mitoKATP Channel Opener Research Articles

The Protective Effect of Uridine in a Rotenone-Induced Model of Parkinson's Disease: The Role of the Mitochondrial ATP-Dependent Potassium Channel.

The effect of the modulators of the mitochondrial ATP-dependent potassium channel (mitoKATP) on the structural and biochemical alterations in the substantia nigra and brain tissues was studied in a rat model of Parkinson's disease induced by rotenone. It was found that, in experimental parkinsonism accompanied by characteristic motor deficits, both neurons and the myelin sheath of nerve fibers in the substantia nigra were affected. Changes in energy and ion exchange in brain mitochondria were also revealed. The nucleoside uridine, which is a source for the synthesis of the mitoKATP channel opener uridine diphosphate, was able to dose-dependently decrease behavioral disorders and prevent the death of animals, which occurred for about 50% of animals in the model. Uridine prevented disturbances in redox, energy, and ion exchanges in brain mitochondria, and eliminated alterations in their structure and the myelin sheath in the substantia nigra. Cytochemical examination showed that uridine restored the indicators of oxidative phosphorylation and glycolysis in peripheral blood lymphocytes. The specific blocker of the mitoKATP channel, 5-hydroxydecanoate, eliminated the positive effects of uridine, suggesting that this channel is involved in neuroprotection. Taken together, these findings indicate the promise of using the natural metabolite uridine as a new drug to prevent and, possibly, stop the progression of Parkinson's disease.

Effects of nicorandil on QT prolongation and myocardial damage caused by citalopram in rats

ABSTRACT Citalopram is a selective serotonin re-uptake inhibitor (SSRI) antidepressant; it exhibits the greatest cardiotoxic effect among SSRIs. Citalopram can cause drug-induced long QT syndrome (LQTS) and ventricular arrhythmias. We investigated the protective effect of nicorandil, a selective mitochondrial KATP (mito-KATP) channel opener, on LQTS and myocardial damage caused by citalopram in male rats. In a preliminary study, we determined that the minimum citalopram dose that prolonged the QT interval was 102 mg/kg injected intraperitoneally. For the main study, rats were divided randomly into five experimental groups: untreated control, normal saline + citalopram, nicorandil + citalopram, 5-hydroxydecanoate (5-HD) + citalopram, 5-HD + nicorandil + citalopram. Biochemical and histologic data from blood and heart tissue samples from six untreated control rats were evaluated. Electrocardiographic parameters including QRS duration, QT interval, corrected QT interval (QTc) and heart rate (HR) were assessed, and biochemical parameters including malondialdehyde, reduced glutathione, glutathione peroxidase, superoxide dismutase were measured. We also performed histomorphologic and immunohistochemical examination of heart tissue. Citalopram prolonged QT-QTc intervals significantly and increased significantly the histomorphologic score and proportion of apoptotic cells, but produced no differences in the oxidant and antioxidant parameters. Nicorandil did not prevent citalopram induced QT-QTc interval prolongation and produced no significant changes in oxidant and antioxidant parameters; however, it did reduce histologic damage and apoptosis caused by citalopram.

Pharmacological Characterization of a Recombinant Mitochondrial ROMK2 Potassium Channel Expressed in Bacteria and Reconstituted in Planar Lipid Bilayers

In the inner mitochondrial membrane, several potassium channels that play a role in cell life and death have been identified. One of these channels is the ATP-regulated potassium channel (mitoKATP). The ROMK2 potassium channel is a potential molecular component of the mitoKATP channel. The current study aimed to investigate the pharmacological modulation of the activity of the ROMK2 potassium channel expressed in Escherichia coli bacteria. ROMK2 was solubilized in polymer nanodiscs and incorporated in planar lipid bilayers. The impact of known mitoKATP channel modulators on the activity of the ROMK2 was characterized. We found that the ROMK2 channel was activated by the mitoKATP channel opener diazoxide and blocked by mitoKATP inhibitors such as ATP/Mg2+, 5-hydroxydecanoic acid, and antidiabetic sulfonylurea glibenclamide. These results indicate that the ROMK2 potassium protein may be a pore-forming subunit of mitoKATP and that the impact of channel modulators is not related to the presence of accessory proteins.

Mitochondrial ATP-Sensitive K+ Channel Opening Increased the Airway Smooth Muscle Cell Proliferation by Activating the PI3K/AKT Signaling Pathway in a Rat Model of Asthma.

Abnormal proliferation of airway smooth muscle cells (ASMCs) leads to airway remodeling and the development of asthma. This study aimed to assess whether mitochondrial ATP-sensitive K+ (mitoKATP) channels regulated the proliferation of ASMCs by regulating the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway in asthmatic rats. Forty-eight Sprague Dawley rats were immunized with ovalbumin-containing alum to establish the asthma models. The ASMCs were isolated and identified by phase-contrast microscopic images and immunohistochemical staining for α-smooth muscle actin. The ASMCs were treated with a potent activator of mitoKATP, diazoxide, or an inhibitor of mitoKATP, 5-hydroxydecanoate (5-HD). Rhodamine-123 (R-123) was used for detecting the mitochondrial membrane potential (Δψm). The proliferation of ASMCs was examined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The protein and mRNA expressions of AKT and p-AKT were detected using western blotting and quantitative real-time PCR. The results showed that diazoxide enhanced the mitoKATP channel opening in ASMCs in the rat model of asthma, while 5-HD impeded it. Diazoxide also increased ASMC proliferation in the rat model of asthma, whereas 5-HD alleviated it. However, LY294002, a PI3K/AKT pathway inhibitor, reversed the functional roles of diazoxide in the proliferation ability of ASMCs in the rat model of asthma. Furthermore, treatment with diazoxide induced the phosphorylation of AKT, and treatment with 5-HD decreased the phosphorylation of AKT in ASMCs in the rat model of asthma. In conclusion, the mitoKATP channel opening increased the proliferation of ASMCs by activating the PI3K/AKT signaling pathway in a rat model of asthma.

Protective Effect of Nicorandil on Cardiac Microvascular Injury: Role of Mitochondrial Integrity.

A major shortcoming of postischemic therapy for myocardial infarction is the no-reflow phenomenon due to impaired cardiac microvascular function including microcirculatory barrier function, loss of endothelial activity, local inflammatory cell accumulation, and increased oxidative stress. Consequently, inadequate reperfusion of the microcirculation causes secondary ischemia, aggravating the myocardial reperfusion injury. ATP-sensitive potassium ion (KATP) channels regulate the coronary blood flow and protect cardiomyocytes from ischemia-reperfusion injury. Studies in animal models of myocardial ischemia-reperfusion have illustrated that the opening of mitochondrial KATP (mito-KATP) channels alleviates endothelial dysfunction and reduces myocardial necrosis. By contrast, blocking mito-KATP channels aggravates microvascular necrosis and no-reflow phenomenon following ischemia-reperfusion injury. Nicorandil, as an antianginal drug, has been used for ischemic preconditioning (IPC) due to its mito-KATP channel-opening effect, thereby limiting infarct size and subsequent severe ischemic insult. In this review, we analyze the protective actions of nicorandil against microcirculation reperfusion injury with a focus on improving mitochondrial integrity. In addition, we discuss the function of mitochondria in the pathogenesis of myocardial ischemia.

Ischemic Postconditioning Reduces NMDA Receptor Currents Through the Opening of the Mitochondrial Permeability Transition Pore and KATP Channel in Mouse Neurons.

Ischemic postconditioning (PostC) is known to reduce cerebral ischemia/reperfusion (I/R) injury; however, whether the opening of mitochondrial ATP-dependent potassium (mito-KATP) channels and mitochondrial permeability transition pore (mPTP) cause the depolarization of the mitochondrial membrane that remains unknown. We examined the involvement of the mito-KATP channel and the mPTP in the PostC mechanism. Ischemic PostC consisted of three cycles of 15s reperfusion and 15s re-ischemia, and was started 30s after the 7.5min ischemic load. We recorded N-methyl-D-aspartate receptors (NMDAR)-mediated currents and measured cytosolic Ca2+ concentrations, and mitochondrial membrane potentials in mouse hippocampal pyramidal neurons. Both ischemic PostC and the application of a mito-KATP channel opener, diazoxide, reduced NMDAR-mediated currents, and suppressed cytosolic Ca2+ elevations during the early reperfusion period. An mPTP blocker, cyclosporine A, abolished the reducing effect of PostC on NMDAR currents. Furthermore, both ischemic PostC and the application of diazoxide potentiated the depolarization of the mitochondrial membrane potential. These results indicate that ischemic PostC suppresses Ca2+ influx into the cytoplasm by reducing NMDAR-mediated currents through mPTP opening. The present study suggests that depolarization of the mitochondrial membrane potential by opening of the mito-KATP channel is essential to the mechanism of PostC in neuroprotection against anoxic injury.

Роль микроРНК, NO-синтаз, киназ, К АТФ -каналов в инфаркт-лимитирующем эффекте адаптации к гипоксии

It has been established that chronic hypoxia (CH) significantly alters the expression of microRNAs in the myocardium. It was shown that miR-138 and miR-184 increase the tolerance of cardiomyocytes to hypoxia, while microRNA 199a 5p and miR-23b decrease the resistance of cardiomyocytes to hypoxia. CH enhanced expression of inducible NO synthase (iNOS) and endothelial NO synthase (eNOS). It was shown the cardioprotective effect of CH is associated with the activation of iNOS. It was found that the infarction-limiting effect of chronic CH depends on mitoKATP channel opening. It was established that the δ and e isoforms of protein kinase C are involved in the cardioprotective effect of adaptation to hypoxia. Chronic hypoxia has been shown to increase expression of kinases: CaMKII, p-ERK1/2, p-p38, p-Akt, hexokinase-1, hexokinase-2. Hypoxia enhances the hexokinase-2 translocation to mitochondria. In animals adapted to hypoxia, it was not detected an increase in the expression of kinases: PKA, p-GSK3β, AMPK and JNK. Presented data are indicated that ERK1/2, MEK1/2 kinases are involved in the cardioprotective effect of adaptation to hypoxia. Myocardial hypertrophy caused by chronic hypoxia is associated with the activation of Rho kinase. The role of PI3K, Akt JNK, PKG, Rho kinase, mTOR, and p38 kinase in the protective effect of adaptation to hypoxia is controversial. The purpose of the review: analysis of data on the role of miRNAs, NO synthase, and kinases in the cardioprotective effect of CH.

Ischemic postconditioning prevents surge of presynaptic glutamate release by activating mitochondrial ATP-dependent potassium channels in the mouse hippocampus.

A mild ischemic load applied after a lethal ischemic insult reduces the subsequent ischemia–reperfusion injury, and is called ischemic postconditioning (PostC). We studied the effect of ischemic PostC on synaptic glutamate release using a whole-cell patch-clamp technique. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) from CA1 pyramidal cells in mouse hippocampal slices. The ischemic load was perfusion of artificial cerebrospinal fluid (ACSF) equilibrated with mixed gas (95% N2 and 5% CO2). The ischemic load was applied for 7.5 min, followed by ischemic PostC 30 s later, consisting of three cycles of 15 s of reperfusion and 15 s of re-ischemia. We found that a surging increase in sEPSCs frequency occurred during the immediate-early reperfusion period after the ischemic insult. We found a significant positive correlation between cumulative sEPSCs and the number of dead CA1 neurons (r = 0.70; p = 0.02). Ischemic PostC significantly suppressed this surge of sEPSCs. The mitochondrial KATP (mito-KATP) channel opener, diazoxide, also suppressed the surge of sEPSCs when applied for 15 min immediately after the ischemic load. The mito-KATP channel blocker, 5-hydroxydecanoate (5-HD), significantly attenuated the suppressive effect of both ischemic PostC and diazoxide application on the surge of sEPSCs. These results suggest that the opening of mito-KATP channels is involved in the suppressive effect of ischemic PostC on synaptic glutamate release and protection against neuronal death. We hypothesize that activation of mito-KATP channels prevents mitochondrial malfunction and breaks mutual facilitatory coupling between glutamate release and Ca2+ entry at presynaptic sites.

Hypoxic Preconditioning Attenuates Reoxygenation-Induced Skeletal Muscle Dysfunction in Aged Pulmonary TNF-α Overexpressing Mice

Aim: Skeletal muscle subjected to hypoxia followed by reoxygenation is susceptible to injury and subsequent muscle function decline. This phenomenon can be observed in the diaphragm during strenuous exercise or in pulmonary diseases such as chronic obstructive pulmonary diseases (COPD). Previous studies have shown that PO2 cycling or hypoxic preconditioning (HPC), as it can also be referred to as, protects muscle function via mechanisms involving reactive oxygen species (ROS). However, this HPC protection has not been fully elucidated in aged pulmonary TNF-α overexpressing (Tg+) mice (a COPD-like model). We hypothesize that HPC can exert protection on the diaphragms of Tg+ mice during reoxygenation through pathways involving ROS/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/extracellular signal regulated kinase (ERK), as well as the downstream activation of mitochondrial ATP-sensitive potassium channel (mitoKATP) and inhibition of mitochondrial permeability transition pore (mPTP).Methods: Isolated Tg+ diaphragm muscle strips were pre-treated with inhibitors for ROS, PI3K, Akt, ERK, or a combination of mitoKATP inhibitor and mPTP opener, respectively, prior to HPC. Another two groups of muscles were treated with either mitoKATP activator or mPTP inhibitor without HPC. Muscles were treated with 30-min hypoxia, followed by 15-min reoxygenation. Data were analyzed by multi-way ANOVA and expressed as means ± SE.Results: Muscle treated with HPC showed improved muscle function during reoxygenation (n = 5, p < 0.01). Inhibition of ROS, PI3K, Akt, or ERK abolished the protective effect of HPC. Simultaneous inhibition of mitoKATP and activation of mPTP also diminished HPC effects. By contrast, either the opening of mitoKATP channel or the closure of mPTP provided a similar protective effect to HPC by alleviating muscle function decline, suggesting that mitochondria play a role in HPC initiation (n = 5; p < 0.05).Conclusion: Hypoxic preconditioning may protect respiratory skeletal muscle function in Tg+ mice during reoxygenation through redox-sensitive signaling cascades and regulations of mitochondrial channels.

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 protein kinase C, PI3 kinase, tyrosine kinases, NO-synthase, KATP channels and MPT pore in the signaling pathway of the cardioprotective effect of chronic continuous hypoxia.

It was established that adaptation to chronic continuous normobaric hypoxia (CCNH) increases cardiac tolerance to ischemia and reperfusion. It was performed coronary artery occlusion (20 min) and reperfusion (3 h) in Wistar rats. CCNH promoted a decrease in the infarct size/area at risk ratio in 2-fold. CCNH promoted an increase in the nitrite/nitrate levels in blood serum and myocardium. Pretreatment with protein kinase C (PKC) inhibitor chelerythrine, NO-synthase (NOS) inhibitor L-NAME, iNOS inhibitor S-methylisothiourea, KATP channel blocker glibenclamide, mitoKATP channel blocker 5-hydroxydecanoic acid abolished the infarct-reducing effect of CCNH. The non-selective tyrosine kinase inhibitor genistein attenuated but not eliminated infarct-sparing effect of CCNH. The nNOS inhibitor 7-nitroindazole, sarcKATP channel blocker HMR 1098, MPT pore inhibitor atractyloside, PI3 kinase inhibitor wortmannin did not reverse infarct-limiting effect of CCNH. It was concluded that infarct-reducing effect of CCNH is mediated via PKC, iNOS activation and mitoKATP channel opening. While nNOS, PI3 kinase, sarcKATP channel, MPT pore are not involved in the development of CCNH-induced cardiac tolerance to impact of ischemia-reperfusion.

Role of mitochondrial ATP-sensitive potassium channels in dexmedetomidine-induced attenuation of myocardial ischemia-reperfusion injury in rats

Objective To evaluate the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in dexmedetomidine-induced attenuation of myocardial ischemia-reperfusion (I/R) injury in rats. Methods Forty pathogen-free healthy male Sprague-Dawley rats, aged 8-12 weeks, weighing 200-350 g, were divided into 5 groups (n=8 each) using a random number table: sham operation group (group S), I/R group, dexmedetomidine group (group DEX), a specific mito-KATP channel blocker 5-hydroxydecanoate (5-HD) group (group 5-HD) and dexmedetomidine plus 5-HD group (group DEX+ 5-HD). Myocardial I/R was produced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 120 min reperfusion in pentobarbital sodium-anesthetized rats.Dexmedetomidine 5 μg/kg was intraperitoneally injected at 15 min prior to reperfusion in group DEX.5-HD 40 mg/kg was intraperitoneally injected at 30 min prior to reperfusion in group 5-HD.In group DEX+ 5-HD, 5-HD 40 mg/kg and dexmedetomidine 5 μg/kg were intraperitoneally injected at 30 and 15 min prior to reperfusion, respectively.The parameters of cardiac function such as left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and the maximum rate of increase or decrease in left ventricular pressure (±dp/dtmax) were recorded before ischemia (T0) and at 60 and 120 min of reperfusion (T1, 2). Blood samples were collected from the carotid artery at the end of reperfusion for determination of the concentrations of creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) in serum.The animals were then sacrificed, and hearts were removed for determination of the myocardial infarct size in the left ventricular myocardial tissues. Results Compared with group S, the LVSP and ±dp/dtmax were significantly decreased, and the LVEDP was increased at T1-2, and the concentrations of CK-MB and cTnI in serum and myocardial infarct size were increased in the other groups (P 0.05). Compared with group DEX, the LVSP and ±dp/dtmax were significantly decreased, and the LVEDP was increased at T1-2, and the concentrations of CK-MB and cTnI in serum and myocardial infarct size were increased in DEX+ 5-HD group (P<0.05). Conclusion The mechanism by which dexmedetomidine attenuates myocardial I/R injury is partially related to promotion of mito-KATP channel opening in rats. Key words: KATP channels; Dexmedetomidine; Myocardial reperfusion injury

MitoKATP channels promote the proliferation of hypoxic human pulmonary artery smooth muscle cells via the ROS/HIF/miR-210/ISCU signaling pathway.

Previous results have indicated that mitochondrial ATP-sensitive potassium (mitoKATP) channels are associated with the hypoxic proliferation of pulmonary artery smooth muscle cells (PASMCs). However, the mechanism underlying the promotive effects of mitoKATP channels on cell proliferation in response to hypoxia remains unknown. mitoKATP channel opening results in a collapse of mitochondrial membrane potential and generation of mitochondrial reactive oxygen species (ROS). As hypoxia-inducible factor-1α (HIF-1α) is a critical oxygen sensor and major transcriptional regulator of the hypoxic adaptive response, the current study assessed whether mitoKATP opening contributes to the chronic proliferation of human PASMCs (hPASMCs) in collaboration with HIF-1α and its downstream targets under hypoxic conditions. The present study demonstrated that there was crosstalk between mitoKATP channels and HIF-1α signaling in PASMCs under hypoxic conditions. The results suggest that mitoKATP channels are involved in the proliferation of PASMCs during hypoxia through upregulation of the ROS/HIF/microRNA-210/iron-sulfur cluster protein signaling pathway.

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

Nicorandil improves post-fatigue tension in slow skeletal muscle fibers by modulating glutathione redox state.

Fatigue is a phenomenon in which force reduction has been linked to impairment of several biochemical processes. In skeletal muscle, the ATP-sensitive potassium channels (KATP) are actively involved in myoprotection against metabolic stress. They are present in sarcolemma and mitochondria (mitoKATP channels). K+ channel openers like nicorandil has been recognized for their ability to protect skeletal muscle from ischemia-reperfusion injury, however, the effects of nicorandil on fatigue in slow skeletal muscle fibers has not been explored, being the aim of this study. Nicorandil (10μM), improved the muscle function reversing fatigue as increased post-fatigue tension in the peak and total tension significantly with respect to the fatigued condition. However, this beneficial effect was prevented by the mitoKATP channel blocker 5-hydroxydecanoate (5-HD, 500μM) and by the free radical scavenger N-2-mercaptopropionyl glycine (MPG, 1mM), but not by the nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100μM). Nicorandil also decreased lipid peroxidation and maintained both reduced glutathione (GSH) levels and an elevated GSH/GSSG ratio, whereas total glutathione (TGSH) remained unaltered during post-fatigue tension. In addition, NO production, measured through nitrite concentrations was significantly increased with nicorandil during post-fatigue tension; this increase remained unaltered in the presence of nicorandil plus L-NAME, nonetheless, this effect was reversed with nicorandil plus MPG. Hence, these results suggest that nicorandil improves the muscle function reversing fatigue in slow skeletal muscle fibers of chicken through its effects not only as a mitoKATP channel opener but also as NO donor and as an antioxidant.

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

Lebetin 2, a Snake Venom-Derived Natriuretic Peptide, Attenuates Acute Myocardial Ischemic Injury through the Modulation of Mitochondrial Permeability Transition Pore at the Time of Reperfusion.

Cardiac ischemia is one of the leading causes of death worldwide. It is now well established that natriuretic peptides can attenuate the development of irreversible ischemic injury during myocardial infarction. Lebetin 2 (L2) is a new discovered peptide isolated from Macrovipera lebetina venom with structural similarity to B-type natriuretic peptide (BNP). Our objectives were to define the acute cardioprotective actions of L2 in isolated Langendorff-perfused rat hearts after regional or global ischemia-reperfusion (IR). We studied infarct size, left ventricular contractile recovery, survival protein kinases and mitochondrial permeability transition pore (mPTP) opening in injured myocardium. L2 dosage was determined by preliminary experiments at its ability to induce cyclic guanosine monophosphate (cGMP) release without changing hemodynamic effects in normoxic hearts. L2 was found to be as effective as BNP in reducing infarct size after the induction of either regional or global IR. Both peptides equally improved contractile recovery after regional IR, but only L2 increased coronary flow and reduced severe contractile dysfunction after global ischemia. Cardioprotection afforded by L2 was abolished after isatin or 5-hydroxydecanote pretreatment suggesting the involvement of natriuretic peptide receptors and mitochondrial KATP (mitoKATP) channels in the L2-induced effects. L2 also increased survival protein expression in the reperfused myocardium as evidenced by phosphorylation of signaling pathways PKCε/ERK/GSK3β and PI3K/Akt/eNOS. IR induced mitochondrial pore opening, but this effect was markedly prevented by L2 treatment. These data show that L2 has strong cardioprotective effect in acute ischemia through stimulation of natriuretic peptide receptors. These beneficial effects are mediated, at least in part, by mitoKATP channel opening and downstream activated survival kinases, thus delaying mPTP opening and improving IR-induced mitochondrial dysfunction.

Nicorandil Inhibits Cyclic Strain-Induced Interleukin-8 Expression in Human Umbilical Vein Endothelial Cells

Background: Nicorandil, a mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channel opener, exerts protective effects on the cardiovascular system. This study examined the effect of nicorandil on cyclic strain-induced interleukin-8 (IL-8) expression in human umbilical vein endothelial cells (HUVECs). Methods: Cultured HUVECs were exposed to cyclic strain in the presence or absence of nicorandil (1-10 μmol/l); we then analyzed IL-8 expression. We also assessed the effects of nicorandil on heme oxygenase-1 (HO-1) expression and cyclic strain-modulated IL-8 expression after HO-1 silencing in HUVECs. Summary: HUVECs exposed to cyclic strain showed increased IL-8 messenger RNA expression and protein secretion. Nicorandil (1-10 μmol/l) inhibited cyclic strain-induced IL-8 expression, whereas 5-hydroxydecanoate (100 μmol/l), a selective inhibitor of the mitoKATP channel, completely reversed the inhibitory effects of nicorandil on cyclic strain-induced IL-8 expression. We demonstrated that nicorandil increased HO-1 expression in HUVECs. In addition, cobalt protoporphyrin (10 μmol/l), an inducer of HO-1 expression, mimicked the effects of nicorandil and inhibited IL-8 expression under cyclic strain, whereas zinc protoporphyrin IX (10 μmol/l), an inhibitor of HO-1 expression, antagonized the effect of nicorandil. HO-1 silencing significantly abrogated the inhibitory effects of nicorandil on cyclic strain-induced IL-8 expression, suggesting that HO-1 plays a role in the mechanism of action of nicorandil. Key Messages: This study is the first to report that nicorandil inhibits cyclic strain-induced IL-8 expression through the induction of HO-1 expression in HUVECs. This finding provides valuable new insight into the molecular pathways contributing to the vasoprotective effects of nicorandil.

Bioenergetic and volume regulatory effects of mitoKATP channel modulators protect against hypoxia-reoxygenation-induced mitochondrial dysfunction.

The mitochondrial ATP-sensitive K(+) (mitoKATP) channel plays a significant role in mitochondrial physiology and protects against ischemic reperfusion injury in mammals. Although fish frequently face oxygen fluctuations in their environment, the role of the mitoKATP channel in regulating the responses to oxygen stress is rarely investigated in this class of animals. To elucidate whether and how the mitoKATP channel protects against hypoxia-reoxygenation (H-R)-induced mitochondrial dysfunction in fish, we first determined the mitochondrial bioenergetic effects of two key modulators of the channel, diazoxide and 5-hydroxydecanoate (5-HD), using a wide range of doses. Subsequently, the effects of low and high doses of the modulators on mitochondrial bioenergetics and volume under normoxia and after H-R using buffers with and without magnesium and ATP (Mg-ATP) were tested. In the absence of Mg-ATP (mitoKATP channel open), both low and high doses of diazoxide improved mitochondrial coupling, but only the high dose of 5-HD reversed the post-H-R coupling-enhancing effect of diazoxide. In the presence of Mg-ATP (mitoKATP channel closed), diazoxide at the low dose improved coupling post-H-R, and this effect was abolished by 5-HD at the low dose. Interestingly, both low and high doses of diazoxide reversed H-R-induced swelling under mitoKATP channel open conditions, but this effect was not sensitive to 5-HD. Under mitoKATP channel closed conditions, diazoxide at the low dose protected the mitochondria from H-R-induced swelling and 5-HD at the low dose reversed this effect. In contrast, diazoxide at the high dose failed to reduce the swelling caused by H-R, and the addition of the high dose of 5-HD enhanced mitochondrial swelling. Overall, our study showed that in the presence of Mg-ATP, both opening of mitoKATP channels and bioenergetic effects of diazoxide were protective against H-R in fish mitochondria, while in the absence of Mg-ATP only the bioenergetic effect of diazoxide was protective.