, respectively; P = not significant). In contrast, IPC and diazoxide treatment reduced the infarct size in ZL hearts (12.7 +/- 2% and 16.3 +/- 6.7%, respectively; P < 0.05). The mitochondrial ATP-activated potassium channel (K(ATP)) antagonist 5-hydroxydecanoic acid inhibited IPC and diazoxide-induced preconditioning in ZL hearts, whereas it had no effect on ZO hearts. Diazoxide elicited reduced depolarization of isolated mitochondria from ZO hearts compared with ZL (73 +/- 9% in ZL vs. 39 +/- 9% in ZO; P < 0.05). Diazoxide also failed to enhance superoxide generation in isolated mitochondria from ZO compared with ZL hearts. Electron micrographs of ZO hearts revealed a decreased number of mitochondria accompanied by swelling, disorganized cristae, and vacuolation. Immunoblots of mitochondrial protein showed a modest increase in manganese superoxide dismutase in ZO hearts. Thus obesity accompanied by IR is associated with the inability to precondition against ischemic cardiac injury, which is mediated by enhanced mitochondrial oxidative stress and impaired activation of mitochondrial K(ATP).     

Effect of combined K(ATP) channel activation and Na(+)/H(+) exchange inhibition on infarct size in rabbits

We tested if combining treatment with cariporide, an Na(+)/H(+) exchange inhibitor, and diazoxide, a mitochondrial ATP-sensitive K(+) (K(ATP)) channel opener, would reduce myocardial infarct size (IS) to a greater extent than either intervention alone. Four groups of rabbits were studied (n = 10 each): cariporide (0.3 mg/kg), diazoxide (10 mg/kg), both drugs, and saline control, given 15 min before a 30-min coronary artery occlusion and 3 h reperfusion. IS in controls comprised 47 +/- 6% of the risk region. Cariporide reduced IS by 55% compared with control (21 +/- 3%), but diazoxide did not significantly reduce IS compared with controls (37 +/- 6%). Combined treatment resulted in an IS of 18 +/- 5%. Also we determined that diazoxide did not potentiate a subthreshold dose of cariporide nor did a mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (5-HD), prevent cariporide from reducing IS. Thus cariporide reduced necrosis by >50% in this model, both in the presence and absence of K(ATP) channel blockade. There was no significant difference in IS reduction between the group receiving cariporide alone and the group receiving combined treatment. Because the effect of cariporide was not blocked by 5-HD, it is unlikely that K(ATP) channels play a role as an end effector in cariporide's mechanism.     

Differential role of sarcolemmal and mitochondrial K(ATP) channels in adenosine-enhanced ischemic preconditioning

Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic preconditioning (IPC) by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. The purpose of this study was to determine whether APC is modulated by ATP-sensitive potassium (K(ATP)) channels and to determine whether this modulation occurs before ischemia or during reperfusion. The role of K(ATP) channels before ischemia (I), during reperfusion (R), or during ischemia and reperfusion (IR) was investigated using the nonspecific K(ATP) blocker glibenclamide (Glb), the mitochondrial (mito) K(ATP) channel blocker 5-hydroxydecanoate (5-HD), and the sarcolemmal (sarc) K(ATP) channel blocker HMR-1883 (HMR). Infarct size was significantly increased (P < 0.05) in APC hearts with Glb-I, Glb-R, and 5-HD-I treatment and partially with 5-HD-R. Glb-I and Glb-R treatment significantly decreased APC functional recovery (P < 0.05 vs. APC), whereas 5-HD-I and 5-HD-R had no effect on APC functional recovery. HMR-IR significantly decreased postischemic functional recovery (P < 0.05 vs. APC) but had no effect on infarct size. These data indicate that APC infarct size reduction is modulated by mitoK(ATP) channels primarily during ischemia and suggest that functional recovery is modulated by sarcK(ATP) channels during ischemia and reperfusion.     

Noninvasive delayed limb ischemic preconditioning attenuates myocardial ischemia-reperfusion injury in rats by a mitochondrial K(ATP) channel-dependent mechanism

We previously demonstrated in rats that noninvasive delayed limb ischemic preconditioning (LIPC) induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb per day for three days confers the same cardioprotective effect as local ischemic preconditioning of the heart, but the mechanism has not been studied in depth. The aim of this project was to test the hypothesis that delayed LIPC enhances myocardial antioxidative ability during ischemia-reperfusion by a mitochondrial K(ATP) channel (mito K(ATP))-dependent mechanism. Rats were randomized to five groups: ischemia-reperfusion (IR)-control group, myocardial ischemic preconditioning (MIPC) group, LIPC group, IR-5HD group and LIPC-5HD group. The MIPC group underwent local ischemic preconditioning induced by three cycles of 5-min occlusion and 5-min reperfusion of the left anterior descending coronary arteries. The LIPC and LIPC-5HD groups underwent LIPC induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb using a modified blood pressure aerocyst per day for three days. All rats were subjected to myocardial ischemia-reperfusion injury. The IR-5HD and LIPC-5HD groups received the mito K(ATP) channel blocker 5-hydroxydecanoate Na (5-HD) before and during the myocardial ischemia-reperfusion injury. Compared with the IR-control group, both the LIPC and MIPC groups showed an amelioration of ventricular arrhythmia, reduced myocardial infarct size, increased activities of total superoxide dismutase, manganese-superoxide dismutase (Mn-SOD) and glutathione peroxidase, increased expression of Mn-SOD mRNA and decreased xanthine oxidase activity and malondialdehyde concentration. These beneficial effects of LIPC were prevented by 5-HD. In conclusion, delayed LIPC offers similar cardioprotection as local IPC. These results support the hypothesis that the activation of mito K(ATP) channels enhances myocardial antioxidative ability during ischemia-reperfusion, thereby contributing, at least in part, to the anti-arrhythmic and anti-infarct effects of delayed LIPC.     

ATP-sensitive potassium channel: a novel target for protection against UV-induced human skin cell damage

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of K(ATP) channels are functionally expressed in HaCaT cells and both non-selective K(ATP) channel opener pinacidil and mitoK(ATP) (mitochondrial K(ATP)) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective K(ATP) channel blocker glibenclamide and selective mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). Also, activation of K(ATP) channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoK(ATP) is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that K(ATP) openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging.     

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。     

Dual roles of mitochondrial K(ATP) channels in diazoxide-mediated protection in isolated rabbit hearts

Whether the mitochondrial ATP-dependent potassium (mK(ATP)) channel is the trigger or the mediator of cardioprotection is controversial. We investigated the critical time sequences of mK(ATP) channel opening for cardioprotection in isolated rabbit hearts. Pretreatment with diazoxide (100 microM), a selective mK(ATP) channel opener, for 5 min followed by 10 min washout before the 30-min ischemia and 2-h reperfusion significantly reduced infarct size (9 +/- 3 vs. 35 +/- 3% in control), indicating a role of mK(ATP) channels as a trigger of protection. The protection was blocked by coadministration of the L-type Ca(2+) channel blockers nifedipine (100 nM) or 5-hydroxydecanoic acid (5-HD; 50 microM) or by the protein kinase C (PKC) inhibitor chelerythrine (5 microM). The protection of diazoxide was not blocked by 50 microM 5-HD but was blocked by 200 microM 5-HD or 10 microM glybenclamide administrated 5 min before and throughout the 30 min of ischemia, indicating a role of mK(ATP) opening as a mediator of protection. Giving diazoxide throughout the 30 min of ischemia also protected the heart, and the protection was not blocked by chelerythrine. Nifedipine did not affect the ability of diazoxide to open mK(ATP) channels assessed by mitochondrial redox state. In electrically stimulated rabbit ventricular myocytes, diazoxide significantly increased Ca(2+) transient but had no effect on L-type Ca(2+) currents. Our results suggest that opening of mK(ATP) channels can trigger cardioprotection. The trigger phase may be induced by elevation of intracellular Ca(2+) and activation of PKC. During the lethal ischemia, mK(ATP) channel opening mediates the protection, independent of PKC, by yet unknown mechanisms.     

Design and synthesis of nitrate esters of aromatic heterocyclic compounds as pharmacological preconditioning agents

Ischemic preconditioning (IPC) constitutes an endogenous protective mechanism in which one or more brief periods of myocardial ischemia and reperfusion render the myocardium resistant to a subsequent more-sustained ischemic insult. Pharmacological preconditioning represents an ideal alternative of IPC. We now describe the design and synthesis of indole, quinoline, and purine systems with an attached pharmacophoric nitrate ester group. The indole and quinoline derivatives 4 and 5 possess structural features of the nitrate containing K(ATP) channel openers. Purine analogues 11 and 12, substituted at the position 6 by a piperidine moiety and at position 9 by an alkyl nitrate, could combine the effects of the nitrate containing K(ATP) channel openers and those of adenosine. Compound 13 bears the nicotinamide moiety of nicorandil instead of nitrate ester. Compounds 4, 5, and 11 reduced infarction and the levels of malondialdehyde (MDA) at reperfusion in anesthetized rabbits. Compounds 12 and 13 did not significantly reduce the infarct size. Analogues 4 and 5 increased cGMP and MDA during ischemia, while combined analogue 4 and mitoK(ATP) blocker 5-hydroxydecanoic acid (5-HD) abrogated this benefit suggesting an action through mitoK(ATP) channel opening. Treatment with derivative 11 combined with 5-HD as well as treatment with 11 and adenosine receptor blocker 8-(p-sulfophenyl)theophylline (SPT) did not abrogate cardioprotection. Compound 11 is a lead molecule for the synthesis of novel analogues possessing a dual mode of action through cGMP-mitoK(ATP) channel opening-free radicals and through adenosine receptors.     

Effective protection by NHE-1 inhibition in ischemic and reperfused heart under preconditioning blockade

We compared the protective effects of ischemic preconditioning (IPC) and the Na(+)/H(+) exchanger-1 (NHE-1) inhibitor cariporide in isolated rat hearts subjected to global ischemia (45 or 90 min) and 30-min reperfusion and determined the protective effects of cariporide under IPC blockade with the mitochondrial ATP-sensitive K(+) channel blocker 5-hydroxydecanoate (5-HD). With 45-min ischemia, both IPC and cariporide equally increased maximum recovery of left ventricular developed pressure twofold (P < 0.05), although recovery was significantly greater with cariporide for the first 15 min of reperfusion. 5-HD almost completely blocked the protective effects of IPC on recovery but had no influence on the salutary effects of cariporide. With 90-min ischemic control, recovery was only 3% of preischemia and was unaffected by IPC, although cariporide increased recovery to approximately 30% (P < 0.05). This was associated with a 37% preservation of viable cardiac cells, whereas no structurally intact cells were found in either IPC or control hearts. Our study shows that NHE-1 inhibition is a more effective cardioprotective strategy than IPC in this model, possibly because of enhanced myocyte salvage, and because protection by NHE-1 inhibition is completely unaffected by IPC blockade with 5-HD.     

Ischemic preconditioning prevents endothelial dysfunction, P-selectin expression, and neutrophil adhesion by preventing endothelin and O2- generation in the post-ischemic guinea-pig heart.

Evidence indicates that ischemia/reperfusion (IR) results in endothelial dysfunction and neutrophil adhesion in the post-ischemic myocardium and that ischemic preconditioning (IPC), superoxide dismutase (SOD), and anti-endothelin-1 (ET-1) interventions prevent these effects. We tested the hypothesis that ET-1-induced superoxide (O(2)(-)) generation mediates endothelial injury and neutrophil accumulation in the IR heart, that IPC protects the endothelium and prevents the adhesion by attenuating post-ischemic ET-1, and thus O(2)(-), generation, and that the mitochondrial ATP-dependent potassium channel (mK(ATP)) triggers the IPC-induced protection. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min wash-out of mK(ATP) opener diazoxide (0.5 microM). Neutrophils were infused to the hearts at 15-25 min of the reperfusion. Coronary flow responses to acetylcholine (ACh) and nitroprusside (SNP) served as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of ET-1 and O(2)(-), P-selectin expression, neutrophil adhesion and functional recoveries were followed during reperfusion. IR augmented ET-1 and O(2)(-) outflow, P-selectin expression, and neutrophil adhesion, and impaired ACh response. These effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK(ATP) blocker) abolished the effects of IPC and diazoxide. SOD (150 U/ml) and tezosentan (5 nM, a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. The preventive effect of IPC, SOD and tezosentan on P-selectin expression preceded their effect on neutrophil adhesion. These data suggest that in guinea-pig heart: (i) ET-1-induced O(2)(-) generation mediates the post-ischemic endothelial dysfunction, P-selectin expression and neutrophil adhesion; (ii) IPC and diazoxide afford protection by attenuating the ET-1, and thus O(2)(-) generation; (iii) the mK(ATP) opening triggers the IPC protection; (iv) endothelial injury promotes post-ischemic neutrophil adhesion, but not vice versa.     

The role of C-reactive protein in ischemia/reperfusion injury and preconditioning in a rat model of myocardial infarction

For the first time the involvement of C-Reactive protein (CRP) in early (acute) and delayed ischemic (IPC) and pharmacological (chemical) preconditioning (CPC) in an in vivo model of rat myocardial infarction was presented. Acute IPC was produced by three 5 minute occlusion (ischemia) periods interspersed with 5 minute reperfusion, followed by 30 minute occlusion of the left coronary artery and 2 hour reperfusion injury. Acute CPC was produced by a k-opioid receptor agonist U50488H (5 mg/kg) applied i.v. 15 minutes before 30 minute ischemia/ 2 hour reperfusion. Delayed preconditioning was produced by 30 minute ischemia/ 2 hour reperfusion, induced 24 hour after either ischemic or pharmacological preconditioning. The myocardial ischemia/reperfusion injury was evaluated on the basis of total and cardiac creatine kinase isoenzyme activity, functional recovery of the heart (ECG), infarct size (% IS/RA) and mortality at the end of the experiments. The results obtained showed that: k-opioid receptor agonist U50488H mimics both the acute and delayed IPC in the above experimental protocol; Both acute IPC and most probably CPC act by opening of K(ATP) channels (the effects were blocked by nonspecific ATP-sensitive K channel blocker glybenclamide), and via activation of protein kinase C (a selective protein kinase C inhibitor chelerythrine blocked the efects); C-reactive protein (CRP) was significantly elevated by 54% in non-preconditioned acute ischemia/reperfusion injury. The elevation was more pronounced (82% increase) 24 hour after non-preconditioned ischemia/reperfusion injury. It reflected very well the increase in cardiac isoenzymes, infarct size and mortality of the rats, and can be used as a marker of the severity of myocardial injury in this model; The increase of CRP was prevented by both IPC and CPC in early, and especially in late preconditioning. This confirms the involvement of CRP as a marker in cardiac ischemic/reperfusion injury. It was concluded that in addition to the established involvement of adenosine, bradykinin, opioid and other receptors, a suppression of myocardial CRP/complement production might be involved in the biological mechanism of preconditioning. This could be a promising perspective in clinical interventions against ischemia/reperfusion injuries of the heart.     

mitoKATP通道参与心肌缺血预处理保护作用的机制

目的：探讨血管紧张素转换酶抑制剂（ACEI）和阈下缺血预处理联合预处理诱导的心肌保护作用中mi-toKatp通道激动后的作用机制：方法：采用离体大鼠心脏Langendorff灌流模型,观察心脏电脱耦联发生时间、细胞膜Na^＋／K^＋-ATPase和Ca^2＋/Mg^2＋-ATPase活性的改变：结果：单独使用卡托普利、或给予大鼠心脏2min缺血／10min复灌作为阈下缺血预处理,均不能改善长时间缺血／复灌引起的心脏收缩功能下降？而卡托普利和阂下缺血预处理联合使用可增高心脏收缩功能。mitoKatp通道特异性阻断剂5-HD可取消这一联合预处理的作用一联合预处理可引起缺血后电脱耦联发生时间延长,缺血心肌细胞膜Na^＋／K^＋-ATPase和Ca^2＋/Mg^2＋-ATPase活性增高;5-HD可取消此作用结论：mitoKatp通道参与了联合预处理延迟缺血引起的细胞间脱耦联和促进细胞膜离子通道稳定性维持的作用。     

Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction

We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (approximately 300 mmHg) elicited protection against ischemia-reperfusion injury (infarction) in multiple distant skeletal muscles subsequently subjected to 4 h of ischemia and 48 h of reperfusion, but the mechanism was not studied. The aim of this project was to test our hypothesis that mitochondrial ATP-sensitive potassium (KATP) (mKATP) channels play a central role in the trigger and mediator mechanisms of hindlimb remote ischemic preconditioning (IPC) of skeletal muscle against infarction in the pig. We observed in the pig that hindlimb remote IPC reduced the infarct size of latissimus dorsi (LD) muscle flaps (8 x 13 cm) from 45 +/- 2% to 22 +/- 3% (n = 10; P < 0.05). The nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg), but not the selective sarcolemmal KATP (sKATP) channel inhibitor HMR-1098 (3 mg/kg), abolished the infarct-protective effect of hindlimb remote IPC in LD muscle flaps (n = 10, P < 0.05) when these drugs were injected intravenously at 10 min before remote IPC. In addition, intravenous bolus injection of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) at the end of hindlimb remote IPC also abolished the infarct protection in LD muscle flaps (n = 10; P < 0.05). Furthermore, intravenous injection of the specific mKATPchannel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of ischemia protected the LD muscle flap against infarction to a similar extent as hindlimb remote IPC, and this infarct-protective effect of BMS-191095 was abolished by intravenous bolus injection of 5-HD (5 mg/kg) at 10 min before or after intravenous injection of BMS-191095 (n = 10; P < 0.05). The infarct protective effect of BMS-191095 was associated with a higher muscle content of ATP at the end of 4 h of ischemia and a decrease in muscle neutrophilic myeloperoxidase activity at the end of 1.5 h of reperfusion compared with the time-matched control (n = 10, P < 0.05). These observations led us to conclude that mKATP channels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction in the pig, and the opening of mKATP channels in ischemic skeletal muscle is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.