Hyperoxia confers myocardial protection in mechanically ventilated rats through the generation of free radicals and opening of mitochondrial ATP-sensitive potassium channels

1. One hour exposure to hyperoxia has been shown previously to limit a subsequent ischaemia-reperfusion injury in spontaneously breathing rats. We tested the cardioprotective effect of a shorter period of hyperoxia during mechanical ventilation and the possible contribution of reactive oxygen species (ROS) and mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels. 2. Mechanically ventilated rats were exposed to normoxia (Fi O2 = 0.3) or hyperoxia (Fi O2 = 1.0) for 30 min and pH, P CO2, PO2, heart rate, airway and blood pressure were measured at baseline and after 30 min mechanical ventilation. Isolated hearts were subsequently subjected to 30 min ischaemia and 120 min reperfusion. Infarct size and left ventricular end-diastolic pressure (LVEDP), developed pressure (LVDP) and coronary flow (CF) were measured. In order to investigate the role of ROS and KATP channels within the mechanism leading to cardioprotection, the free radical scavenger N-acetylcysteine (NAC; 150 mg/kg) was infused in mechanically ventilated rats and the KATP channel blockers glibenclamide (200 mmol/L) or 5-hydroxydecanoate (10 mmol/L) were infused in isolated hearts immediately before ischaemia. 3. No differences were detected in P CO2, pH, heart rate, airway and blood pressure between the groups. However, the PO2 in hyperoxic groups was significantly higher compared with that in normoxic groups (P < 0.01). After 30 min ischaemia, we found that hyperoxic preconditioning significantly improved CF (P < 0.01), LVDP (P < 0.01) and LVEDP (P < 0.01) and reduced the extent of infarct size in the reperfused heart compared with the normoxic group (P < 0.01). When rats were pretreated either with NAC before hyperoxic ventilation or with K(ATP) channel blockers before ischaemia, myocardial protection was abolished. 4. Hyperoxic mechanical ventilation, prior to ischaemia, reduces myocardial reperfusion injury. This is likely to occur through the induction of oxidative stress, which leads to myocyte mitoKATP channel opening.     

Sevoflurane post-conditioning protects against myocardial reperfusion injury by activation of phosphatidylinositol-3-kinase signal transduction

The mechanisms underlying myocardial protection by sevoflurane post-conditioning are unclear. In the present study, we tested two hypotheses: (i) that sevoflurane post-conditioning produces cardioprotection via a phosphatidylinositol-3-kinase (PI3-K)-dependent pathway; and (ii) combining sevoflurane and ischaemic post-conditioning offers an additional benefit against reperfusion injury. Rat isolated perfused hearts were exposed to 25 min ischaemia followed by 90 min reperfusion. Sevoflurane post-conditioning was induced by administration of sevoflurane (3.0 vol%) for 15 min from the onset of reperfusion. In some groups, 15 micromol/L LY294002, a selective PI3-K inhibitor, was coadministrated with sevoflurane. Other groups of hearts were exposed to ischaemic post-conditioning or combined sevoflurane plus ischaemic post-conditioning in the presence and absence of LY294002. After 15 min reperfusion, phosphorylation of Akt and glycogen synthase kinase 3beta (GSK3beta) was determined by Western blot analysis. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining and subsarcolemmal mitochondrial lesions were assessed by electron microscopy after 90 min reperfusion. Sevoflurane post-conditioning significantly decreased infarct size compared with control hearts (31 +/- 2 vs 42 +/- 3%, respectively; P < 0.05), diminished mitochondrial lesions and increased phosphorylation of Akt and GSK3beta, as did ischaemic post-conditioning. However, combined sevoflurane plus ischaemic post-conditioning did not further improve the cardioprotective effects compared with either intervention alone. Sevoflurane-mediated cardioprotection was abolished or inhibited by 15 micromol/L LY294002. In conclusion, sevoflurane acts during early reperfusion after ischaemia to salvage the myocardium by activating PI3-K. The combination of sevoflurane plus ischaemic post-conditioning does not offer any additional benefit over either intervention alone.     

ISCHAEMIC POST-CONDITIONING PROTECTS BOTH ADULT AND AGED SPRAGUE-DAWLEY RAT HEART FROM ISCHAEMIA–REPERFUSION INJURY THROUGH THE PHOSPHATIDYLINOSITOL 3-KINASE–AKT AND GLYCOGEN SYNTHASE KINASE-3β PATHWAYS

• 1 Numerous studies have demonstrated that ischaemic post‐conditioning (IPoC) protects adult rats from myocardial ischaemia‐reperfusion (I/R) injury. Recent evidence suggests compromised cardioprotection by IPoC in aged mice. The present study was designed to test the hypothesis that IPoC protects against I/R injury in aged hearts, potentially through a phosphatidylinositol 3‐kinase (PI3‐K)–Akt‐ and glycogen synthase kinase (GSK)‐3β‐dependent mechanism.
• 2 Hearts from adult (3–4 months) or aged (16–18 months) Sprague‐Dawley rats were subjected in vivo to 30 min ischaemia followed by 3 h reperfusion. Ischaemic post‐conditioning (four cycles of 10 s reperfusion–10 s ischaemia) was applied at the beginning of reperfusion, either alone or in combination with the PI3‐K inhibitor LY294002 (0.3 mg/kg). Infarct size and the phosphorylation of Akt and GSK‐3β were determined.
• 3 Ischaemic post‐conditioning reduced infarct size in both adult and aged rat hearts. This protection was accompanied by a significant increase in phosphorylation of Akt and GSK‐3β. LY294002 abolished the IPoC‐induced phosphorylation of Akt and GSK‐3β, as well as the infarct‐limiting effect of IPoC in adult and aged rats. In addition, IPoC significantly attenuated plasma concentrations of creatine kinase and lactate dehydrogenase after reperfusion in both adult and aged rats.
• 4 In conclusion, IPoC, at the onset of reperfusion, reduces myocardial infarct size in both adult and aged rat hearts, potentially through a PI3‐K‐, Akt‐ and GSK‐3β‐dependent mechanism.

     

Mechanisms mediating the cardioprotective effects of rapamycin in ischaemia-reperfusion injury

1. In the present study, the temporal and concentration-dependent cardioprotective effects of rapamycin against ischaemia-reperfusion (I/R) injury, as well as the underlying mechanisms, were investigated. 2. Rat Langendorff-perfused isolated hearts were exposed to 40 min global ischaemia followed by 120 min reperfusion. Hearts were perfused with different concentrations of rapamycin before and after ischaemia. Myocardial injury was assessed in terms of infarct size and the release of lactate dehydrogenase (LDH) and creatine kinase (CK). The phosphorylation of Akt, extracellular signal-regulated kinase (ERK) 1/2 and endothelial nitric oxide synthase (eNOS) was determined at the end of reperfusion. 3. When administered prior to ischaemia, 25, 50 and 100 nmol/L rapamycin significantly reduced infarct size compared with control (40.1 ± 1.5, 26.3 ± 4.1 and 21.2 ± 3.4 vs 52.5 ± 4.5%, respectively) without affecting the recovery of ventricular function. No reduction in infarct size was observed when 50 nmol/L rapamycin was administered 10 or 120 min into the reperfusion period. 4. Rapamycin (50 nmol/L) enhanced the phosphorylation of Akt kinase but did not affect the phosphorylation of ERK1/2 or eNOS at the end of reperfusion. The cardioprotective effect of rapamycin was blocked by the phosphatidylinositol 3-kinase (Akt) inhibitor LY294002 (15 nmol/L). 5. In conclusion, rapamycin mediates cardioprotection prior to ischaemia and after reperfusion. This protection may involve activation of the phosphatidylinositol 3-kinase pathway.     

Roles of KATP channels in delayed cardioprotection and intracellular Ca(2+) in the rat heart as revealed by kappa-opioid receptor stimulation with U50488H

The effect of preconditioning with U50488 H (UP), a selective kappa-opioid receptor (kappa-OR) agonist, on infarct size and intracellular Ca2+ ([Ca2+]i) in the heart subjected to ischaemic insults were studied and evaluated. U50488 H administered intravenously reduced the infarct size 18-48 h after administration in isolated hearts subjected to regional ischaemia/reperfusion (I/R). The effect was dose dependent. A peak effect was reached at 10 mg x kg-1 U50488 H and at 24 h after administration. The effect of 10 mg x kg-1 U50488 H at 24 h after administration was abolished by nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, indicating the effect was kappa-OR mediated. The infarct reducing effect of U50488 H was attenuated when a selective blocker of mitochondrial (5-hydroxydecanoic acid, 5-HD) or sarcolemmal (HRM-1098) ATP-sensitive potassium channel (KATP) was coadministered with U50488 H 24 h before ischaemia or when 5-HD was administered just before ischaemia. U50488 H also attenuated the elevation in [Ca2+]i and reduction in electrically induced [Ca2+]i transient in cardiomyocytes subjected to ischaemic insults. The effects were reversed by blockade of KATP channel, which abolished the protective effect of preconditioning with U50488 H. The results indicated that mitochondrial KATP channel serves as both a trigger and a mediator, while sarcolemmal KATP channel as a trigger only, of delayed cardioprotection of kappa-OR stimulation. The effects of these channels may result from prevention/attenuation of [Ca2+]i overload induced by ischaemic insults.     

Sevoflurane post-conditioning protects isolated rat hearts against ischemia-reperfusion injury via activation of the ERK1/2 pathway

Aim:

To investigate the role of extracellular signal-regulated kinases (ERKs) in sevoflurane post-conditioning induced cardioprotection in vitro.

Methods:

Isolated rat hearts were subjected to 30 min ischemia followed by 120 min reperfusion (I/R). Sevoflurane post-conditioning was carried out by administration of O₂-enriched gas mixture with 3% sevoflurane (SEVO) for 15 min from the onset of reperfusion. Cardiac functions, myocardial infarct size, myocardial ATP and NAD⁺ contents, mitochondrial ultrastructure, and anti-apototic and anti-oncosis protein levels were measured.

Results:

Sevoflurane post-conditioning significantly improved the heart function, decreased infarct size and mitochondria damage, and increased myocardial ATP and NAD⁺ content in the I/R hearts. Furthermore, sevoflurane post-conditioning significantly increased the levels of p-ERK and p-p70S6K, decreased the levels of porimin, caspase-8, cleaved caspase-3, and cytosolic cytochrome c in the I/R hearts. Co-administration of the ERK1/2 inhibitor PD98059 (20 μmol/L) abolished the sevoflurane-induced protective effects against myocardial I/R.

Conclusion:

Sevoflurane post-conditioning protects isolated rat hearts against myocardial I/R injury and inhibits cell oncosis and apoptosis via activation of the ERK1/2 pathway.     

Contribution of both the sarcolemmal K(ATP) and mitochondrial K(ATP) channels to infarct size limitation by K(ATP) channel openers: differences from preconditioning in the role of sarcolemmal K(ATP) channels

The roles of sarcolemmal ATP-sensitive K+ (sarcK(ATP)) and mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels in the cardioprotection induced by K(ATP) channel openers remain unclear, though the mitoK(ATP) channel has been proposed to be involved as a subcellular mediator in cardioprotection afforded by ischemic preconditioning (PC). In the present study, selective inhibitors of the sarcK(ATP) and mitoK(ATP) channels were used to examine the role of each channel subtype in infarct size limitation by KATP channel openers. Isolated rabbit hearts were perfused in the Langendorff mode with monitoring of the activation recovery interval (ARI) and subjected to 30-min global ischemia/2-h reperfusion to induce infarction. Before ischemia, hearts received 10 microM pinacidil, 100 microM diazoxide, or PC with or without preceding infusion of a sarcK(ATP) channel-selective blocker (5 microM HMR1098) or a mitoK(ATP) channel-selective blocker (100 microM 5-hydroxydecanoate, 5-HD). ARI, an index of action potential duration, was shortened from 118+/-3 ms to 77+/-5 ms after 10 min of ischemia in untreated control hearts. Pinacidil shortened ARI before ischemia from 113+/-2 ms to 78+/-5 ms and enhanced the ARI shortening during ischemia. Diazoxide did not affect ARI before ischemia but accelerated ischemia-induced shortening of ARI. Infarct size as a percentage of the left ventricle (%IS/LV) was reduced by pinacidil and diazoxide from the control value of 47.2+/-4.0% to 4.5+/-1.5% and 5.2+/-1.2%, respectively. HMR1098 significantly inhibited the shortening of ARI by ischemia, pinacidil and diazoxide and partially blocked infarct size limitation by these K(ATP) channel openers (%IS/LV=32.6+/-4.2% and 23.4+/-5.3%, respectively). Infusion of 5-HD did not modify the change in ARI caused by the K(ATP) channel openers but completely abolished cardioprotection (%IS/LV=46.0+/-6.2% with pinacidil and 57.2+/-7.0% with diazoxide). PC with two episodes of 5-min ischemia limited %IS/LV to 21.6+/-4.0%, and this protection was not inhibited by HMR1098. Neither HMR1098 nor 5-HD alone modified infarct size. In conclusion, both sarcK(ATP) and mitoK(ATP) channels may contribute to the anti-infarct tolerance afforded by pinacidil and diazoxide.     

Cardioprotective effects of (2S,3R,4S)-N'-benzyl-N"-cyano-N-(3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-benzopyran-4-yl)-guanidine (KR-31372) in rats and dogs

The cardioprotective effects of (2S,3R,4S)-N'-benzyl- N"-cyano-N-(3,4-dihydro-2-dimethoxymethyl-3-hydro- xy-2-methyl-6-nitro-2H-benzopyran-4-yl)-guanidine (KR-31372) were evaluated against ischemic/reperfusion injury in isolated rat hearts in vitro and in anesthetized rats and dogs in vivo. In isolated perfused rat hearts subjected to a 30-min global ischemia/30-min reperfusion, KR-31372 (1-10 microM) significantly improved severe contracture (end-diastolic pressure and time to contracture), markedly reduced reperfusion lactate dehydrogenase release, and enhanced the recovery of reperfusion contractile function (left ventricular developed pressure and double product) in a concentration-dependent manner compared with the vehicle-treated group. In anesthetized rats subjected to a 45-min coronary occlusion and a 90-min reperfusion, intravenous KR-31372 dose-dependently reduced infarct size from 58.6% to 48.5, 48.1 and 39.6% at 0.3, 1.0 and 3.0 mg/kg, respectively (p < 0.05). In anesthetized beagle dogs that underwent a 1.5-hour occlusion followed by a 5-hour reperfusion, KR-31372 (2 mg/kg, i.v.) markedly reduced infarct size from 57.0% in controls to 28.0% (p < 0.05). The cardioprotective effects of KR-31372 on contractile function in globally ischemic rat hearts and on reperfusion injury in anesthetized rats were significantly reversed by pretreatment with selective adenosine triphosphate-sensitive potassium (K(ATP)) channel blockers, sodium 5-hydroxydecanoate and glibenclamide. Taken together, these results indicate that KR-31372 possesses potent cardioprotective effects in rats and dogs and its effects may be mediated by activation of mitochondrial K(ATP) channels.     

Heat stress-induced protection of endothelial function against ischaemic injury is abolished by ATP-sensitive potassium channel blockade in the isolated rat heart

The protection conferred by heat stress (HS) against myocardial ischaemia-reperfusion injury, in terms of mechanical function preservation and infarct size reduction, is well documented and mechanisms underlying these effects have been extensively explored. However, the effect of HS on coronary circulation is less known. The aim of this study was thus to investigate the role of ATP-sensitive potassium (K(ATP)) channels in the protection against ischaemic injury afforded by HS to the coronary endothelial function. Twenty-four hours after whole body hyperthermia (42 degrees C for 15 min, H groups) or sham anaesthesia (Sham groups), isolated perfused rat hearts were subjected to a 15 min stabilization period followed by a 30 min infusion of either 0.3 microM glibenclamide (Gli, a K(ATP) channel blocker) or its vehicle (V). Hearts were then exposed to a low-flow ischaemia (30 min)-reperfusion (20 min) (I/R) or normally perfused (50 min), after which coronaries were precontracted with 0.1 microM U-46619. Finally, the response to the endothelium-dependent vasodilator, 5-hydroxytryptamine (5-HT, 10 microM) was compared to that of the endothelium-independent vasodilator, sodium nitroprusside (SNP, 3 microM). In hearts from Sham-V and Sham-Gli groups, I/R selectively diminished 5-HT-induced vasodilatation without affecting the vasodilatation to SNP. In V-treated groups, prior HS preserved the vasodilatation produced by 5-HT. This HS-induced protection was abolished by Gli treatment. In conclusion, these results suggest that K(ATP) channel activation contributes to the preservation of coronary endothelial function conferred by heat stress against ischaemic insult.     

Cytochrome P450 2J3/epoxyeicosatrienoic acids mediate the cardioprotection induced by ischaemic post-conditioning, but not preconditioning, in the rat

1. Cytochrome P450 (CYP) epoxygenases and their arachidonic acid metabolites play a protective role against ischaemia-reperfusion injury. In the present study, we investigated whether endogenous CYP2J3/epoxyeicosatrienoic acid (EET) mediates the cardioprotective effects of ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPost). 2. Male Wistar rats were subjected to two cycles of IPC, consisting of 5 min ischaemia and 5 min reperfusion, followed by 45 min occlusion and 2 h reperfusion; IPost consisted of three cycles of 30 s reperfusion and 30 s re-occlusion at the onset of reperfusion. The selective CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 3 mg/kg) was administered 10 min before ischaemia or during ischaemia 10 min before reperfusion started. Cardiac function was measured continuously with a angiocatheter connected to a fluid-filled pressure transducer and myocardial infarct size was assessed by triphenyl tetrazolium chloride staining at the end of the experiment. 3. Subjecting rats to IPC and IPost similarly improved cardiac function and reduced myocardial infarct size. Interestingly, IPost, but not IPC, significantly increased CYP2J3 mRNA (1.75 ± 0.22 vs 1.0; P < 0.05) and protein (1.62 ± 0.22 vs 1.0; P < 0.05), as well as 11,12-EET synthesis compared to I/R (6.2 ± 0.2 vs 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01). Administration of MS-PPOH before ischaemia significantly decreased 11,12-EET synthesis in both IPC and IPost compared with I/R rats (2.1 ± 0.2, 3.2 ± 0.3 and 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01), but decreased the cardioprotective effects, as evidenced by cardiac function and myocardial infarct size, of IPost only. 4. These data indicate that endogenous activation of CYP2J3/EET may be an essential trigger leading to the protective effects of IPost, but not IPC, in the rat heart.     

Multiple signalling pathways underlie the protective effect of levosimendan in cardiac myocytes

Levosimendan is a cardiovascular drug for the treatment of acute and decompensated heart failure. The current weight of evidence on the cardioprotective effects of levosimendan originates from whole heart models and there is no information on the mechanism whereby signalling pathways are activated. In the present study, we investigated the effect of levosimendan on ischaemia/reperfusion injury and the underlying mechanism in cardiac myocytes. Pretreatment with levosimendan reversed the effects of ischaemia and ischaemia/reperfusion on cell viability and enhanced phosphorylation of Akt, p38-mitogen activated protein kinase (MAPK) and extracellular signal-regulated kinases 1/2 (ERK1/2). Inhibitors of these kinases and the blocker of the mitochondrial K(ATP) channels, 5-hydroxydecanoate, completely abolished the protection afforded by levosimendan. Levosimendan stimulated the phosphorylation of Akt, ERK1/2 and p38-MAPK with different kinetics and the activation of these pathways was dependent on the opening of the mitochondrial K(ATP) channels and the production of oxygen free radicals. The levosimendan-induced phosphorylation of ERK1/2 and Akt was reduced by inhibitors of epidermal growth factor receptor and Src. On the other hand, inhibition of the protein kinase A (PKA) pathway reduced phosphorylation of p38-MAPK. Furthermore, p38-MAPK was activated when a phosphodiesterase inhibitor or a selective PKA activator was used. Overall, our results suggest that levosimendan regulates the wiring of the natural salvaging pathways to execute the prosurvival signals. This network includes Akt, ERK1/2 and p38-MAPK. Opening of mitochondrial K(ATP) channels and the subsequent production of oxygen free radicals, the epidermal growth factor receptor/Src, and the cAMP/PKA pathways seem to mediate this response.     

Preservation of mitochondrial function by diazoxide during sustained ischaemia in the rat heart

1. A possible mechanism for the action of the K(ATP) channel opener diazoxide on the improvement of energy metabolism of ischaemic/reperfused hearts was examined. 2. Isolated, perfused rat hearts were subjected to 40 min ischaemia followed by 60 min reperfusion. Diazoxide at concentrations of 3 to 30 microM was present in the perfusion buffer for the last 15 min of pre-ischaemia. 3. Treatment of the perfused heart with diazoxide enhanced the post-ischaemic recovery of rate-pressure product, attenuated the post-ischaemic rise in left ventricular end-diastolic pressure, and suppressed the release of creatine kinase and purine nucleosides and bases from the reperfused heart. Treatment of the heart with diazoxide also restored myocardial ATP and creatine phosphate and attenuated the decrease in mitochondrial oxygen consumption rate after reperfusion. This attenuation was maintained at the end of ischaemia as well as at the end of reperfusion. 4. In another set of experiments, myocardial skinned bundles were incubated for 30 min under hypoxic conditions in the presence and absence of diazoxide, and then the mitochondrial oxygen consumption rate was determined. Hypoxia induced a decrease in the mitochondrial oxygen consumption rate of the skinned bundles to approximately 40% of the pre-hypoxic value. In contrast, treatment of the bundles with 30 microM diazoxide preserved the normal mitochondrial oxygen consumption rate during hypoxia. This effect was abolished concentration-dependently by the combined treatment with either the K(ATP) channel blocker glibenclamide or 5-hydroxydecanoate. 5. These results suggest that diazoxide is capable of attenuating ischaemia/reperfusion injury of isolated perfused hearts due to preservation of mitochondrial function during ischaemia.     

Cardiomyocyte mitochondrial KATP channels participate in the antiarrhythmic and antiinfarct effects of KATP activators during ischemia and reperfusion in an intact anesthetized rabbit model

Recent evidence suggests that the mitochondrial K(ATP) channels may be involved as a subcellular mediator in cardioprotection afforded by ischemic and pharmacological preconditioning by K(ATP) activators. The present study investigated the effects of administration of non-hypotensive doses of ATP-sensitive K(+) channel (K(ATP)) openers, nicorandil (NIC) and pinacidil (PIN), and specific blockers of mitochondrial (5-hydroxydecanoate) and sarcolemmal (1-[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl-3-methyl-thiourea, HMR 1883) K(ATP) channels prior to and during coronary occlusion and post-ischemic reperfusion on survival rate, ischemia- and reperfusion-induced arrhythmias and myocardial infarct size in anesthetized rabbits. In Group I, myocardial ischemia-induced arrhythmias were provoked by tightening a ligature over the left main coronary artery for 30 min. In Group II, arrhythmias were induced by reperfusion following a 20 min ligation of the same artery. Both in Group I and Group II, early iv administration of NIC (0.47 mg/kg), PIN (0.1 mg/kg), HMR 1883 (3 mg/kg)/NIC and HMR 1883/PIN just prior to and during ischemia increased survival rate (75%, 86%, 75% and 75%, respectively, vs. 55% in the control in Group I; 75%, 75%, 75% and 67%, respectively, vs. 50% in the control in Group II), significantly decreased the incidence and severity of life-threatening arrhythmias and significantly decreased myocardial infarct size. However, late iv administration of NIC or PIN just prior to reperfusion did not increase survival rate nor confer any antiarrhythmic or cardioprotective effects. The antiarrhythmic and cardioprotective effects were abolished by pretreating rabbits with 5-hydroxy-decanoate (5 mg/kg, iv bolus). In the present study, higher levels of malondialdehyde and lower levels of reduced glutathione and superoxide dismutase in necrotic zone of myocardium in all subgroups in Group II suggest little anti-free radical property of NIC and PIN. Therefore, it may be assumed that mitochondrial K(ATP) channel opening leads to mitochondrial generation and release of ROS providing for IPC and antiarrhythmic activity. The mitochondrial rather than sarcolemmal K(ATP) channel may represent a potential site of cardioprotection and antiarrhythmic activity.     

Efficacy of ischaemic preconditioning in the eNOS overexpressed working mouse heart model

We recently demonstrated that exogenous nitric oxide (NO) acts as a trigger for preconditioning in the isolated rat heart model. There is however little data concerning the effects of elevated cardiac endothelial nitric oxide synthase (eNOS) expression on myocardial tolerance to ischaemia. Similarly, the effects of gender and eNOS overexpression on ischaemic preconditioning is unknown. We hypothesized that: 1) eNOS overexpression increases myocardial tolerance to ischaemia, and, 2) eNOS overexpressed hearts cannot be preconditioned, since the hearts are already maximally protected. Male and female wild-type and transgenic mice that overexpress eNOS exclusively in cardiac myocytes were perfused in the working heart mode with a modified Krebs-Henseleit buffer at a pre-load of 12.5 mm Hg and afterload of 50 mm Hg. Cardiac output, coronary flow, peak aortic systolic pressure and total work were determined before hearts were preconditioned by 4x5 min cycles of ischaemia/reperfusion, and then subjected to 20 min total global ischaemia, followed by reperfusion. Reperfusion function and myocardial infarct size were used as endpoints. Pre-ischaemic mechanical function (rate pressure product and cardiac output) was similar for wild-type and transgenic mice of both sexes. The eNOS overexpressed hearts had smaller infarcts than the hearts from their wild-type littermates (26.9+/-1.4% vs. 37.0+/-2.1% for controls, P<0.05). Preconditioning the eNOS overexpressed hearts resulted in infarct sizes comparable with control non-preconditioned hearts (27.5+/-2.0% vs. 26.9+/-1.4% for controls). Myocardial cGMP levels were elevated during sustained ischaemia in the transgenic hearts when compared with wild-type hearts (22.43+/-1.63 pmol/g ww vs 16.54+/-1.48 pmol/g ww, P<0.05). Preconditioning also elevated myocardial cGMP levels during sustained ischaemia in the wild-type hearts (26.77+/-2.81 pmol/g ww, P<0.05). We conclude that: 1) basal mechanical function is similar for both wild-type and transgenic mice of both sexes, 2) reperfusion function and infarct size was also similar for both sexes under both control conditions and after preconditioning, 3) the transgenic mice are more tolerant of ischaemia as reflected by their smaller myocardial infarcts, and, 4) the eNOS overexpressed mouse heart cannot be preconditioned regardless of whether mechanical function or infarct size is used as an end-point. These hearts may be maximally protected against ischaemia/reperfusion injury by their elevated endogenous NO levels.     

Cardioprotection by L-glutamate during postischaemic reperfusion: reduced infarct size and enhanced glycogen resynthesis in a rat insulin-free heart model

1. Previously, we found that administration of high-dose L-glutamate during postischaemic reperfusion improves haemodynamic recovery and enhances glycogen resynthesis. In the present study, we investigated whether the same effect occurs in an insulin-free model and whether glutamate administration reduces infarct size. Further, we studied whether the cardioprotective effect of glutamate depends on preserved glutamate transamination and K(ATP) channel activity. 2. In a rat isolated, insulin-free, perfused heart model, we compared the effects of administration of L-glutamate (10 mmol/L) during either 45 min no-flow regional ischaemia plus 120 min reperfusion or reperfusion alone on infarct size and left ventricular (LV) recovery. The effect of glutamate on glycogen metabolism was studied in a model of 30 min global no-flow ischaemia and 60 min reperfusion. In both models, the effects of inhibition of glutamate transamination and K(ATP) channel activity were examined by adding amino-oxyacetate (an aminotransferase inhibitor; 0.1 mmol/L) and glibenclamide (a K(ATP) blocker; 10 mmol/L), respectively. 3. Administration of L-glutamate reduced infarct size by 60% (P < 0.01) and improved postischaemic LV function (developed pressure and rate pressure product; P < 0.05). L-Glutamate increased glycogen content after 60 min reperfusion by 65% (P < 0.01). Amino-oxyacetate, as well as glibenclamide, abolished the glutamate-mediated reduction in infarct size, haemodynamic improvement and glycogen resynthesis during reperfusion. 4. In conclusion, L-glutamate administration from the start of postischaemic reperfusion exerts cardioprotective effects, including reduced infarct size, improved haemodynamic recovery and enhanced glycogen resynthesis. These effects depend on preserved transamination of glutamate and K(ATP) channel activity, but not on insulin administration.     

Selective mitochondrial K(ATP) channel activation results in antiarrhythmic effect during experimental myocardial ischemia/reperfusion in anesthetized rabbits

We investigated the effects of administration of non-hypotensive doses of ATP-sensitive K+ channel (K(ATP)) openers (nicorandil and aprikalim), and a specific mitochondrial K(ATP) channel blocker (5-hydroxydecanoate) prior to and during coronary occlusion as well as prior to and during post-ischemic reperfusion on survival rate, ischemia/reperfusion-induced arrhythmias and myocardial infarct size in anesthetized albino rabbits. Arrhythmias were induced by reperfusion following a 20 min ligation of the left main coronary artery with a releaseable silk ligature. Early intervention by intravenous infusion of nicorandil (100 microg/kg bolus+10 microg/kg/min) or aprikalim (10 microg/kg bolus+0.1 microg/kg/min) just before and during ischemia increased survival rate (86% and 75% vs. 55% in the control group), significantly decreased the incidence and severity of life-threatening arrhythmias and myocardial infarct size. The antiarrhythmic and cardioprotective effects of both nicorandil and aprikalim were abolished by pretreating the rabbits with 5-hydroxydecanoate (5 mg/kg, i.v. bolus). In conclusion, intervention by intravenous administration of nicorandil and aprikalim (through the selective activation of mitochondrial K(ATP) channels) increased survival rate and exhibited antiarrhythmic and cardioprotective effects during coronary occlusion and reperfusion in anesthetized rabbits when administered prior to and during coronary occlusion.     

Reduction of myocardial infarct size by SM-198110, a novel Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange inhibitor,in rabbits

The effects of 3-[2-({[amino(imino)methyl]amino}carbonyl)-4-chloro-1H-indol-1-yl]-1-propanesulphonic acid monohydrate (SM-198110), a novel potent Na⁺/H⁺ exchange inhibitor, and cariporide (Hoe642), another Na⁺/H⁺ exchange inhibitor, were studied in a myocardial ischaemia and reperfusion injury model. Anaesthetized rabbits were subjected to occlusion of the coronary artery for 30 min followed by reperfusion for 5 h. SM-198110 or cariporide was administered before ischaemia and before reperfusion. We also assessed the anti-necrotic effect of SM-198110 when given before reperfusion, both alone and together with glibenclamide, a K_ATP channel blocker, 5-hydroxydecanoate (5-HD), a mitochondrial K_ATP channel-selective blocker and 8-(p-sulphophenyl)-theophylline (8-SPT), an adenosine receptor blocker. The infarct size was reduced dose-dependently by i.v. administration of SM-198110 before ischaemia, with a significant reduction in serum creatine phosphokinase activity. Infarct sizes, normalized to the size of the area-at-risk (means±SE) were: vehicle 56.6±3.7%; low-dose SM-198110 39.2±6.3%; mid-dose 32.8±7.4% (P<0.05); high-dose 22.1±6.7% (P<0.01). This anti-necrotic effect of SM-198110 was achieved without significant haemodynamic changes. Cariporide given before ischaemia also reduced infarct size significantly and dose-dependently. SM-198110 administered before reperfusion also resulted in a dose-dependent reduction in the infarct size. Infarct sizes were: vehicle 56.6±3.7%; low-dose SM-198110 44.5±5.7%; mid-dose 36.3±6.6% (P<0.01); high-dose 34.7±3.8% (P<0.01). In contrast, cariporide given before reperfusion did not reduce infarct sizes significantly. The anti-necrotic effect of SM-198110 was observed even when given 10 min after the beginning of reperfusion. Glibenclamide and 5-HD abolished the anti-necrotic effect of treatment before reperfusion with SM-198110. However, the co-administration of 8-SPT with SM-198110 did not affect infarct size. These results suggest that, in addition to Na⁺/H⁺ exchange inhibition, mitochondrial and/or sarcolemmal K_ATP channels contribute to the anti-necrotic effect of SM-198110 when the latter is given before reperfusion.     

Preconditioning effects of levosimendan in a rabbit cardiac ischemia-reperfusion model

The preconditioning effects of levosimendan were investigated on ischemia-reperfusion induced morphological and functional cardiac damage. Langendorff-perfused rabbit hearts were reserved as controls or subjected either to global myocardial ischemic preconditioning or to perfusion with levosimendan (0.1 micromol/l) for two 5-minute cycles. After a washout period, all hearts were then subjected to 30 minutes of global ischemia and 120 minutes of drug-free reperfusion. Intraventricular pressure and coronary flow were measured, and infarct size determined after nitroblue-tetrazolium staining on completion of the experiments. Levosimendan pretreatment resulted in a significantly smaller elevation from the preischemic level in left ventricular end-diastolic pressure during reperfusion (37 +/- 17 mm Hg) compared with controls (56 +/- 14 mm Hg) and ischemia-preconditioned hearts (53 +/- 34 mm Hg). The left ventricular developed pressure-representing the functional recovery of the heart after ischemia-that was significantly improved by levosimendan pretreatment (38 +/- 6% vs 16 +/- 5% in controls, P < 0.05). In addition, contractility and relaxability parameters (+dP/dt and -dP/dt, respectively) were better preserved in the levosimendan hearts. The volume of infarcted myocardium after global ischemia-reperfusion was significantly (P < 0.05) decreased by both ischemic preconditioning (38 +/- 2%) or levosimendan pretreatment (45 +/- 2%) versus controls (52 +/- 2%). The results of this study suggest that levosimendan pretreatment is capable of decreasing infarct size in an ischemia-reperfusion model and improving recovery of cardiac function following ex vivo global ischemia.     

Sufentanil limits the myocardial infarct size by preservation of the phosphorylated connexin 43

Sufentanil, with a potent analgesia effect, has been wildly used in anesthesia and analgesia, especially for the cardiovascular surgeries. The aim of the study was to evaluate whether sufentanil provides cardioprotection and the effect of connexin 43 on the cardiac infarct size reduction. Sufentanil post-conditioning (bolus injection at 0.1, 0.3, 1, 3, 10 μg/kg) or ischemic post-conditioning (3 cycles of a 10s reperfusion alternating with a 10s ischemia) was induced in an intact rat heart model of ischemia-reperfusion injury. Both ischemic and sufentanil post-conditioning reduced the myocardial infarct size compared with control group. The infarct size limitation of sufentanil was dose-dependent, 1 μg/kg has the optimal effect and increasing dosage could not afford further cardioprotection. Connexin 43 underwent dephosphorylation in response to ischemia-reperfusion measured by Western blot at the anterior myocardium tissues of left ventricle while sufentanil preserved the phosphorylation of connexin 43. The results demonstrated that sufentanil limits myocardial infarct size which is similar with ischemic post-conditioning at the dosage of 1 μg/kg. Preservation of phosphorylation of connexin 43 plays an important role in the cardioprotection of ischemic and sufentanil post-conditioning.     

Cardioprotective effects of bms-180448, a prototype mitokatp channel opener, and the role of salvage kinases, in the rat model of global ischemia and reperfusion heart injury

To investigate the involvement of reperfusion-induced salvage kinases (RISK) as possible signaling molecules for the cardioprotective effects of BMS-180448, a prototype mitochondrial ATP-sensitive K+ (mitoK(ATP)) channel opener, we measured its cardioprotective effects in a rat model of ischemia/reperfusion (I/R) heart injury, together with western blotting analysis of five different signaling proteins. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, BMS-180448 (1, 3 and 10 microM) significantly increased reperfusion left ventricular developed pressure (LVDP) and 30-min reperfusion double product (heart rate x LVDP) in a concentration-dependent manner, while decreasing left ventricular end-diastolic pressure (LVEDP) throughout reperfusion period in a concentration-dependent manner. SDS-PAGE/western blotting analysis of left ventricle reperfused for 30 min revealed that BMS-180448 significantly decreased phospho-GSK3beta at high concentration, whereas it tended to increase slightly phospho-eNOS and phospho-p70S6K with concentration. However, BMS-180448 had no effect on phospho-Akt and phospho-Bad. These results suggest that the cardioprotective effects of BMS-180448 against I/R heart injury may result from direct activation of mitoK(ATP) channel in cardiomyocytes, with the minimal role of RISK pathway in the activation of this channel and the cardioprotective effects of BMS-180448.