Role Of mitoKATP Channels Research Articles

Role of mito-KATP channel in pinacidil postconditioning-induced reduction of myocardial ischemia-reperfusion injury in rats: an in vitro experiment

Objective To evaluate the role of mitochondrial KATP (mito-KATP) channel in pinacidil postconditioning-induced reduction of myocardial ischemia-reperfusion (I/R) injury in rats. Methods SPF healthy male Sprague-Dawley rats, aged 16-20 weeks, weighing 250-300 g, were anesthetized with pentobarbital.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 36.5-37.5 ℃.Thirty-two Langendorff-perfused hearts were divided into 4 groups (n=8 each) using a random number table: control group (group C), group I/R, pinacidil postconditioning group (group P) and 5-hydroxy decanoic acid plus pinacidil postconditioning group (group 5-HD+ P). Myocardial ischemia was induced by interrupting perfusion for 40 min followed by 60 min reperfusion.Immediately after onset of reperfusion, hearts were perfused with K-H solution containing 50 μmol/L pinacidil for 2 min and then with K-H solution for 58 min in group P, hearts were perfused with K-H solution containing 100 μmol/L 5-HD for 5 min, with K-H solution containing 50 μmol/L pinacidil for 2 min and then with K-H solution for 53 min in group 5-HD+ P.The heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and the maximum rate of increase in left ventricular pressure (+ dp/dtmax) were recorded at 20 min of equilibration (T1) and at the end of reperfusion(T2). Myocardial tissues were obtained at T2 for determination of myocardial infarct size and for examination of myocardial ultrastructure and Flameng scoring of the mitochondria was performed. Results Compared with group C, the HR, LVDP and + dp/dtmax were significantly decreased, and the LVEDP, myocardial infarct size and mitochondrial Flameng score were increased at T2 in group I/R (P 0.05). Compared with group P, the HR, LVDP and + dp/dtmax were significantly decreased and the LVEDP, myocardial infarct size and mitochondrial Flameng score were increased at T2 (P<0.05), and the pathological changes of myocardium were accentuated in group 5-HD+ P. Conclusion The whole mechanism by which pinacidil postconditioning reduces myocardial I/R injury is related to promoting opening of mito-KATP channel in rats. Key words: Myocardial reperfusion injury; Pinacidil; Mitochondria; KATP channels

EFFECTS OF NICORANDIL ON ELECTRON TRANSPORT CHAIN ACTIVITY IN SKELETAL MUSCLE MITOCHONDRIA

In skeletal muscle, pharmacological modulation of the mitochondrial ATP‐sensitive K+ channel (mitoKATP) represents an strategy in myoprotection against metabolic stress. The role of mitoKATP channel in different tissues has been proposed by its participation in several signaling routes that promotes the production of ATP, affecting the transfer of energy between mitochondria and cellular ATPases. However, it has been reported that the action of activators of mitoKATP channels besides acting on the channels, could also have effects on different structures at mitochondrial level. Nicorandil has been recognized as a selective mitoKATP channel opener, making it a useful tool for determining the importance of this mitochondrial site. It is considered an effective drug for the treatment against ischemia‐reperfusion injury and fatigue in skeletal muscle, however its effects on mitochondrial respiration remains unclear. Hence, the aim of this study was to explore the effects of nicorandil on the activity of the electron transport chain (ETC) in isolated mitochondria derived from chicken skeletal muscle.Mitochondria from pectoralis muscle of 2‐week‐old Arbor Acres chickens were isolated by differential centrifugation. The rate of oxygen consumption was determined at room temperature using a Clark‐type oxygen electrode coupled to an oxygen monitor YSI 5300 and a computer for data acquisition. The determinations started after adding 10 mM glutamate/malate as respiratory substrate for complex I (state 4) and 1 mM ADP was added to determine oxygen consumption in the phosphorylating state (state 3). The activities of the ETC complexes were (I–IV) were assayed spectrophotometrically in a Perkin Elmer Lambda 18UV/vis spectrophotometer using 0.5 mg/mL mitochondria resuspended in 50 mM KH2PO4buffer. NADH‐oxidoreductase (complex I) activity was evaluated by measuring the oxidation of NADH at an absorbance of 340 nm; Succinate‐DCIP oxidoreductase (complex II) activity was measured at 600 nm by following the reduction of 2,6‐dichlorophenolindophenol (DCIP). Antimycin A‐sensitive succinate‐cytochrome oxidoreductase (complex III) activity was followed by measuring at 550 nm the reduction of cytochrome. Cytochrome oxidase (complex IV) activity was evaluated by measuring the oxidation of reduced cytochrome at 550 nm.Nicorandil (10 μM) significantly decreased respiration (31.59 ± 6.15 %.) compared with control condition; this effect on oxygen consumption in the respiratory state 3 is associated with changes in the enzymatic activity of complexes II, III and IV. These findings suggest and question the assumption that sensitivity to nicorandil implies involvement of mitoKATP channels. Therefore, we suggest that effects pharmacological by nicorandil may be related to its effects on respiratory chain complexes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of mito-KATP channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose deprivation and restoration-induced pyroptosis in primary rat cardiomyocytes

Objective To evaluate the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose deprivation and restoration (OGD/R)-induced pyroptosis in primary rat cardiomyocytes. Methods Cardiomyocytes of newborn Sprague-Dawley rats (<48 h after birth) were cultured in vitro and seeded in 6-well dishes (2 cm in diameter) or in 96-well plates.The cells were divided into 6 groups (n=15 each) using a random number table: control group (group C), OGD/R group (group O), sevoflurane postconditioning group (group Sev), sevoflurane postconditioning plus 5-hydroxydecanoate (5-HD) group (group SH), 5-HD group (group H) and OGD/R plus 5-HD group (group HO). The cardiomyocytes were subjected to oxygen-glucose deprivation for 4 h followed by restoration of oxygen-glucose supply for 24 h. After oxygen-glucose restoration, the cardiomyocytes in the culture media were exposed to 2% sevoflurane for 1 h to perform sevoflurane postconditioning.At 1 h before oxygen-glucose deprivation, a specific mito-KATP channel blocker 5-HD 100 μmol/L was added to the culture media.Cardiomyocytes were cultured in normal culture atmosphere in group C. Cardiomyocytes were collected at 24 h of oxygen-glucose restoration.Cell pyroptosis was detected by double flow cytometry AlexaFour488 (caspase-1 FLICA staining) and TMR red (DNA staining) staining.The pyroptosis rate was calculated.The cell survival rate was measured by methyl thiazolyl tetrazolium assay.The content of reactive oxygen species (ROS) in mitochondria was determined by 2′, 7′-dichlorofluorescin diacetate assay.The mitochondrial membrane potential (MMP) was measured by using JC-1 fluorescent probe.The expression of interleukin-1beta (IL-1β) was determined by Western blot. Results Compared with group C, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group O (P<0.05). Compared with group O, the pyroptosis rate and ROS content were significantly decreased, the cell survival rate and MMP were increased, and the expression of IL-1β was down-regulated in group Sev (P<0.05). Compared with group Sev, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group SH (P<0.05). Compared with group SH, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group HO (P<0.05). Conclusion The mechanism by which sevoflurane postconditioning inhibits OGD/R-induced pyroptosis in primary rat cardiomyocytes is probably associated with increasing mito-KATP channel opening. Key words: Anesthetics, inhalation; Anoxia; Myocytes, cardiac; Cell death; Postconditioning

Effect of sevoflurane postconditioning on mitochondrial connexin 43 during myocardial ischemia-reperfusion in rats and the role of mito-KATP channels: an in vitro experiment

Objective To investigate the effect of sevoflurane postconditioning on mitochondrial connexin 43 (Cx43) during myocardial ischemia-reperfusion (I/R) in isolated rat hearts and the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in it. Methods Forty-five adult male Sprague-Dawley rats, weighing 200-250 g, were used in the study.Their hearts were excised and retrogradely perfused with K-H solution in a Langendorff apparatus.The 45 isolated hearts were assigned into 5 groups (n=9 each) using a random number table: control group (group C), I/R group, sevoflurane postconditioning group (group Sev), and sevoflurane postconditioning plus 5-hydroxydecanoate (5-HD, a specific mito-KATP channel blocker) group (group Sev+ 5-HD) and I/R plus 5-HD group (group I/R+ 5-HD). The hearts were subjected to 20 min of global ischemia followed by 90 min of reperfusion to establish the model of myocardial I/R injury.From the beginning of reperfusion, the hearts were perfused with K-H solution saturated with 3% sevoflurane for 15 min in group Sev, with K-H solution saturated with 3% sevoflurane and containing 100 μmol/L 5-HD in group Sev+ 5-HD, and with K-H solution containing 100 μmol/L 5-HD in group 5-HD.The heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP) and the maximum rate of increase and decrease of ventricular pressure (±dp/dtmax) were recorded at the end of equilibration (T1) and 30 and 60 min of reperfusion (T2, 3). At 90 min of reperfusion, the myocardial infarct size was measured by TTC staining, and the expression of total Cx43 (tCx43) and phosphorylated Cx43 (p-Cx43) in mitochondria was determined by Western blot analysis.The percentage of myocardial infarct size and p-Cx43/tCx43 ratio were calculated. Results Compared with group C, the HR, LVDP and ±dp/dtmax were significantly decreased, and the LVEDP was increased at T2, 3, and the percentage of myocardial infarct size was increased in the other 4 groups, the expression of mitochondrial tCx43 and p-Cx43 was significantly down-regulated in I/R, Sev+ 5-HD and 5-HD groups (P 0.05). Compared with group I/R, the HR, LVDP and ±dp/dtmax were significantly increased, and the LVEDP was decreased at T2, 3, the percentage of myocardial infarct size was decreased, and the expression of mitochondrial tCx43 and p-Cx43 was up-regulated in group Sev (P 0.05). Compared with group Sev, the HR, LVDP and ±dp/dtmax were significantly decreased, and the LVEDP was increased at T2, 3, the percentage of myocardial infarct size was increased, and the expression of mitochondrial tCx43 and p-Cx43 was down-regulated in group Sev+ 5-HD (P 0.05). Conclusion The mechanism by which sevoflurane postconditioning attenuates myocardial I/R injury may be related to induction of mito-KATP channel opening and up-regulation of the expression of mitochondrial Cx43 in cardiomyocytes of rats. Key words: Anesthetics, inhalation; Ischemic postconditioning; Myocardial reperfusion injury; Mitochondria; Connexin 43; KATP channels

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.

MitoKATP-Channel Opener Protects against Neuronal Death in Rat Venous Ischemia

Mitochondrial adenosine triphosphate-dependent potassium (mitoKATP) channels are present in the brain, and several reports have shown their neuroprotective, preconditioning effect against an ischemic insult. The role of mitoKATP channels in the penumbra area has not been studied thoroughly. In a model of venous ischemia, widespread penumbra-like low flow areas are created, which are susceptible to cortical spreading depression. Thus, we studied effects of mitoKATP channels on infarct size in this model. Male Wistar rats were subjected to two-vein occlusion by photochemical thrombosis of two adjacent cortical veins combined with KCl-induced cortical spreading depression. The rats were assigned to four experimental groups pretreated intraventricularly 15 minutes before two-vein occlusion with 1) vehicle, 2) the mitoKATP channel opener diazoxide (2 mmol/L), 3) diazoxide (2 mmol/L) plus the selective mitoKATP channel blocker 5-hydroxydecanoate (5-HD; 100 mmol/L), or 4) 5-HD alone (100 mmol/L). Regional cerebral blood flow (laser Doppler scanning) and brain cell swelling (impedance) were monitored acutely. Infarct volume was assessed 7 days after ischemia. Pretreatment with diazoxide significantly reduced the infarct volume from 6.2 +/- 0.7 mm3 to 3.8 +/- 0.4 mm3, whereas regional cerebral blood flow in the vicinity of the two veins was comparable in both groups 70 minutes after two-vein occlusion. Effects of diazoxide were abolished by 5-HD, whereas 5-HD alone even increased infarct volume. These results suggest that the opening of mitoKATP channels plays a major role in brain protection under penumbra-like conditions, as shown in this venous occlusion model.