mitoKATP Channel Research Articles

Oxytocin protects rat heart against ischemia–reperfusion injury via pathway involving mitochondrial ATP-dependent potassium channel

Cardiac preconditioning represents the most potent and consistently reproducible method of rescuing heart tissue from undergoing irreversible ischemic damage. One of the major goals of the current cardiovascular research is to identify a reliable cardioprotective intervention that can salvage ischemic myocardium. The aim of the present study is to evaluate the oxytocin (OT)-induced cardioprotection and the signaling pathway involved with mitochondrial ATP-dependent potassium (mitoKATP) channel in the anesthetized rat heart. Animals were divided into six groups ( n = 6): (1) IR; hearts were subjected to 25 min ischemia and 120 min reperfusion, (2) OT; oxytocin was administered (0.03 μg/kg i.p.) 25 min prior to ischemia, (3) ATO+OT; atosiban (ATO) was used as an OT-selective receptor antagonist (1.5 μg/kg i.p.) 10 min prior to OT administration, (4) ATO; atosiban was used 35 min prior to ischemia, (5) 5HD + OT; 5-hydroxydecanoic acid (5HD) was used as a specific inhibitor of mitoKATP channel (10 mg/kg i.v.) 10 min prior to OT administration, (6) 5HD; 5HD was used 35 min prior to ischemia. Then infarct size, ventricular arrhythmia and creatine kinase-MB isoenzyme (CK-MB) plasma level were measured. Hemodynamic parameters were recorded throughout the experiment. OT administration significantly decreased infarct size, CK-MB plasma level, severity and incidence of ventricular arrhythmia as compared to IR group. Administration of atosiban and 5HD abolished the cardiopreconditioning effect of OT. This study demonstrates that cardioprotective effects of OT are mediated through opening the mitoKATP channels.

Role of cyclooxygenase-2 and mitochondrial KATP channels in sufentanil preconditioning-induced delayed cardioprotection against myocardial ischemia-reperfusion injury in rats

Objective To investigate the role of cyclooxygenase-2(COX-2)and mitochondrial adenosine tuiphosphate sensitive potassium channels (mito-KATP channels) in sufentanil preconditioning-induced delayed cardiopreteetion against myocardial ischemia-reperfnsion (I/R) injury in rats. Methods Seventy-two adult male Wistsr rats weighing 250-300 g were randomly divided into 6 groups ( n =12 each). Group Ⅰ,Ⅱ,Ⅲ were preconditioned with intraperitoneal (IP) normal saline (NS) 1 ml/kg while group Ⅳ,Ⅴ,Ⅵ with IP sufentanil 20 μg/kg at 24 h before myocardial ischemia. Group Ⅱ and Ⅴ were given IP NS-398 ( COX-2 inhibitor) 5 mg/kg at 30 rain before myocardial ischemla while group Ⅲ and Ⅵ were given intravenous 5-HD (mito-KATP channelblocker) 10 mg/kg at 10 min before ischemia or before being killed. Six animals in each group underwent 45 min myocardial ischemia followed by 120 min reperfusion, while the other six animals in each group were killed immediately before ischemia for determination of myocardial COX-2 expression and myocardial PGF2 and PGF1α content. Myocardial ischemia was induced by occlusion of left anterior descending branch (LAD) of coronary artety for 45 rain followed by 120 min reperfusion. MAP and HR were recorded immediately before ischemia (T0), at 15, 30, 45 rain of ischemia (T1-3) and at 30, 60, 90, 120 vain of reperfusion (T4-7). Heart rate-blood pressure product (RPP) was calculated. Arterial blood samples were obtained at T0.3 and T7 for measurement of plasma CK-MB activity. The animals were killed at the end of 120 nan reperfusion. The hearts were removed for determination of myocardial infarct area (IA) and area at risk (AAR). LA/AAR was calculated. Results There was no significant difference in HR, MAP and RPP at all time points among the 6 groups. Preconditioning with sufentanil significantly decreased plasma CK-MB activity at T3 and T7 and IA/AAR in group Ⅳ as compared with group Ⅰ.Myocardial COX-2 expression was up-regulated and PGE2 and PGF1α, contents were elevated by sufentanil preconditioning in group Ⅳ as eomared with control group (Ⅰ). In group Ⅴ and Ⅳ preconditioning with NS-398/5-HD significantly increased plasma CK-MB concentration and IS/AAB as compared with group Ⅳ, indicating involvement of COX-2 and mito-KATP channels in the sufentanil-induced delayed cardioprotection.The myocardial PGE2 and PGF1α contents were significantly reduced in group Ⅴ as compared with group Ⅳ. There was no significant difference in the myocardial COX-2 expression among group Ⅳ, Ⅴ and Ⅵ. Conclusion Both COX-2 and mito-KATP channels are involved in sufentanil preconditioning-induced delayed cardiopmtection. Key words: Prostaglandin-endoperoxide synthases; KATP channels; Sufentanil; Ischemic preconditioning, myocardial; Myocardial reperfuaion injury

Connexin 43 acts as a cytoprotective mediator of signal transduction by stimulating mitochondrial KATP channels in mouse cardiomyocytes

Potassium (K+) channels in the inner mitochondrial membrane influence cell function and survival. Increasing evidence indicates that multiple signaling pathways and pharmacological actions converge on mitochondrial ATP-sensitive K+ (mitoKATP) channels and PKC to confer cytoprotection against necrotic and apoptotic cell injury. However, the molecular structure of mitoKATP channels remains unresolved, and the mitochondrial phosphoprotein(s) that mediate cytoprotection by PKC remain to be determined. As mice deficient in the main sarcolemmal gap junction protein connexin 43 (Cx43) lack this cytoprotection, we set out to investigate a possible link among mitochondrial Cx43, mitoKATP channel function, and PKC activation. By patch-clamping the inner membrane of subsarcolemmal murine cardiac mitochondria, we found that genetic Cx43 deficiency, pharmacological connexin inhibition by carbenoxolone, and Cx43 blockade by the mimetic peptide 43GAP27 each substantially reduced diazoxide-mediated stimulation of mitoKATP channels. Suppression of mitochondrial Cx43 inhibited mitoKATP channel activation by PKC. MitoKATP channels of interfibrillar mitochondria, which do not contain any detectable Cx43, were insensitive to both PKC activation and diazoxide, further demonstrating the role of Cx43 in mitoKATP channel stimulation and the compartmentation of mitochondria in cell signaling. Our results define a role for mitochondrial Cx43 in protecting cardiac cells from death and provide a link between cytoprotective stimuli and mitoKATP channel opening, making Cx43 an attractive therapeutic target for protection against cell injury.

Pre‐ and postconditioning with the mitochondrial ATP‐sensitive potassium (mitoKATP) channel opener BMS‐191095 protects cerebral endothelial cells against oxygen glucose deprivation (OGD)

We have shown that the selective mitoKATP channel opener BMS‐191095 protects cultured rat cortical neurons against lethal OGD via acute and delayed preconditioning. We have tested if BMS‐191095 also protects rat brain endothelial cells (RBEC) against lethal OGD, in vitro. Primary RBECs were pretreated with BMS‐191095 (10, 20, 30μM) for 1 day or 1 hour (h) before and 1h immediately after 2h of OGD. OGD reduced viability from 100% to 53±1% but 1 day pretreatment and 1h post‐treatment dose dependently increased viability to 76±2% and 74±1%, respectively (n=16/group; p<0.05). 1h pretreatment was ineffective. Protection could be abolished using the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin (47±1% and 54±1%; n=16‐16), but not with the selective mitoKATP channel blocker 5‐hydroxydecanoate (66±1% and 71±1%; n=16‐16). Western blot analysis showed a rapid phosphorylation of Akt and ERK 1/2 which peaked at 15 minutes after BMS‐191095 application. Treatment for 24h did not change manganese superoxide dismutase and endothelial nitric oxide synthase levels. In summary, BMS‐191095 prevents OGD‐induced death of RBECs likely by mechanisms involving the PI3K‐Akt and ERK 1/2 pathways.

The phosphatidylinositol-3 kinase/Akt pathway mediates geranylgeranylacetone-induced neuroprotection against cerebral infarction in rats

Previous studies demonstrated the cytoprotective effect of geranylgeranylacetone (GGA), a heat shock protein inducer, against ischemic insult. Phosphatidylinositol-3 kinase/Akt (PI3K/Akt) is thought to be an important factor that mediates neuroprotection. However, the signaling pathways in the brain in vivo after oral GGA administration remain unclear. We measured and compared infarction volumes to investigate the effect of GGA on cerebral infarction induced by permanent middle cerebral artery occlusion in rats. We evaluated the effects of pretreatment with 5-hydroxydecanoate (5HD), a specific mitochondrial ATP-sensitive potassium (mitoK ATP) channel inhibitor; diazoxide (DZX), a selective mitoK ATP channel opener and wortmannin (Wort), a specific PI3K inhibitor of GGA-induced neuroprotection against infarction volumes. To clarify the relationship between PI3K/Akt activation and neuroprotection, we used immunoblot analysis to determine the amount of p-Akt proteins present after GGA administration with or without Wort treatment. Neuroprotective effects of GGA (pretreatment with a single oral GGA dose (800 mg/kg) 48 h before ischemia) were prevented by 5HD, DZX and Wort pretreatment, which indicates that the selective mitoK ATP channel and the PI3K/Akt pathway may mediate GGA-dependent protection. Oral GGA-induced p-Akt and GGA pretreatment enhanced ischemia-induced p-Akt, both of which were prevented by Wort pretreatment. These results suggest that a single oral dose of GGA induces p-Akt and that GGA plays an important role in neuroprotection against cerebral ischemia through the mitoK ATP channel opening.

Involvement of mitochondrial ATP-sensitive potassium channels in etomidate preconditioning-induced protection in human myeloid HL-60 cells

Exposure of HL-60 cells, a human myeloid cell line, to 500 μM etomidate for 24 h reduced cell viability and increased nitric oxide production and mitochondrial permeability transition pore (mPTP) opening. Preconditioning (1 h) with 1 μM etomidate 4 h before exposure to the 500 μM dose of etomidate attenuated those detrimental effects. The mitochondrial ATP-sensitive potassium channel (mitoK ATP channel) inhibitor 5-hydroxydecanoic acid reduced the etomidate preconditioning effects. The mitoK ATP channel opener diazoxide attenuated the mPTP opening caused by the large dose of etomidate. Our results suggest that etomidate can induce a preconditioning effect that may involve mitoK ATP channel activation.

Sevoflurane pre- and post-conditioning protect the brain via the mitochondrial KATP channel

This study aimed to evaluate whether exposure to sevoflurane at the onset of reperfusion provides protection similar to sevoflurane preconditioning and whether the effect depends on mitochondrial potassium ATP-dependent channel (mitoK(ATP)) in a rat model of focal cerebral ischaemia. Adult Wistar male rats were subjected to focal cerebral ischaemia for 1 h followed by 24 h or 7 days of reperfusion. Preconditioning consisted of 15 min exposure to sevoflurane at 1 minimum alveolar concentration (2.6%) 72 h before ischaemia. Post-conditioning was performed by exposure to sevoflurane immediately at the onset of reperfusion or by a delayed exposure 5 min after the onset of reperfusion. The role of the mitoK(ATP) channel was assessed by i.p. injection of the selective blocker 5-hydroxydecanoate before each sevoflurane administration or by the mitoK(ATP) channel opener, diazoxide (DZX), given in place of sevoflurane. Cerebral infarct size, neurological deficit score, and motor coordination were evaluated 24 h and 7 days after reperfusion. Sevoflurane preconditioning and early post-conditioning reduced both cerebral infarct size and neurological defect score at 24 h of reperfusion whereas the sole sevoflurane post-conditioning improved motor coordination. At 7 days, only infarct volume remained lower in pre- and post-conditioned animals. Neuroprotection mediated by sevoflurane was lost when it was given 5 min after the onset of reperfusion and was abolished by inhibition of mitoK(ATP). DZX alone mimicked sevoflurane-induced pre- and post-conditioning. The pretreatment with sevoflurane or its early administration at reperfusion provides neuroprotection via mitoK(ATP) in a rat model of focal cerebral ischaemia.

Mitochondrial ATP-sensitive K+channels, protectors of the heart

ATP-sensitive K+ (KATP) channels were first identified in the sarcolemma of cardiac myocytes as inwardly rectifying K+ channels that were inhibited by intracellular ATP (Noma, 1983). It was proposed that KATP channels would have a cardioprotective effect during ischaemia by shortening action potential duration and thereby decreasing Ca2+ influx into myocytes. Then it was found that K+ channel opener compounds (KCOs), that are known to activate sarcolemmal KATP channels, exert cardioprotective effects under ischaemia–reperfusion (Grover et al. 1989; Grover & Garlid, 2000). However, several groups of investigators found that KCOs exerted cardioprotection at concentrations below those causing action potential shortening (Yao & Gross, 1994; Grover et al. 1995a,b; Garlid et al. 1997), indicating that KCOs may have targets other than sarcolemmal KATP channels. Alternative targets for KCOs include mitochondrial KATP (mitoKATP) channels (Grover & Garlid, 2000).

Fatigue in Mouse Muscle Fibers: Role of Mitochondrial ATP-Sensitive Potassium Channels

Aim. The role of mitochondrial KATP (mitoKATP) channels on muscle fatigue was assessed in adult mouse skeletal muscle fibers.Methods. Muscle fatigue was produced by eliciting short repetitive tetani. Isometric tension and the rate of production of reactive oxygen species (ROS) were measured at room temperature (20-22°C) using a force transducer and the fluorescent indicator CM-H2DCFDA.Results. We found that opening mitoKATP channels with diazoxide (100 ∈μM) significantly reduced muscle fatigue. Fatigue tension was 34% higher in diazoxide-treated fibers relative to controls. This effect was blocked by the mitoKATP channel blocker 5-Hydroxydecanoate (5-HD), by the protein kinase C (PKC) inhibitor chelerythrine, and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester hydrochloride (L-NAME). We found that the rate of ROS production in muscle fibers incubated with diazoxide under non-fatigue and fatigue conditions was similar to control experiments, but that the increase in the rate of ROS production during recovery from fatigue was greatly reduced.Conclusions. A physiological role of mitoKATP channels on muscle fatigue is proposed.Diazoxide, an opener of mitoKATP channels, reduces muscle fatigue possibly through a preservation of mitochondrial volume and function.This work was supported in part by CONACYT grants 82667 (JS) and 60880 (MCG).

Immunolocalization of mitoKATP subunits in human osteoblast-like cells

The mitochondrial ATP-dependent K channel (mitoKATP) has been shown to play a role in cellular protection against apoptosis, or programmed cell death. This channel has been identified and characterized in a number of cell and tissue types but to date the possible existence of mitoKATP in osteoblastic cells has not been investigated. The aim of this investigation was to establish whether the mitochondria of human osteosarcoma-derived osteoblasts (SaOS-2 cells) contain the putative mitoKATP subunits Kir6.1 and Kir6.2. Ultrathin sections of SaOS-2 cells were prepared for transmission electron microscopy using an adaptation of the Tokuyasu method, and immunolabelled using goat anti-Kir6.1 or anti-Kir6.2 antisera as the primary label, and a 10nm colloidal gold-conjugated donkey anti-goat secondary antibody. The suitability of the antisera and the immunostaining protocol were confirmed by using a sample of rat cardiac muscle as a positive control. Ultrastructural analysis revealed that SaOS-2 cells contain Kir6.2 but not Kir6.1, and that Kir6.2 is present in the mitochondria, but in extremely low abundance. These findings suggest that human osteoblast-like cells might contain mitoKATP channels in which Kir6.2 is the pore-forming subunit, although it appears that these channels are likely to be present in extremely low abundance.

5-Hydroxydecanoate and coenzyme A are inhibitors of native sarcolemmal KATP channels in inside-out patches

Background 5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K + (mitoK ATP) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5‑HD + CoA + ATP → 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K ATP (sarcK ATP) channel activity. Methods Single sarcK ATP channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK ATP channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A. Results 5-HD inhibited (IC 50 ∼ 30 μM) K ATP channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 μM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 μM coenzyme A (CoA) inhibited sarcK ATP channels. Using giant inside-out patches, which are weakly sensitive to “contaminating” CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK ATP channel activation by either metabolic inhibition or rilmakalim. General significance SarcK ATP channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at “directly” inhibiting sarcK ATP and mitoK ATP channels. However, in intact cells, 5-HD fails to inhibit sarcK ATP channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.

Opening of the mitoKATP channel and decoupling of mitochondrial complex II and III contribute to the suppression of myocardial reperfusion hyperoxygenation

Diazoxide, a mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener, protects the heart from ischemia-reperfusion injury. Diazoxide also inhibits mitochondrial complex II-dependent respiration in addition to its preconditioning effect. However, there are no prior studies of the role of diazoxide on post-ischemic myocardial oxygenation. In the current study, we determined the effect of diazoxide on the suppression of post-ischemic myocardial tissue hyperoxygenation in vivo, superoxide (O(2)(-*)) generation in isolated mitochondria, and impairment of the interaction between complex II and complex III in purified mitochondrial proteins. It was observed that diazoxide totally suppressed the post-ischemic myocardial hyperoxygenation. With succinate but not glutamate/malate as the substrate, diazoxide significantly increased ubisemiquinone-dependent O(2)(-*) generation, which was not blocked by 5-HD and glibenclamide. Using a model system, the super complex of succinate-cytochrome c reductase (SCR) hosting complex II and complex III, we also observed that diazoxide impaired complex II and its interaction with complex III with no effect on complex III. UV-visible spectral analysis revealed that diazoxide decreased succinate-mediated ferricytochrome b reduction in SCR. In conclusion, our results demonstrated that diazoxide suppressed the in vivo post-ischemic myocardial hyperoxygenation through opening the mitoK(ATP) channel and ubisemiquinone-dependent O(2)(-*) generation via inhibiting mitochondrial complex II-dependent respiration.

Diazoxide supplemented Celsior solution improves hypothermic heart preservation effect in rat through activation of mitochondrial ATP-sensitive potassium channel

Successful heart preservation is critical for clinical heart transplantation, but even the most successful heart preservation method can only preserve the heart in a time range of approximately 4 h. In the present study, diazoxide (DE)-containing cardioplegia solution was used as the hypothermic heart preservation solution, and hearts were preserved for different time periods before reperfusion. Five groups were randomly divided into the control group, DE groups with different concentrations of DE (15, 30, and 45 μM), and the 5-HD + DE group containing 30 μM DE and 100 μM of 5-hydroxydecanoate (5-HD). Each group was further divided according to the storage time: 3 h and 8 h subgroups. Hemodynamic parameters were monitored continually and myocardial injury markers lactate dehydrogenase (LDH) and creatine kinase (CK) were measured before arrest and during reperfusion. After reperfusion, myocardium water content, myocardium apoptosis, and myocardial mitochondrial superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured. All data were analyzed by independent samples t-test. Our results showed that diazoxide treatment improved impaired hemodynamics parameters and decreased the leakage of myocardial enzymes during reperfusion, compared to the control group. Moreover, diazoxide treatment significantly alleviated myocardial edema, decreased the number of TUNEL-positive cardiomyocytes, prevented the loss of mitochondrial SOD activity, and depressed the mitochondrial MDA content. This study indicated that mitoKATP channel opener diazoxide could significantly enhance myocardial protection during long-term hypothermic preservation, and the inhibition of oxidative stress-induced myocardium apoptosis seems to be the mechanism involved in the protective action on the mitoKATP channel.

Molecular Identification and Functional Characterization of a Mitochondrial Sulfonylurea Receptor 2 Splice Variant Generated by Intraexonic Splicing

Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel but its composition is not fully understood. We hypothesized that the mitoK(ATP) channel contains a sulfonylurea receptor (SUR)2 regulatory subunit and aimed to identify the molecular structure. Western blot analysis in cardiac mitochondria detected a 55-kDa mitochondrial SUR2 (mitoSUR2) short form, 2 additional short forms (28 and 68 kDa), and a 130-kDa long form. RACE (Rapid Amplification of cDNA Ends) identified a 1.5-Kb transcript, which was generated by a nonconventional intraexonic splicing (IES) event within the 4th and 29th exons of the SUR2 mRNA. The translated product matched the predicted size of the 55-kDa short form. In a knockout mouse (SUR2KO), in which the SUR2 gene was disrupted, the 130-kDa mitoSUR2 was absent, but the short forms remained expressed. Diazoxide failed to induce increased fluorescence of flavoprotein oxidation in SUR2KO cells, indicating that the diazoxide-sensitive mitoK(ATP) channel activity was associated with 130-kDa-based channels. However, SUR2KO mice displayed similar infarct sizes to preconditioned wild type, suggesting a protective role for the remaining short form-based channels. Heterologous coexpression of the SUR2 IES variant and Kir6.2 in a K(+) transport mutant Escherichia coli strain permitted improved cell growth under acidic pH conditions. The SUR2 IES variant was localized to mitochondria, and removal of a predicted mitochondrial targeting sequence allowed surface expression and detection of an ATP-sensitive current when coexpressed with Kir6.2. We identify a novel SUR2 IES variant in cardiac mitochondria and provide evidence that the variant-based channel can form an ATP-sensitive conductance and may contribute to cardioprotection.

Variable effects of the mitoKATP channel modulators diazoxide and 5-HD in ATP-depleted renal epithelial cells

The role of mitochondrial K(ATP) (mitoK(ATP)) channels in renal ischemia-reperfusion injury is controversial with studies showing both protective and deleterious effects. In this study, we compared the effects of the putative mitoK(ATP) opener, diazoxide, and the mitoK(ATP) blocker, 5-hydroxydecanoate (5-HD) on cytotoxicity and apoptosis in tubular epithelial cells derived from rat (NRK-52E) and pig (LLC-PK1) following in vitro ischemic injury. Following ATP depletion-recovery, there was a significant increase in cytotoxicity in both NRK cells and LLC-PK1 cells although NRK cells were more sensitive to the injury. Diazoxide treatment attenuated cytotoxicity in both cell types and 5-HD treatment-increased cytotoxicity in the sensitive NRK cells in a superoxide-dependant manner. The protective effect of diazoxide was also reversed in the presence of 5-HD in ATP-depleted NRK cells. The ATP depletion-mediated increase in superoxide was enhanced by both diazoxide and 5-HD with the effect being more pronounced in the cells undergoing 5-HD treatment. Further, ATP depletion-induced activation of caspase-3 was decreased by diazoxide in NRK cells. In order to determine the signaling pathways involved in apoptosis, we examined the activation of Erk and JNK in ATP-depleted NRK cells. Diazoxide-activated Erk in ATP-depleted cells, but did not have any effect on JNK activation. In contrast, 5-HD did not impact Erk levels but increased JNK activation even under controlled conditions. Further, the use of a JNK inhibitor with 5-HD reversed the deleterious effects of 5-HD. This study demonstrates that in cells that are sensitive to ATP depletion-recovery, mitoK(ATP) channels protect against ATP depletion-mediated cytotoxicity and apoptosis through Erk- and JNK-dependant mechanisms.

Anti-ischemic properties of a new spiro-cyclic benzopyran activator of the cardiac mito-KATP channel

Many activators of KATP channels exhibit cardioprotective effects, mainly mediated by channels expressed on mitochondria (mito-KATP). Previous results showed anti-ischemic effects of the spiro-cyclic derivative A, on isolated rat hearts. In this work this molecule was more extensively studied and diazoxide was used as reference mito-KATP opener. The studies were performed on an in vivo rat model of myocardial infarct and on heart-derived H9c2 cells exposed to an anoxic environment. The mechanism of action was further investigated on isolated rat heart mitochondria. In the model of myocardial infarct compound A and diazoxide produced significant cardioprotective effects, antagonised by the selective mito-KATP blocker 5-hydroxydecanoic acid (5-HD). Compound A, like diazoxide, produced modest and non-significant hypotensive responses, while the hyperglycaemic effects of diazoxide were not observed for the new compound. Protective effects of compound A and diazoxide were also recorded in H9c2 cells and again were inhibited by 5-HD. Compound A and diazoxide caused swelling of cardiac mitochondria, in agreement with the profile of mito-KATP openers. Both compounds evoked concentration-dependent Ca2+-release from Ca2+-preloaded mitochondria, prevented mitochondrial Ca2+-uptake and caused mitochondrial membrane depolarisation. These effects were antagonised by ATP, the endogenous KATP inhibitor. In conclusion, compound A exhibits a promising profile of an anti-ischemic agent, with a mechanism likely to be linked to the activation of mito-KATP channels, and, because of its chemical characteristics such as structural rigidity and chirality due to the spiro-cyclic moiety, represents an interesting template for development of analogues further improved in activity and selectivity.

Mitochondrial ATP-sensitive potassium-channel: A novel target of neuroprotection in cerebral hypoperfusion

Chronic cerebral hypoperfusion, a mild ischemic condition is associated with advancing age and severity of dementia; however, no undisputed therapy has been established to alleviate related neurological symptoms. Considerable interest has recently been generated from studies, in which the activation of mitochondrial (mito)KATP channels has been shown to protect against hypoxic/ischemic stress in the brain. Diazoxide has been identified as a mitoKATP channel opener, and a potentially neuroprotective compound.

Predictive models, based on classification algorithms, for compounds potentially active as mitochondrial ATP-sensitive potassium channel openers

Heart mitochondrial ATP-sensitive potassium channel (mito-K ATP channels) are deeply implicated in the self-defense mechanism of ischemic preconditioning. Therefore, exogenous molecules activating these channels are considered as a promising pharmacological tool to reduce the myocardial injury deriving from ischemia/reperfusion events. In our laboratory, a series of 4-spiro-substituted benzopyran derivatives were earlier synthesized and some of them exhibited anti-ischemic properties. In this study, the above compounds are exploited in order to develop QSAR models, based on classification approaches, capable of discriminating between the ones acting as cardioprotective agents and those that are unable to elicit such a property. Molecules belonging to the whole dataset were subjected to CODESSA and E-Dragon calculations in order to compute a large number of molecular descriptors enabling the construction of classification models. Based on the two program packages used, two different experiments were carried out, with the aim of identify batteries of models to be exploited for designing new cardioprotective agents from libraries of new chemical entities. Both model batteries satisfy the rigorous criteria adopted for the validation, either when tested on the training and test set, according to the most straightforward protocol, and when tested on an additional prediction set. They were proven to ensure successful applications in the field of cardioprotective agent design.

Role of mitochondrial ATP-sensitive potassium channels on fatigue in mouse muscle fibers

The role of mitochondrial K ATP (mitoK ATP) channels on muscle fatigue was assessed in adult mouse skeletal muscle bundles. Muscle fatigue was produced by eliciting short repetitive tetani. Isometric tension and the rate of production of reactive oxygen species (ROS) were measured at room temperature (20–22 °C) using a force transducer and the fluorescent indicator CM-H 2DCFDA. We found that opening mitoK ATP channels with diazoxide (100 μM) significantly reduced muscle fatigue. Fatigue tension was 34% higher in diazoxide-treated fibers relative to controls. This effect was blocked by the mitoK ATP channel blocker 5-hydroxydecanoate (5-HD), by the protein kinase C (PKC) inhibitor chelerythrine, and by the nitric oxide (NO) synthase inhibitor N G-nitro- l-arginine methyl ester hydrochloride ( l-NAME) but was not accompanied by a change in the rate of ROS production during fatigue. A physiological role of mitoK ATP channels on muscle fatigue is proposed.

Hyperthermia-Induced Cardioprotection Is Potentiated by Ischemic Postconditioning in Rats

We tested the hypothesis that the protective effects of hyperthermia (HT) could be augmented by ischemic postconditioning (PostC) via enhancement of reperfusion-induced Akt phosphorylation. The role of the mitoKATP channel as an effecter to protect hearts against ischemia/reperfusion injury was also investigated. In isolated perfused heart experiments using a Langendorff apparatus, 30 min of no-flow global ischemia was followed by 120 min of reperfusion. Ischemic PostC, 5 cycles of 10-sec reperfusion/10-sec ischemia, was achieved at the initial moment of reperfusion. Hyperthermia (HT, 43 degrees C for 20 min) was applied 24 hr before ischemia onset. Ischemic PostC alone did not show significant protection, but HT did. The HT-induced protection in terms of infarct size, recovery of left ventricular performance, amount of released creatine kinase and apoptosis were enhanced by ischemic PostC. These protective effects were consistent with the levels of Akt phosphorylation 7 min after reperfusion and were completely blocked by the pretreatment with the phosphatidylinositol 3-kinase inhibitor wortmannin. HT-induced protection was also completely abolished by concomitant perfusion with 5-hydroxydecanoate (5HD, 100 microM), an inhibitor of the mitochondrial ATP-sensitive potassium (mitoKATP) channel. However, the potentiated protection by ischemic PostC remained, even in the presence of 5HD. In conclusion, ischemic PostC could potentiate the protective effects of HT possibly via enhancement of reperfusion-induced Akt phosphorylation. Although the opening of the mitoKATP channel is predominantly involved as an effecter in HT-induced protection, potentiated protection by ischemic PostC may involve mechanisms other than the mitoKATP channel.