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.     

Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion

Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts (n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A(1), A(2), and A(3)) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A(1)/A(3)) and MRS-1191/MRS-1220 (A(3)) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion (P < 0.05 vs. APC). DPCPX (A(1)) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.     

Effects of 5-hydroxydecanoate and ischemic preconditioning on the ischemic-reperfused heart of fed and fasted rats

This investigation aimed to assess whether the mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) could abolish the protection conferred by fasting and ischemic preconditioning (IPC) and to ascertain whether these effects are associated with glycogen breakdown and glycolytic activity. Langendorff perfused hearts of fed and 24-h fasted rats were exposed to 25 min ischemia plus 30 min reperfusion. IPC was achieved by a 3 min ischemia plus a 5 min reperfusion cycle. 5-HD (100 microM) perfusion begun 5 min before IPC or 13 min before sustained ischemia in the non preconditioned groups. Fasting improved the reperfusion recovery of contraction, decreased the contracture and the lactate production, increased glycogenolysis and did not affect the percentage of viable tissue. 5-HD abolished the effects of fasting on the contractile recovery but did not affect the contracture. 5-HD decreased the lactate production in the fed group, increased the preischemic glycogen content in both nutritional groups and did not affect the ischemic glycogen fall. IPC improved the contractile function but prevented the contracture only in the fed group, reduced lactate accumulation and glycogenolysis and evoked an increase of the viable tissue. 5-HD abolished the effects of IPC on the contractile recovery and did not affect its effect on the contracture, lactate production, glycogenolysis and viable tissue. These data suggest that the mitocondrial ATP-sensitive potassium channel is involved in the effects of fasting and IPC on the contractile function but the other cardioprotective and metabolic effects appear evoked through other mechanisms. Also suggest that besides the inhibition of the mitochondrial potassium channel, other mechanisms mediate the effects of 5-HD.     

Receptor and non-receptor-dependent mechanisms of cardioprotection with adenosine

The relative roles of mitochondrial (mito) ATP-sensitive K(+) (mitoK(ATP)) channels, protein kinase C (PKC), and adenosine kinase (AK) in adenosine-mediated protection were assessed in Langendorff-perfused mouse hearts subjected to 20-min ischemia and 45-min reperfusion. Control hearts recovered 72 +/- 3 mmHg of ventricular pressure (50% preischemia) and released 23 +/- 2 IU/g lactate dehydrogenase (LDH). Adenosine (50 microM) during ischemia-reperfusion improved recovery (149 +/- 8 mmHg) and reduced LDH efflux (5 +/- 1 IU/g). Treatment during ischemia alone was less effective. Treatment with 50 microM diazoxide (mitoK(ATP) opener) during ischemia and reperfusion enhanced recovery and was equally effective during ischemia alone. A(3) agonism [100 nM 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide], A(1) agonism (N(6)-cyclohexyladenosine), and AK inhibition (10 microM iodotubercidin) all reduced necrosis to the same extent as adenosine, but less effectively reduced contractile dysfunction. These responses were abolished by 100 microM 5-hydroxydecanoate (5-HD, mitoK(ATP) channel blocker) or 3 microM chelerythrine (PKC inhibitor). However, the protective effects of adenosine during ischemia-reperfusion were resistant to 5-HD and chelerythrine and only abolished when inhibitors were coinfused with iodotubercidin. Data indicate adenosine-mediated protection via A(1)/A(3) adenosine receptors is mitoK(ATP) channel and PKC dependent, with evidence for a downstream location of PKC. Adenosine provides additional and substantial protection via phosphorylation to 5'-AMP, primarily during reperfusion.     

Measurements of 1,2-diacylglycerol and ceramide in hearts subjected to ischemic preconditioning

An accumulation of recent evidence suggests that the mechanism in ischemic preconditioning (IPC) may involve the activation of protein kinase C (PKC) regulatory pathway. In this study, we examined whether the content of 1,2-diacylglycerol (1,2-DAG) and ceramide, which are intracellular second messengers regulating PKC activity, change during IPC in isolated perfused rat hearts, and whether the observed change in 1,2-DAG is accompanied with alteration in its fatty acid composition. Hearts subjected to IPC, consisting of 5-min transient global ischemia followed by 5-min reperfusion, presented a significant functional recovery during subsequent 40-min reperfusion following 40-min global ischemia compared with non-preconditioned hearts. An increase in 1,2-DAG content was observed in hearts subjected to 5-min transient ischemia compared with non-ischemic control hearts, however this was not seen in hearts harvested after 5-min reperfusion following 5-min ischemia. While fatty acid composition in 1,2-DAG was virtually unchanged in hearts subjected to 5-min ischemia, saturated 1,2-DAG decreased and monounsaturated/polyunsaturated 1,2-DAG increased in hearts reperfused for 5-min following 5-min ischemia compared with the non-ischemic control hearts. Ceramide mass did not change significantly, suggesting that the contribution of ceramide may be small in IPC. These data are in concert with the hypothesis that 1,2-DAG is a second messenger in IPC and the changes in fatty acid composition of 1,2-DAG may add new insight concerning signal transduction pathway in IPC.     

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.     

Na(+)/H(+) exchange inhibition prevents endothelial dysfunction after I/R injury

Whereas inhibition of the Na(+)/H(+) exchanger (NHE) has been demonstrated to reduce myocardial infarct size in response to ischemia-reperfusion injury, the ability of NHE inhibition to preserve endothelial cell function has not been examined. This study examined whether NHE inhibition could preserve endothelial cell function after 90 min of regional ischemia and 180 min of reperfusion and compared this inhibition with ischemic preconditioning (IPC). In a canine model either IPC, produced by one 5-min coronary artery occlusion (1 x 5'), or the specific NHE-1 inhibitor eniporide (EMD-96785, 3.0 mg/kg) was administered 15 min before a 90-min coronary artery occlusion followed by 3 h of reperfusion. Infarct size (IS) was determined by 2,3,5-triphenyl tetrazolium chloride staining and expressed as a percentage of the area-at-risk (IS/AAR). Endothelial cell function was assessed by measurement of coronary blood flow in response to intracoronary acetylcholine infusion at the end of reperfusion. Whereas neither control nor IPC-treated animals exhibited a significant reduction in IS/AAR or preservation of endothelial cell function, animals treated with the NHE inhibitor eniporide showed a marked reduction in IS/AAR and a significantly preserved endothelial cell function (P < 0.05). Thus NHE-1 inhibition is more efficacious than IPC at reducing IS/AAR and at preserving endothelial cell function in dogs.     

Ischemic preconditioning does not protect via blockade of electron transport.

Ischemic preconditioning (IPC) before sustained ischemia decreases myocardial infarct size mediated in part via protection of cardiac mitochondria. Reversible blockade of electron transport at complex I immediately before sustained ischemia also preserves mitochondrial respiration and decreases infarct size. We proposed that IPC would attenuate electron transport from complex I as a potential effector mechanism of cardioprotection. Isolated, Langendorff-perfused rat hearts underwent IPC (3 cycles of 5-min 37 degrees C global ischemia and 5-min reperfusion) or were perfused for 40 min without ischemia as controls. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated. IPC did not decrease ADP-stimulated respiration measured in intact mitochondria using substrates that donate reducing equivalents to complex I. Maximally expressed complex I activity measured as rotenone-sensitive NADH:ubiquinone oxidoreductase in detergent-solubilized mitochondria was also unaffected by IPC. Thus the protection of IPC does not occur as a consequence of a partial decrease in complex I activity leading to a decrease in integrated respiration through complex I. IPC and blockade of electron transport both converge on mitochondria as effectors of cardioprotection; however, each modulates mitochondrial metabolism during ischemia by different mechanisms to achieve cardioprotection.     

Involvement of the mitochondrial calcium uniporter in cardioprotection by ischemic preconditioning

The objective of the present study was to determine whether the mitochondrial calcium uniporter plays a role in the cardioprotection induced by ischemic preconditioning (IPC). Isolated rat hearts were subjected to 30 min of regional ischemia by ligation of the left anterior descending artery followed by 120 min of reperfusion. IPC was achieved by two 5-min periods of global ischemia separated by 5 min of reperfusion. IPC reduced the infarct size and lactate dehydrogenase release in coronary effluent, which was associated with improved recovery of left ventricular contractility. Treatment with ruthenium red (RR, 5 μM), an inhibitor of the uniporter, or with Ru360 (10 μM), a highly specific uniporter inhibitor, provided cardioprotective effects like those of IPC. The cardioprotection induced by IPC was abolished by spermine (20 μM), an activator of the uniporter. Cyclosporin A (CsA, 0.2 μM), an inhibitor of the mitochondrial permeability transition pore, reversed the effects caused by spermine. In mitochondria isolated from untreated hearts, both Ru360 (10 μM) and RR (1 μM) decreased pore opening, while spermine (20 μM) increased pore opening which was blocked by CsA (0.2 μM). In mitochondria from preconditioned hearts, the opening of the pore was inhibited, but this inhibition did not occur in the mitochondria from hearts treated with IPC plus spermine. These results indicate that the mitochondrial calcium uniporter is involved in the cardioprotection conferred by ischemic preconditioning.     

Ischemic and anesthetic preconditioning reduces cytosolic [Ca2+] and improves Ca(2+) responses in intact hearts

Ca(+) loading during reperfusion after myocardial ischemia is linked to reduced cardiac function. Like ischemic preconditioning (IPC), a volatile anesthetic given briefly before ischemia can reduce reperfusion injury. We determined whether IPC and sevoflurane preconditioning (SPC) before ischemia equivalently improve mechanical and metabolic function, reduce cytosolic Ca(2+) loading, and improve myocardial Ca(2+) responsiveness. Four groups of guinea pig isolated hearts were perfused: no ischemia, no treatment before 30-min global ischemia and 60-min reperfusion (control), IPC (two 2-min occlusions) before ischemia, and SPC (3.5 vol%, two 2-min exposures) before ischemia. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured at the left ventricular (LV) free wall with the fluorescent probe indo 1. Ca(2+) responsiveness was assessed by changing extracellular [Ca(2+)]. In control hearts, initial reperfusion increased diastolic [Ca(2+)] and diastolic LV pressure (LVP), and the maximal and minimal derivatives of LVP (dLVP/dt(max) and dLVP/dt(min), respectively), O(2) consumption, and cardiac efficiency (CE). Throughout reperfusion, IPC and SPC similarly reduced ischemic contracture, ventricular fibrillation, and enzyme release, attenuated rises in systolic and diastolic [Ca(2+)], improved contractile and relaxation indexes, O(2) consumption, and CE, and reduced infarct size. Diastolic [Ca(2+)] at 50% dLVP/dt(min) was right shifted by 32-53 +/- 8 nM after 30-min reperfusion for all groups. Phasic [Ca(2+)] at 50% dLVP/dt(max) was not altered in control but was left shifted by -235 +/- 40 nM [Ca(2+)] after IPC and by -135 +/- 20 nM [Ca(2+)] after SPC. Both SPC and IPC similarly reduce Ca(2+) loading, while augmenting contractile responsiveness to Ca(2+), improving postischemia cardiac function and attenuating permanent damage.     

Influence of ischemic preconditioning on intracellular sodium,pH, and cellular energy status in isolated perfused heart

The possible relationships between intracellular Na(+) (Na(i)(+)), bioenergetic status and intracellular pH (pH(i)) in the mechanism for ischemic preconditioning were studied using (23)Na and (31)P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na(i) during ischemia in the preconditioned hearts was characterized by an increase in Na(i)(+) at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P(i) and betaATP/P(i) ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na(i)(+) during reperfusion in the preconditioned hearts suggest activation of Na(+)/H(+) exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.     

Cardiac preconditioning with 4-h, 17 degrees C ischemia reduces [Ca(2+)](i) load and damage in part via K(ATP) channel opening

Brief ischemia before normothermic ischemia protects hearts against reperfusion injury (ischemic preconditioning, IPC), but it is unclear whether it protects against long-term moderate hypothermic ischemia. We explored in isolated guinea pig hearts 1) the influence of two 2-min periods of normothermic ischemia before 4 h, 17 degrees C hypothermic ischemia on cardiac cytosolic [Ca(2+)], mechanical and metabolic function, and infarct size, and 2) the potential role of K(ATP) channels in eliciting cardioprotection. We found that IPC before 4 h moderate hypothermia improved myocardial perfusion, contractility, and relaxation during normothermic reperfusion. Protection was associated with markedly reduced diastolic [Ca(2+)] loading throughout both hypothermic storage and reperfusion. Global infarct size was markedly reduced from 36 +/- 2 (SE)% to 15 +/- 1% with IPC. Bracketing ischemic pulses with 200 microM 5-hydroxydecanoic acid or 10 microM glibenclamide increased infarct size to 28 +/- 3% and 26 +/- 4%, respectively. These results suggest that brief ischemia before long-term hypothermic storage adds to the cardioprotective effects of hypothermia and that this is associated with decreased cytosolic [Ca(2+)] loading and enhanced ATP-sensitive K channel opening.     

大鼠肢体缺血预处理对肝缺血/再灌注损伤的保护作用

目的：研究肢体缺血预处理对大鼠肝缺血/再灌注损伤是否具有保护作用。方法：雄性SD大鼠32只,随机分为对照组（S组）;缺血/再灌注组（I/R组）;经典缺血预处理组（IPC组）;肢体缺血预处理组（远端缺血预处理组,RPC组）。S组仅行开腹,不作其他处理;IPC组以肝缺血5min作预处理;RPC组以双后肢缺血5min,反复3次作预处理,2个预处理组及I/R组均行肝缺血1h再灌注3h。取血用于血清谷丙转氨酶（ALT）与血清谷草转氨酶（AST）检测。切取肝组织用于测定湿干比（W/D）、中性粒细胞（PMN）计数及观察显微、超微结构的变化。结果：与I/R组比较,IPC组,RPC组ALT,AST,W/D值,及PMN计数均明显降低（P〈0.01）,肝脏的显微及超微结构损伤减轻。结论：肢体缺血预处理对大鼠肝脏I/R损伤有明显的保护作用,强度与经典缺血预处理相当,其机制可能与抑制肝脏炎症反应、减轻肝脏水肿、改善肝组织微循环有关。     

Ischemic preconditioning-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contraction-induced myocardial injury

Dystrophin is an integral membrane protein involved in the stabilization of the sarcolemmal membrane in cardiac muscle. We hypothesized that the loss of membrane dystrophin during ischemia and reperfusion is responsible for contractile force-induced myocardial injury and that cardioprotection afforded by ischemic preconditioning (IPC) is related to the preservation of membrane dystrophin. Isolated and perfused rat hearts were subjected to 30 min of global ischemia, followed by reperfusion with or without the contractile blocker 2,3-butanedione monoxime (BDM). IPC was introduced by three cycles of 5-min ischemia and 5-min reperfusion before the global ischemia. Dystrophin was distributed exclusively in the membrane of myocytes in the normally perfused heart but was redistributed to the myofibril fraction after 30 min of ischemia and was lost from both of these compartments during reperfusion in the presence or absence of BDM. The loss of dystrophin preceded uptake of the membrane-impermeable Evans blue dye by myocytes that occurred after the withdrawal of BDM and was associated with creatine kinase release and the development of contracture. Although IPC did not alter the redistribution of membrane dystrophin induced by 30 min of ischemia, it facilitated the restoration of membrane dystrophin during reperfusion. Also, myocyte necrosis was not observed when BDM was withdrawn after complete restoration of membrane dystrophin. These results demonstrate that IPC-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contractile force-induced myocardial injury and suggest that the cardioprotection conferred by IPC can be enhanced by the temporary blockade of contractile activity until restoration of membrane dystrophin during reperfusion.     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

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.     

Na+/H+ exchange inhibitors: a new class of cardioprotectors

The Na+/H+ exchanger (NHE) extrudes intracellular H+ in exchange for Na+ in an electroneutral process. Of the 6 mammalian exchanger isoforms identified to date, the NHE-1 is believed to be the molecular homologue of the sarcolemma Na+/H+ transporter. The exchanger is activated primarily by a reduction in intracellular pH, although such activation is subject to modulation by a variety of endogenous mediators (catecholamines, thrombin, endothelin) through receptor-mediated mechanisms. A large body of animal studies using both in vitro and in vivo models indicates that the inhibition of the sarcolemma NHE-1 attenuates myocardial injury in ischemia and reperfusion. Cardioprotective effects of NHE-1 inhibition involve a reduced susceptibility to severe ventricular arrhythmia, augmentation of contractile function recovery, and limitation of infarction size during reperfusion. Such protection is likely to arise partly from attenuation of "Ca2+ overload" in ischemic cardiomyocytes, which has been causally linked with all these pathologic phenomena. A marked benefit that has been observed with cariporide (HOE-642) and its structurally related congener HOE-694 in patients with acute myocardial infarction and in cardiac surgery demonstrates that selective NHE-1 inhibitors represent a novel and effective class of cardioprotectors.     

Contribution of endocannabinoids in the endothelial protection afforded by ischemic preconditioning in the isolated rat heart

The aim of the present study was to assess the contribution of endogenous cannabinoids in the protective effect of ischemic preconditioning on the endothelial function in coronary arteries of the rat. Isolated rat hearts were exposed to a 30-min low flow ischemia (1 ml/min) followed by 20-min reperfusion, after which the response to the endothelium-dependent vasodilator, serotonine (5-HT), was compared with that of the endothelium-independent vasodilator, sodium nitroprusside (SNP). In untreated hearts, ischemia-reperfusion diminished selectively 5-HT-induced vasodilatation, compared with time-matched sham hearts, the vasodilatation to SNP being unaffected. A 5-min zero-flow preconditioning ischemia in untreated hearts preserved the vasodilatation produced by 5-HT. Blockade of either CB(1)-receptors with SR141716A or CB(2)-receptors with SR144528 abolished the protective effect of preconditioning on the 5-HT vasodilatation. Perfusion with either palmitoylethanolamide or 2-arachidonoylglycerol 15 min before and throughout the ischemia mimicked preconditioning inasmuch as it protected the endothelium in a similar fashion. This protection was blocked by SR144528 in both cases, whereas SR141716A only blocked the effect of PEA. The presence of CB(1) and CB(2)-receptors in isolated rat hearts was confirmed by Western blots. In conclusion, the data suggest that endogenous cannabinoids contribute to the endothelial protective effect of ischemic preconditioning in rat coronary arteries.     

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.     

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.