Mitochondrial KATP Channel Blocker Research Articles

Effect of esaxerenone on ischemia and reperfusion injury in rat hearts.

In myocardial infarction, the addition of mineralocorticoid receptor blockers (MRBs) to standard therapies, such as angiotensin-converting enzyme inhibitors or beta-blockers, reportedly reduces mortality and cardiac events. We investigated whether the non-steroidal MRB esaxerenone has cardioprotective effects and its protective mechanisms. Isolated rat hearts were Langendorff-perfused (constant pressure, 80 mmHg) with oxygenated Krebs-Henseleit bicarbonate buffer and reperfused for 60 min; afterwards, recovery of function (left ventricular pressure, measured with an intraventricular balloon) and myocardial injury were measured. In a preliminary study, we determined the optimal concentration of esaxerenone required for myocardial protection. Next, esaxerenone was administered in the pre- and post-ischemic phases to determine the optimal timing of administration. In addition, we assessed coronary flow response to acetylcholine (ACh) with and without esaxerenone. We examined whether esaxerenone-induced cardioprotection was prevented by targeting putative components in the preconditioning manner (the mitochondrial ATP-sensitive potassium (KATP) channel). Myocardial protection by esaxerenone was observed when esaxerenone was administered before ischemia but not after ischemia. The coronary flow response to ACh was significantly better in the esaxerenone group than in the control group. The cardioprotective effect of esaxerenone was eliminated by the mitochondrial KATP channel blocker, 5-hydroxy decanoate. This study confirmed the myocardial protective effect of the pre-ischemic administration of esaxerenone. Esaxerenone may contribute to coronary endothelial protection and exert pharmacological preconditioning via the mitochondrial KATP channel.

Effect of the selective mitochondrial KATP channel opener nicorandil on the QT prolongation and myocardial damage induced by amitriptyline in rats.

The aim of this study is to evaluate the protective effect of nicorandil, a selective mitochondrial KATP channel opener, on QT prolongation and myocardial damage induced by amitriptyline. The dose of amitriptyline (intraperitoneal, i.p.) that prolong the QT interval was found 75 mg/kg. Rats were randomized into five groups the control group, amitriptyline group, nicorandil (selective mitochondrial KATP channel opener, 3 mg/kg i.p.) + amitriptyline group, 5-hdyroxydecanoate (5-HD, selective mitochondrial KATP channel blocker, 10 mg/kg i.p.) + amitriptyline group and 5-HD + nicorandil + amitriptyline group. Cardiac parameters, biochemical and histomorphological/immunohistochemical examinations were evaluated. p < 0.05 was accepted as statistically significant. Amitriptyline caused statistically significant prolongation of QRS duration, QT interval and QTc interval (p < 0.05). It also caused changes in tissue oxidant (increase in malondialdehyde)/anti-oxidant (decrease in glutathione peroxidase) parameters (p < 0.05), myocardial damage and apoptosis (p < 0.01 and p < 0.001). While nicorandil administration prevented amitriptyline-induced QRS, QT, QTc prolongation (p < 0.05), myocardial damage and apoptosis (p < 0.05), it did not affect the changes in oxidative parameters (p > 0.05). Our results suggest that nicorandil, a selective mitochondrial KATP channel opener, plays a protective role in amitriptyline-induced QT prolongation and myocardial damage. Mitochondrial KATP channel opening and anti-apoptotic effects may play a role in the cardioprotective effect of nicorandil.

Optimizing Nicorandil for Spinal Cord Protection in a Murine Model of Complex Aortic Intervention

There are currently no clinically utilized pharmacological agents for the induction of metabolic tolerance to spinal cord ischemia-reperfusion injury in the setting of complex aortic intervention. Nicorandil, a nitric oxide donor and ATP-sensitive potassium (KATP) channel opener, has shown promise in neuroprotection. However, the optimized clinical application of the drug and its mechanism of neuroprotection remains unclear. We hypothesized that 3-days pretreatment would confer the most effective neuroprotection, mediated by mitochondrial KATP channel activation. Spinal cord injury was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Time course: mice received 0.1 mg/kg nicorandil for 10 min, 4 hours, and 3 consecutive days prior to ischemia compared with control. Dose challenge: mice received 3-days nicorandil pretreatment comparing 0.1 mg/kg, 1.0 mg/kg, 5.0 mg/kg, and saline administration. Mitochondrial KATP channel blocker 5-hydroxy-decanoate (5HD) was co-administered to elucidate mechanism. Limb motor function was evaluated, and viable anterior horn neurons quantified. Nicorandil pretreatment at 4 hours and 3 days before ischemia demonstrated significant motor function preservation; administration 10 minutes before ischemia showed no neuroprotection. All nicorandil doses showed significant motor function preservation. Three days administration of Nicorandil 1.0 mg/kg was most potent. Neuroprotection was completely abolished by 5HD co-administration. Histological analysis showed significant neuron preservation with nicorandil pretreatment, which was attenuated by 5HD co-administration. Three days administration of Nicorandil 1.0 mg/kg showed near-total motor function preservation in a murine spinal cord ischemia-reperfusion model, mediated by the mitochondrial KATP channel.

Abstract 13107: Nitroglycerin Pretreatment Induces Metabolic Tolerance to Spinal Cord Ischemia-reperfusion Injury Through Nitric Oxide-mediated Mitochondrial Katp Channel Activation

Introduction: Pharmacologic induction of metabolic tolerance to spinal cord ischemia-reperfusion injury (SCI) after thoracoabdominal aortic intervention is not well established. We previously demonstrated that nicorandil pretreatment preserved motor function in a murine SCI model via direct mitochondrial ATP-sensitive potassium (KATP) channel activation and nitric oxide (NO) donation. However, the independent role of NO-mediated neuroprotection has not been elucidated. Hypothesis: Nitroglycerin pretreatment will induce neuroprotection through NO-mediated KATP channel activation. Methods: SCI was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Pretreatment constituted intraperitoneal injection 3 consecutive days before injury. Experimental groups: sham (no pretreatment or ischemia, n=10), SCI control (normal saline, n=20), nitroglycerin 1 mg/kg (n=18), nitroglycerin 1 mg/kg + 5-hydroxydecanonate 5 mg/kg (5HD, mitochondrial KATP channel blocker, n=13), 5HD 5 mg/kg (n=10), nitroglycerin 1 mg/kg + carboxy-PTIO (cPTIO) 1 mg/kg (NO scavenger, n=16), and cPTIO 1 mg/kg (n=10). Limb motor function and the number of viable neurons within the anterior horn of the spinal cord were evaluated. Results: Compared to SCI control, motor function was significantly preserved in the nitroglycerin pretreatment group at every time point after ischemia. In the nitroglycerin+5HD and nitroglycerin+cPTIO groups, motor preservation was significantly attenuated compared to nitroglycerin pretreatment (p&lt;0.001). Histological analysis showed significant neuron preservation in the nitroglycerin pretreatment group compared with SCI control (p=0.011). This preservation was completely attenuated with 5HD or cPTIO co-administration (p=0.001). Conclusions: Nitroglycerin pretreatment significantly preserved motor function in a murine SCI model through NO-mediated KATP channel activation.

ATP-sensitive K+ channels maintain resting membrane potential in interstitial cells of Cajal from the mouse colon

To investigate the role of ATP-sensitive K+(KATP) channels on pacemaker activity in interstitial cells of Cajal (ICC), whole-cell patch clamping, RT-PCR, and intracellular Ca2+([Ca2+]i) imaging were performed in cultured colonic ICC. Pinacidil (a K+ channel opener) hyperpolarized the membrane and inhibited the generation of pacemaker potential, and this effect was reversed by glibenclamide (a KATP channel blocker). RT-PCR showed that Kir 6.1 and SUR2B were expressed in Ano-1 positive colonic ICC. Glibenclamide depolarized the membrane and increased pacemaker potential frequency. However, 5-hydroxydecanoic acid (a mitochondrial KATP channel blocker) had no effects on pacemaker potentials. Phorbol 12-myristate 13-acetate (PMA; a protein kinase C activator) blocked the pinacidil-induced effects, and PMA alone depolarized the membrane and increased pacemaker potential frequency. Cell-permeable 8-bromo-cyclic AMP also increased pacemaker potential frequency. Recordings of spontaneous intracellular Ca2+([Ca2+]i) oscillations showed that glibenclamide increased the frequency of [Ca2+]i oscillations. In small intestinal ICC, glibenclamide alone did not alter the generation of pacemaker potentials, and Kir 6.2 and SUR2B were expressed in Ano-1 positive ICC. Therefore, KATP channels in colonic ICC are activated in resting state and play an important role in maintaining resting membrane potential.

Effects of ATP-sensitive potassium channel blockers on vascular hyporeactivity, mesenteric blood flow, and survival in lipopolysaccharide-induced septic shock model.

In this study, the possible therapeutic effects of various ATP-sensitive potassium channel (KATP) blockers (glibenclamide, repaglinide, 5-HD, HMR-1098) have been tested in experimental septic shock model. Rats were given lipopolysaccharide (1 mg·kg(-1)) to create experimental shock model and 4 h later, under 400 mg·kg(-1) chloral hydrate anesthesia, parameters such as blood pressure, mesenteric blood flow, the response of mesenteric circulation to phenylephrine (vasoconstrictor stimulation), and organ and oxidative damage were analyzed. Also 75 mg·kg(-1) lethal dose of lipopolysaccharide was given to mice and effects of KATP blockers on survival have been tested. Non-selective blocker glibenclamide with sulphonylurea structure and sarcolemmal KATP channel blocker HMR-1098, which have the similar chemical structure, have improved the pathological parameters such as decrease in mesenteric blood flow, vascular hyporeactivity, but could not prevent the decrease in blood pressure, and oxidative and organ damage that were observed in the shock model. Also, both blockers have decreased the mortality rate from 80% to 40%-50%. Similar (preventive) therapeutic effects were not observed with non-selective blocker repaglinide and mitochondrial KATP channel blocker 5-HD, which were non-sulphonylurea structure. As a result, only KATP channel blockers that have sulphonylurea structure can be a new therapeutic approach in septic shock.

ATP-sensitive K⁺ channels contribute to the protective effects of exogenous hydrogen sulfide against high glucose-induced injury in H9c2 cardiac cells.

Hyperglycemia, as well as diabetes mellitus, has been shown to impair ATP-sensitive K+ (KATP) channels in human vascular smooth muscle cells. Hydrogen sulfide (H2S) is also known to be an opener of KATP channels. We previously demonstrated the cardioprotective effects exerted by H2S against high-glucose (HG, 35 mM glucose)-induced injury in H9c2 cardiac cells. As such, we hypothesized that KATP channels play a role in the cardioprotective effects of H2S against HG-induced injury. In this study, to examine this hypothesis, H9c2 cardiac cells were treated with HG for 24 h to establish a model of HG-induced insults. Our findings revealed that treatment of the cells with HG markedly decreased the expression level of KATP channels. However, the decreased expression of KATP channels was reversed by the treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) for 30 min prior to exposure to HG. Additionally, the HG-induced cardiomyocyte injuries, including cytotoxicity, apoptosis, oxidative stress and mitochondrial damage, were ameliorated by treatment with NaHS or 100 µM diazoxide (a mitochondrial KATP channel opener) or 50 µM pinacidil (a non-selective KATP channel opener) for 30 min prior to exposure to HG, as indicated by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). Notably, treatment of the H9c2 cardiac cells with 100 µM 5-hydroxydecanoic acid (5-HD, a mitochondrial KATP channel blocker) or 1 mM glibenclamide (Gli, a non-selective KATP channel blocker) for 30 min prior to treatment with NaHS and exposure to HG significantly attenuated the above-mentioned cardioprotective effects exerted by NaHS. Notably, treatment of the cells with 500 µM N-acetyl‑L‑cysteine (NAC, a scavenger of ROS) for 60 min prior to exposure to HG markedly reduced the HG-induced inhibitory effect on the expression of KATP channels. Taken together, our results suggest that KATP channels play an important role in the cardioprotective effects of exogenous H2S against HG-induced injury. This study also provides novel data demonstraring that there is an antagonistic interaction between ROS and KATP channels in HG-exposed H9c2 cardiac cells.

Retraction Note to: Postconditioning promotes the cardiac repair through balancing collagen degradation and synthesis after myocardial infarction in rats.

Postconditioning (Postcon) reduces infarct size. However, its role in modulation of cardiac repair after infarction is uncertain. This study tested the hypothesis that Postcon inhibits adverse cardiac repair by reducing degradation of extracellular matrix (ECM) and synthesis of collagens via modulating matrix metalloproteinase (MMP) activity and transforming growth factor (TGF) β1/Smad signaling pathway. Sprague–Dawley rats were subjected to 45 min ischemia followed by 3 h, 7 or 42 days of reperfusion, respectively. In acute studies, four cycles of 10/10 s Postcon significantly reduced infarct size, which was blocked by administration of a mitochondrial KATP channel blocker, 5-hydroxydecanoate (5-HD) at reperfusion. In chronic studies, Postcon inhibited MMP activity and preserved ECM from degradation as evidenced by reduced extent of collagen-rich scar and increased mass of viable myocardium. Along with a reduction in collagen synthesis and fibrosis, Postcon significantly down-regulated expression of TGFβ1 and phospho-Smad2/3, and up-regulated Smad7 as compared to the control, consistent with a reduction in the population of α-smooth muscle actin expressing myofibroblasts within the infarcted myocardium. At 42 days of reperfusion, echocardiography showed significant improvements in left ventricular end-diastolic volume and ejection fraction. The wall thickness of the infarcted middle anterior septum in the Postcon was also significantly greater than that in the control. The beneficial effects of Postcon on cardiac repair were comparable to preconditioning and still evident after a blockade with 5-HD. These data suggest that Postcon is effective to promote cardiac repair and preserve cardiac function; protection is potentially mediated by inhibiting ECM degradation and collagen synthesis.

MP43-19 EFFECT OF PHARMACOLOGICAL POST CONDITIONING ON ISCHEMIA-REPERFUSION INJURY IN THE RAT TESTIS

INTRODUCTION AND OBJECTIVES: The effects of administered KATP channel openers or blockers during ischemia are still controversial in many organs/tissues. To examine whether the administration of KATP channel openers or blockers before or during ischemia ameliorates IR injury in the testis. METHODS: Eight-week-old male SD rats were subjected to 2 h right testicular ischemia followed by 24 h reperfusion. The selective mitochondrial (mito) KATP channel blocker, 5-hydroxydecanoate (5-HD) (40 mg/kg), non-selective KATP channel blocker glibenclamide (Glc) (5 mg/kg) or selective mito KATP channel opener diazoxide (DZ) (10 mg/kg), non-selective KATP channel opener cromakalim (Crom) (300 mg/kg) were administered intraperitoneally 15 min prior to the ischemia or 45 min before the induction of reperfusion. Tissue damage was evaluated by malondialdehyde concentration, myeloperoxidase activity, histological evaluation and TUNEL assay in the testis. RESULTS: There was a significant reduction in oxidative stress, neutrophil infiltration, histological score and apoptosis in the IR model in the testis when DZ and Crom were administrated before ischemia. However, this reduction was not seen with the administration of DZ or Crom during ischemia. In addition, there was a significant reduction in the testicular IR injury with the administration of Glc, but not 5-HD during ischemia. CONCLUSIONS: The pre-conditioning effect on IR injury in the testis was confirmed by treatment with DZ. The administration of non-selective KATP channel blocker Glc during ischemia ameliorated against the testicular IR injury. On the other hand, the selective mito KATP channel blocker, 5-HD and KATP channel openers during ischemia did not reduce the testicular IR injury. These data suggest that opening of KATP channel before ischemia, and blocking of the sarcolemmal KATP channel during the testicular ischemia may have a protective effect. Glc could be an effective drug to manage the post-ischemic injury after testicular torsiondetorsion injury. Source of Funding: A grant in aid from the Ministry of Education, Science, and Culture of Japan (#23118)

Blocking of the ATP sensitive potassium channel ameliorates the ischaemia‐reperfusion injury in the rat testis

There is increasing evidence that the effects of administered ATP sensitive potassium (KATP ) channel openers or blockers during ischaemia are still controversial in many organs/tissues. Testicular torsion detorsion which causes ischaemia-reperfusion (IR) injury, cannot be predicted, thus an effective drug should be administered during or after the ischaemia. The aim of this study was to examine whether the administration of KATP channel openers or blockers during ischaemia ameliorates IR injury in the testis. Eight-week-old male Sprague-Dawley rats were subjected to 2 h right testicular ischaemia followed by 24 h reperfusion. The selective mitochondrial (mito) KATP channel blocker, 5-hydroxydecanoate (5-HD) (40 mg/kg), the non-selective KATP channel blocker glibenclamide (5 mg/kg), the selective mito KATP channel opener diazoxide (10 mg/kg) and the non-selective KATP channel opener cromakalim (300 μg/kg) were administered intraperitoneally 15 min prior to the ischaemia or 75 min after the induction of ischaemia. Tissue damage was evaluated by malondialdehyde concentration, myeloperoxidase activity, histological evaluation and TdT-mediated dUTP nick end labelling assay in the testis. There was a significant increase in oxidative stress, neutrophil infiltration, histological damage and apoptosis in the testicular IR model. A significant reduction in the testicular IR injury was observed with the administration of glibenclamide, but not 5-HD, diazoxide or cromakalim during ischaemia. The administration of non-selective KATP channel blocker glibenclamide ameliorated the testicular IR injury. On the other hand, the selective mito KATP channel blocker, 5-HD and KATP channel openers did not reduce the testicular IR injury. These data suggest that blocking of the membrane KATP channel may have a protective effect during the testicular ischaemia. Glibenclamide could be an effective drug to manage the post-ischaemic injury caused by the testicular torsion-detorsion.

Roles of endothelial nitric oxide synthase (eNOS) and mitochondrial permeability transition pore (MPTP) in epoxyeicosatrienoic acid (EET)-induced cardioprotection against infarction in intact rat hearts

We previously demonstrated that 11,12 and 14,15-epoxeicosatrienoic acids (EETs) produce cardioprotection against ischemia-reperfusion injury in dogs and rats. Several signaling mechanisms have been implicated in the cardioprotective actions of the EETs; however, their mechanisms remain largely elusive. Since nitric oxide (NO) plays a significant role in cardioprotection and EETs have been demonstrated to induce NO production in various tissues, we hypothesized that NO is involved in mediating the EET actions in cardioprotection. To test this hypothesis, we used an in vivo rat model of infarction in which intact rat hearts were subjected to 30-min occlusion of the left coronary artery and 2-hr reperfusion. 11,12-EET or 14,15-EET (2.5mg/kg) administered 10min prior to the occlusion reduced infarct size, expressed as a percentage of the AAR (IS/AAR), from 63.9±0.8% (control) to 45.3±1.2% and 45.5±1.7%, respectively. A nonselective nitric oxide synthase (NOS) inhibitor, L-NAME (1.0mg/kg) or a selective endothelial NOS inhibitor, L-NIO (0.30mg/kg) alone did not affect IS/AAR but they completely abolished the cardioprotective effects of the EETs. On the other hand, a selective neuronal NOS inhibitor, nNOS I (0.03mg/kg) and a selective inducible NOS inhibitor, 1400W (0.10mg/kg) did not affect IS/AAR or block the cardioprotective effects of the EETs. Administration of 11,12-EET (2.5mg/kg) to the rats also transiently increased the plasma NO concentration. 14,15-EET (10μM) induced the phosphorylation of eNOS (Ser1177) as well as a transient increase of NO production in rat cardiomyoblast cell line (H9c2 cells). When 11,12-EET or 14,15-EET was administered at 5min prior to reperfusion, infarct size was also reduced to 42.8±2.2% and 42.6±1.9%, respectively. Interestingly, L-NAME (1.0mg/kg) and a mitochondrial KATP channel blocker, 5-HD (10mg/kg) did not abolish while a sarcolemmal KATP channel blocker, HMR 1098 (6.0mg/kg) and a mitochondrial permeability transition pore (MPTP) opener, atractyloside (5.0mg/kg) completely abolished the cardioprotection produced by the EETs. 14,15-EET (1.5mg/kg) with an inhibitor of MPTP opening, cyclosporin A (CsA, 1.0mg/kg) produced a greater reduction of infarct size than their individual administration. Conversely, an EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 2.5mg/kg) completely abolished the cardioprotective effects of CsA, suggesting a role of MPTP in mediating the EET actions. Taken together, these results suggest that the cardioprotective effects of the EETs in an acute ischemia-reperfusion model are mediated by distinct mediators depending on the time of EET administration. The cardioprotective effects of EETs administered prior to ischemia were regulated by the activation of eNOS and increased NO production, while sarcKATP channels and MPTP were involved in the beneficial effects of the EETs when administered just prior to reperfusion.

Baicalein Preconditioning Protects Cardiomyocytes from Ischemia-Reperfusion Injury via Mitochondrial Oxidant Signaling

Previous studies suggest baicalein, in addition to its antioxidant effects, protects against hypoxia/reoxygenation injury via its pro-oxidant properties. We hypothesize that a brief period of baicalein treatment prior to ischemia/reperfusion (I/R) may trigger preconditioning protection via a mitochondrial pro-oxidant mechanism. Using an established chick cardiomyocyte model of I/R, cells were preconditioned with baicalein (10 μM) for 10 min followed by 10-min wash prior to I/R. Intracellular oxidants were measured using 2', 7'-dichlorofluorescin diacetate (DCFH/DA). Cell viability was assessed by propidium iodide and apoptosis determined by DNA fragmentation. Baicalein induced a transient but significant increase of DCF fluorescence within the 10-min preconditioning period, and led to significant reduction of cell death (38.9 ± 1.8% vs. 58.7 ± 1.2% in I/R control, n = 6, p < 0.001) and DNA fragmentation after I/R. Cotreatment with N-acetylcysteine (500 μM), mitochondrial complex III electron transport chain inhibitor myxothiazol (1 μM), mitochondrial KATP channel blocker 5-hydroxydecanoate-Na (5-HD, 500 μM) or anion channel inhibitor 4', 4'-diisothiocyanato-stilbene-2, 2'-disulfonic acid (DIDS, 200 μM) resulted in significant abrogation of oxidant increase during induction as well as the protection conferred by baicalein preconditioning. These results suggest that baicalein preconditioning exhibits significant anti-apoptotic protection against cardiomyocyte I/R injury by mitochondrial oxidant signaling, which was in part mediated by mitochondrial KATP channel and anion channel opening.

Effects of Ischemic and Sevoflurane-induced Preconditioning on Myocardial Infarction and Arrhythmias in Rabbits In Vivo

Objectives: The present study aimed to investigate whether ischemic or sevoflurane-induced preconditioning exerts infarct size limiting effects and depresses ischemia-reperfusion arrhythmias through opening of mitochondrial KATP channels in rabbits in vivo. Methods: Rabbits anesthetized with ketamine and xylazine given intramuscularly underwent 30 min of left anterior descending coronary artery (LAD) occlusion followed by 3 hrs of reperfusion. Before this, rabbits were randomized into one of five groups. Control rabbits received no intervention before 30 min LAD occlusion and 3 h reperfusion (Group-C). The ischemia-preconditioned (IP) rabbits underwent 5 min LAD occlusion followed by 10 min of reperfusion before prolonged ischemia-reperfusion (Group-IP). In the sevoflurane (S)–preconditioned group, 30 min of sevoflurane exposure at a 1.5% end-tidal concentration was followed by 15 min of washout before prolonged ischemia-reperfusion (Group-S). The selective mitochondrial KATP channel blocker, 5-hydroxy-decanoate (5-HD, 5 mg/kg) was given intravenously 10 min before ischemic preconditioning and sevoflurane exposure, respectively (Group-5-HD-IP, Group-5-HD-S). An electrocardiogram was recorded throughout the experiment via lead 2 of the standard electrocardiogram. At the end of the 3 hrs reperfusion period, area at risk (R) and infarct size (I) were measured. Results: RPP decreased in Group-5-HD-IP and Group-5-HD-S compared with Group-S at 30 min after ischemia. The ratio of R to left ventricular mass showed no significant difference among all groups. I/R values of each group were 51.6 ± 3.0% in Group-C, 33.3 ± 4.7% in Group-IP, 36.6 ± 4.8% in Group-S, 48.9 ± 5.2% in Group-5-HD-IP, 54.8 ± 4.2% in Group-Group-5-HD-S. There was no significant difference in duration of arrhythmias during myocardial ischemia and reperfusion among 5 groups. Conclusion: Ischemic preconditioning and sevoflurane-induced preconditioning exert infarct size limiting effects through opening of mitochondrial KATP channels. However, ischemic preconditioning and sevoflurane-induced preconditioning do not have antiarrhythmic effects. This suggests that the opening of mitochondrial KATP channels does not cause antiarrhythmic effects.

Postconditioning with glucagon like peptide-2 reduces ischemia/reperfusion injury in isolated rat hearts: role of survival kinases and mitochondrial KATP channels

We recently reported that heart expresses functional receptors for the anorexigenic glucagon-like peptide (GLP)-2. Activation of these cardiac receptors affected basal heart performance through extracellular regulated kinase (ERK1/2) activation. Since ERK1/2 is considered one of the prosurvival kinases of postconditioning cardioprotective pathways, we hypothesized that GLP-2 directly protects the heart against ischemia/reperfusion (I/R) injury via prosurvival kinases. Wistar rat hearts were retrogradely perfused on a Langendorff perfusion apparatus. After 40-min stabilization, hearts underwent 30-min global ischemia and 120-min reperfusion (I/R group). In GLP-2 group, the hearts received 20-min GLP-2 (10(-7)M) infusion at the beginning of the 120-min reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated by nitroblue-tetrazolium staining. Compared with the I/R group, GLP-2-treated hearts showed a significant reduction of infarct size and of postischemic diastolic LVP (index of contracture), together with a sharp improvement of developed LVP recovery (index of contractility). The protective effects were abolished by co-infusion with phosphatidylinositol 3-kinase inhibitor, Wortmannin (WT), the ERK1/2 inhibitor, PD98059, or the mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate. GLP-2 effects were accompanied by increased phosphorylation of protein kinase B (PKB/Akt), ERK1/2 and glycogen synthase kinase (GSK3β). After 7-min reperfusion, WT blocked Akt and GSK3β phosphorylation. After 30-min reperfusion, WT inhibited phosphorylation of all kinases. In conclusion, the data suggest that GLP-2, given in early reperfusion, as postconditioning, protects against myocardial I/R injury, limiting infarct size, and improving post-ischemic mechanical recovery. It seems that the GLP-2-protection of rat heart involves multiple prosurvival kinases and mitochondrial K(ATP) channels.

Protective effects of diazoxide against Aβ25–35-induced PC12 cell apoptosis due to prevention of endoplasmic reticulum stress

Accumulated amyloid-β (Aβ) is a well-known cause of neuronal apoptosis in Alzheimer's disease and exerts its action partly by inducing mitochondrial dysfunction. Previous studies have suggested a neuroprotective role for mitochondrial ATP-sensitive potassium (KATP) channel openers against Aβ damages, but the molecular details were unclear. Recent evidence indicates that endoplasmic reticulum (ER) stress also plays an important role in the process of cell apoptosis. It remains to be determined whether KATP channel openers mediate their potential neuroprotective role by inhibiting ER stress pathways. The mRNA and protein expression levels of caspase-12, an ER-specific caspase, were observed. Here we showed that in response to the treatment with Aβ₂₅₋₃₅ (10 μM) for 24 h the mRNA and protein expression levels of caspase-12 were significantly upregulated; however, this change could be partly reversed by pretreatment with diazoxide (1 mM) for 1 h. This effect was negated by 5-hydroxydecanoate, a selective mitochondrial KATP channel blocker. Our results indicate that the cytoprotective efficacy of diazoxide under Aβ₂₅₋₃₅-induced insults is mediated, at least in part, by inhibition of ER stress. Demonstration of the neuroprotective action of diazoxide provides additional insights into the pathogenic mechanisms of Aβ₂₅₋₃₅ toxicity and defines possible molecular targets for therapeutic intervention.

A possible subcellular mechanism underlying the “French paradox”: the opening of mitochondrial KATP channels

A reduction in the risk of coronary heart disease has been associated to moderate red wine consumption. We tested whether a nonalcoholic red wine extract would open mitochondrial K(ATP) channels in guinea pig myocytes. The opening of mitochondrial K(ATP) channels was assessed by endogenous flavoprotein fluorescence. Red wine extract (100 μg·mL(-1)) increased flavoprotein oxidation (10.9% ± 1.2%, n = 20). This effect was prevented by the mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (500 µmol·L(-1); 0.3% ± 1.1%, n = 13), confirming the hypothesis that red wine extract opens mitochondrial K(ATP) channels.

Isoflurane preconditioning increases endothelial cell tolerance to in-vitro simulated ischaemia

Isoflurane preconditioning has been shown to protect endothelial cells against lipopolysaccharide and cytokine induced injury. This study was designed to determine whether isoflurane preconditioning increased endothelial cell tolerance to ischaemia. Bovine pulmonary arterial endothelial cells were exposed or not exposed to various concentrations of isoflurane for 1 h. After a 30-min isoflurane-free period, cells were subjected to oxygen-glucose deprivation (OGD) for 3 h and reoxygenation for 1 h. Lactate dehydrogenase release from cells was used to measure cell injury. In some experiments, various protein kinase C (PKC) inhibitors and ATP-sensitive potassium channel (K(ATP) channel) inhibitors were present from 30 min before isoflurane treatment to the end of isoflurane treatment. Isoflurane preconditioning dose-dependently decreased the OGD induced lactate dehydrogenase release. This protection was inhibited by 2 µM chelerythrine, a general PKC inhibitor, or 10 µM Gö6976, an inhibitor for the conventional PKCs. This protection was also inhibited by 0.3 µM glybenclamide, a general K(ATP) channel inhibitor, and 500 µM 5-hydroxydecanoate, a mitochondrial K(ATP) channel blocker. In addition, pretreatment with 100 µM diazoxide, a K(ATP) channel activator, for 1 h also reduced OGD induced endothelial cell injury. This diazoxide induced protection was inhibited by chelerythrine. The results suggest that isoflurane preconditioning induces endothelial protection against in-vitro simulated ischemia. This protection may be mediated at least in part by conventional PKCs and mitochondrial K(ATP) channels. The results also indicate that PKCs may be downstream of K(ATP) channels in causing endothelial protection.

The Cardiac α1c Subunit is Down Regulated by Pharmacological Preconditioning

Background and Purpose: It is well established that Pharmacological Preconditioning (PP), achieved with openers of mitochondrial KATP channels like diazoxide, leads to cardioprotection against subsequent ischemia. However, the changes in Ca2+ homeostasis during PP are poorly understood. Here, we investigate the effects of PP on the L-type Ca2+ channel of the adult heart.Experimental approach: Preincubation with diazoxide (100μM) for 90 min was used to induce PP in two preparations: Isolated hearts from rat Wistar and enzymatically dissociated rat ventricular myocytes. Cardiomyocytes were voltage-clamped to measure L-type Ca2+ currents (ICa) with the whole-cell patch-clamp technique and Ca2+ signals and ROS production were measured with the fluorescent probes, Fluo 3-AM and CM-H2DCFDA, respectively. The levels of the α1c subunit, obtained from diazoxide preconditioned hearts, were measured in the membrane fraction of rat ventricles by Western blot. The ROS scavenger NAC was used to examine the role of ROS on the L-type Ca2+ channel after PP in both preparations.Results: Diazoxide induced PP was accompanied by a significant downregulation of the α1c subunit in the membrane fraction and by a reversible reduction in the amplitude of ICa and Ca2+ transients. These effects were complete within 90min and were prevented by NAC. Diazoxide significantly increased ROS production in cardiomyocytes. The reduction of ICa and Ca2+ transients by PP were prevented by the mitochondrial KATP channel blocker 5-HD.Conclusions: Pharmacological preconditioning induced with diazoxide, leads to downregulation of the α1c subunit of the L-type Ca2+ channel. This reduces the influx of Ca2+ through these channels and may contribute to attenuate the overload of Ca2+ during reperfusion.Grants: CONACYT: 60880 (J.S) and 82667 (M.C.G).

Postconditioning with levosimendan reduces the infarct size involving the PI3K pathway and KATP-channel activation but is independent of PDE-III inhibition

Reperfusion injury is strongly involved in the loss of functional heart tissue in patients after acute myocardial infarction. Various signal transduction pathways to reduce infarct size during reperfusion have been characterized. However, so far in the clinical setting no standard therapies are applied due to the lack of suitable drugs. Levosimendan, a calcium sensitizer, has been shown to improve survival in cardiogenic shock after infarction. Focus of the present study was to address the question, whether a bolus application of levosimendan prior to reperfusion is able to reduce the infarct size. A well-characterized model, the in vivo rat model, was used and levosimendan applied 5 min prior to reperfusion after 30-min occlusion of the left coronary artery followed by a 30-min reperfusion period. This pharmacological postconditioning was compared to the ischemic postconditioning with three times occlusion/reperfusion periods of 30 s each. To further address the question if in this in vivo model the phosphatidylinositol 3-kinase (PI3K) pathway may be involved, the PDE-III inhibiting property of levosimendan was compared to the PDE-III inhibitor enoximone. Ischemic postconditioning significantly reduced the infarct size from 48 +/- 2 to 32 +/- 1% of the area at risk (P < 0.05). Similarly, levosimendan decreased infarct size down to 29 +/- 3%. The combination of ischemic postconditioning and pharmacological postconditioning using levosimendan did not result in a further reduction of the infarct size. Both, the mitochondrial KATP-channel blocker 5-hydroxydecanoate (5-HD) and the PI3K inhibitor wortmannin abolished the protection afforded by levosimendan completely, while the inhibitors alone did not influence the infarct size in control hearts. Pharmacological postconditioning with enoximone did not result in any infarct size reduction. Postconditioning with levosimendan significantly increased the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3beta (GSK-3beta) at 5 min of reperfusion, an effect which could be blocked completely by the additional administration of wortmannin. In conclusion, levosimendan applied prior to reperfusion in acute myocardial infarction significantly reduces the infarct size in an in vivo rat model. This protection involves the PI3K pathway and the activation of mitochondrial KATP-channels, but is independent of PDE-III inhibition. This finding may open new possibilities for the treatment of patients with acute myocardial infarction using levosimendan, which is an already established therapy in cardiogenic shock. Whether the reduction of mortality in cardiogenic shock by levosimendan may in part be based on this postconditioning effect remains to be elucidated in clinical setting.

Effects of activation of μ-, κ1-, δ1-, δ2-, and ORL1-receptors on heart resistance to the pathogenic action of delayed ischemia and reperfusion

Different subtypes of opioid receptors (OR) were activated in rats in vivo to study the activation effect on the heart’s resistance to ischemia and reperfusion. It has been established that administration of deltorphin II, a selective δ2-OR agonist, lowered the infarct size/area at risk index (IS/AAR) by 23%. Naltrexone, naloxone methiodide (an OR inhibitor not penetrating the blood-brain barrier (BBB)), and naltriben (δ2-antagonist) eliminated the cardioprotective effect of deltorphin II, while BNTX (a δ1-antagonist) produced no effect on the cardioprotective action of the δ2-agonist. The infarct-reducing effect of deltorphin II was eliminated by administration of chelerythrine (a protein kinase C (PKC) inhibitor), glibenclamide (a KATP-channels inhibitor), and 5-hydroxydecanoate (a mitochondrial KATP-channel blocker). Administration of other opioids did not reduce the IS/AAR index. It has been established that all the deltorphins manifest antiarrhythmic potency. Other opioids do not produce any effect on the incidence of arrhythmia occurrences. The antiarrhythmic effect of deltorphin II was eliminated by preliminary administration of naltrexone, naloxone methiodide, and naltriben, but BNTX did not affect the δ2-agonist’s anti-arrhythmic effect. The preliminary administration of chelerythrine, a PKC inhibitor, eliminated the δ2 agonist’s antiarrhythmic action. However, glibenclamide and 5-hydroxydecanoate did not alter the antiarrhythmic effect by deltorphin II. Therefore, activation of the peripheral δ2-ORs reduces the infarct size and prevents the onset of arrhythmias. The antiarrhythmic effect of the δ2-OR stimulation is mediated by activating PKC and opening the mitochondrial KATP-channels. PKC participates in the antiarrhythmic effect of the δ2-OR activation, but this effect does not depend on the condition of KATP-channels.