Activation Of mitoKATP Channels Research Articles

Activation of mitoKATP channels induces penile vasodilation and inhibits mitochondrial respiration and ROS production: Role of NO

ObjectiveMitochondrial ATP-sensitive K+ (mitoKATP) channels are involved in neuronal and cardiac protection from ischemia and oxidative stress. Penile erection is a neurovascular event mediated by relaxation of the erectile tissue via nitric oxide (NO) released from nerves and endothelium. In the present study, we investigated whether mitoKATP channels play a role in the control of penile vascular tone and mitochondrial dynamics, and the involvement of NO. MethodsThe effect of the selective mitoKATP activator BMS191095 was examined on vascular tone, on mitochondrial bioenergetics by real-time measurements with Agilent Seahorse and on ROS production by MitoSOX fluorescence in freshly isolated microarteries. ResultsBMS191095 and diazoxide relaxed penile arteries, BMS191095 being one order of magnitude more potent. BMS191095-induced relaxations were reduced by mechanical endothelium removal and by inhibitors of the nitric oxide synthase (NOS) and PI3K enzymes. The NO-dependent component of the relaxation to BMS191095 was impaired in penile arteries from insulin resistant obese rats. The blockers of mitoKATP channel 5-HD, sarcolemma KATP (sarcKATP) channel glibenclamide, and large conductance Ca2+-activated K+ (BKCa) channel iberiotoxin, inhibited relaxations to BMS191095 and to the NO donor SNAP. BMS191095 reduced the mitochondrial bioenergetic profile of penile arteries and attenuated mitochondrial ROS production. Blockade of endogenous NO impaired and exogenous NO mimicked, respectively, the inhibitory effects of BMS191095 on basal respiration and oxygen consumed for ATP synthesis. Exogenous NO exhibited dual inhibitory/stimulatory effects on mitochondrial respiration. ConclusionsThese results demonstrate that selective activation of mitoKATP channels causes penile vasodilation, attenuates ROS production and inhibits mitochondrial respiration in part by releasing endothelial NO. These mechanisms couple blood flow and metabolism in penile arterial wall and suggest that activation of vascular mitoKATP channels may protect erectile tissue against ischemic injury.

Effects of Pb2+ and Cd2+ Heavy Metal Ions on the Rat Liver Mitochondrial ATP-Dependent Potassium Channel

In the article, rat liver of Pb2+ and Cd2+ ionsthe effect on mitoKATP-channel activity in mitochondria was studied.Inhibition of mitoKATF-channel activity was observed experimentally in the presence of 200 μM concentration of ATP in the incubation medium of mitochondria isolated from rat liver.In the absence of ATP in the incubation medium, 8.0 µM concentration of Pb2+ and Cd2+ salts was noted to reduce the permeability of liver mitoKATP-channel activity in both energized and de-energized states compared to the control.

Uridine increases endurance and improves the rehabilitation of experimental animals after physical performance

BACKGROUND: The pharmacological correction of metabolic processes, providing an increase in the efficiency and duration of physical performance and contributing to rapid rehabilitation, is an important component of the regulation of adaptation. Previously, we found that the pyrimidine nucleoside uridine exhibits antihypoxic properties, activates mitochondrial K+ATP channels (mitoKATP), normalizes energy metabolism, reduces lipid peroxidation, activates the antioxidant system, and increases glycogen content. The substance with such properties was assumed to increase endurance and improve recovery after physical performance.
 AIM: To examine the effect of uridine on the endurance of experimental animals in the forced swimming test under different intensities of physical performance and their rehabilitation.
 MATERIALS AND METHODS: Experiments were performed on male Wistar rats (350380 g) and male outbred mice (2530 g). In the first series, the effect of uridine on the rats endurance was studied in the forced swimming test with a load of 5%, 7%, or 10% of the animal weight. In the second series, the effect of uridine on the first phase of recovery was evaluated in a three-load swimming test. Mice with 10% load were subjected to a swimming test three times, after which the trail index the ratio of time of the third trail to the first trail was determined. The frequency of animals with low, medium, and high recovery ability was estimated. Uridine 30 mg/kg or physiological saline (control) was administered 30 min before testing, 5-hydroxidecanoate (5-HD, mitoKATP blocker) 5 mg/kg 45 min before testing, and mexidol (reference drug) 200 mg/kg 50 min before testing.
 RESULTS: Uridine increased the critical swimming duration by 58% and 44% at 5% and 7% exercise, respectively, in comparison with control. At 7% load, the drug increased the period before the appearance of the first signs of fatigue by 100%. After the blockade of mitoKATP channels, the effect of uridine decreased by 40% in the presence of fatigue and 24% in critical swimming duration. In the three-load swimming test, uridine increased the trail index by 1.5 times, which was comparable to the effect of mexidol, and increased the number of animals with a high ability to recover by 2.6 times. The use of uridine after mitoKATP channel blockade did not lead to a decrease of its positive effect and the blockade of channels with 5-HD did not affect rehabilitation.
 CONCLUSIONS: Uridine increases the endurance of rats with a medium load in the forced swimming test and the rehabilitation of mice in the three-load swimming test. It also increases the number of animals with a high ability to recover after a swimming performance. The mechanism of its effects was realized both through the activation of mitoKATP channels and, probably, the stimulation of glycogenesis.

Interaction of nitric oxide synthase and mitochondrial ATP-sensitive potassium channels in protective impacts of combination therapy with irisin-preconditioning and melatonin-postconditioning in myocardial ischemia/reperfusion in aging

ObjectivesThe main challenge for cardioprotection in patients with cardiac ischemia–reperfusion (I/R) damage is the existence of negatively-acting multiple risk factors such as aging. This work aimed to evaluate the efficacy of combined therapy with irisin and melatonin on cardiac I/R injury in aged rats by focusing on the involvement of mitochondrial ATP-sensitive potassium (mitoKATP) channels, and the eNOS/NO pathway. MethodsMale aged Wistar rats (78 rats, 400–450 g) were experienced LAD ligation for 30 min followed by 3 h or 2 weeks reperfusion. Irisin (0.5 mg/kg/day) was administered for one week before ischemic insult. Melatonin (20 mg/kg) was administered 10 min before the onset of reperfusion. The releases of CK and LDH, mitochondrial function, inflammatory and apoptosis responses, the expression of eNOS, the levels of NO, cardiac function, and infarct size (IS) were evaluated. ResultsCombined therapy with irisin and melatonin significantly improved heart function, reduced IS, and limited the releases of CK and LDH (P < 0.01 for all). This combined therapy improved mitochondrial function, decreased inflammatory cytokines and apoptosis response, and increased eNOS/NO activity (P < 0.05 for all). Monotherapies had no significant impacts. Inhibition of mitoKATP channels and eNOS enzyme by 5HD and L-NAME, respectively, reversed the cardioprotective effects of combination therapy (P < 0.05), however, the effect of 5HD was more potent as compared to L-NAME (P < 0.05). ConclusionsIt can be suggested that combined therapy with irisin and melatonin can be proposed as a potential candidate to mitigate the I/R severity of aged hearts via activation of mitoKATP channels partly through the eNOS/NO signaling pathway.

The 40-Hz White Light-Emitting Diode (LED) Improves the Structure-Function of the Brain Mitochondrial KATP Channel and Respiratory Chain Activities in Amyloid Beta Toxicity.

It has been described that using noninvasive exposure to 40-Hz white light LED reduces amyloid-beta, a peptide thought to initiate neurotoxic events in Alzheimer's disease (AD). However, the mechanisms remain to be identified. Since AD impairs mitochondrial potassium channels and respiratory chain activity, the objectives of the current study were to determine the effect of 40-Hz white light LED on structure-function of mitoKATP channel and brain mitochondrial respiratory chain activity, production of reactive oxygen species (ROS), and ΔΨm in AD. Single mitoKATP channel was considered using a channel incorporated into the bilayer lipid membrane and expression of mitoKATP-Kir6.1 subunit as a pore-forming subunit of the channel was determined using a western blot analysis in Aβ1-42 toxicity and light-treated rats. Our results indicated a severe decrease in mito-KATP channel permeation and Kir6.1 subunit expression coming from the Aβ1-42-induced neurotoxicity. Furthermore, we found that Aβ1-42-induced neurotoxicity decreased activities of complexes I and IV and increased ROS production and ΔΨm. Surprisingly, light therapy increased channel permeation and mitoKATP-Kir6.1 subunit expression. Noninvasive 40-Hz white light LED treatment also increased activities of complexes I and IV and decreased ROS production and ΔΨm up to ~ 70%. Here, we report that brain mito-KATP channel and respiratory chain are, at least in part, novel targets of 40-Hz white light LED therapy in AD.

Ginsenoside-Mc1 reduces cerebral ischemia-reperfusion injury in hyperlipidemia through mitochondrial improvement and attenuation of oxidative/endoplasmic reticulum stress

The neuroprotective effect of ginsenoside-Mc1 (GMc1) in hyperlipidemic rats in the setting of cerebral ischemiareperfusion injury (I/RI), as well as the role of mitochondrial ATP-sensitive potassium (mitoKATP) channels and oxidative/ endoplasmic reticulum (ER) stress, was investigated. Hyperlipidemia (8 weeks) was induced by a high-fat diet in Sprague Dawley rats. GMc1 (10 mg/kg, i.p.) was given to hyperlipidemic rats daily for one month before I/RI. Rat brains were subjected to 2 h of local ischemia followed by 24 h reperfusion. The cerebral infarcted injury was measured by triphenyltetrazolium chloride staining and the levels of oxidative stress indicators were detected by ELISA and spectrophotometry. A fluorometric technique was employed to evaluate mitochondrial function. Western blotting was used to detect changes in the expression of ER stress proteins. GMc1 reduced cerebral infarct volume in hyperlipidemic rats in comparison to untreated ones (P&lt;0.01). GMc1 reduced cerebral infarct volume in hyperlipidemic rats as compared to untreated rats (P&lt;0.01). GMc1 significantly decreased mitochondrial membrane depolarization, mitochondrial reactive oxygen species (mitoROS) and malondialdehyde levels (P&lt;0.01), while increasing the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GPx) (P&lt;0.001). GMc1 administration reduced the expression of ER stress markers, including phosphorylated (p)-endoplasmic reticulum kinase (PERK), p-eukaryotic translation initiation factor 2 subunit 1 (elF2?), and C/EBP homologous protein (CHOP). Inhibition of mitoKATP channels with hydroxydecanoate significantly eliminated the protective impacts of GMc1 in hyperlipidemic rats subjected to cerebral I/RI. The neuroprotective effect of GMc1 preconditioning was remarkably improved by increasing mitoKATP channel activity and decreasing oxidative and ER stress levels in hyperlipidemic rats, implying that this compound could be an appropriate candidate for reducing cerebral I/RI in comorbidities.

Nicorandil Affects Mitochondrial Respiratory Chain Function by Increasing Complex III Activity and ROS Production in Skeletal Muscle Mitochondria.

Adenosine triphosphate (ATP)-dependent potassium channels openers (KATP) protect skeletal muscle against function impairment through the activation of the mitochondrial KATP channels (mitoKATP). Previous reports suggest that modulators of the mitochondrial KATP channels have additional effects on isolated mitochondria. To determine whether the KATPchannel opener nicorandil has non-specific effects that explain its protective effect through the mitochondrial function, chicken muscle mitochondria were isolated, and respiration rate was determined pollarographically. The activity of the electron transport chain (ETC) complexes (I-IV) was measured using a spectrophotometric method. Reactive oxygen species(ROS)levels and lipid peroxidation were assessed using flow cytometry and thiobarbituric acid assay, respectively. Both KATP channel opener nicorandil and KATP channel blocker 5-hydroxydecanoate (5-HD) decreased mitochondrial respiration; nicorandil increased complex III activity and decreased complex IV activity. The effects of nicorandil on complex III were antagonized by 5-HD. Nicorandil increased ROS levels, effect reverted by either 5-HD or the antioxidant N-2-mercaptopropionyl glycine (MPG). None of these drugs affected lipid peroxidation levels. These findings suggest that KATPchannel opener nicorandil increases mitochondrial ROS production from complex III. This results by partially blocking electron flow in the complex IV, setting electron carriers in a more reduced state, which is favored by the increase in complex III activity by nicorandil. Overall, our study showed that nicorandil like other mitochondrial KATP channel openers might not act through mitoKATP channel activation.

The Protective Effect of Uridine on Metabolic Processes in the Rat Myocardum during its Ischemia/Reperfusion Injury

The experimental study of the cardioprotective effect of uridine, the metabolic precursor of the endogenous activator of mitochondrial ATP-dependent K+-channels (mitoKATP-channels), was performed using the model of myocardial ischemia/reperfusion injury (I/RP) in rats. Ischemia for 30 min followed by reperfusion for 120 min resulted in a significant decrease in the ATP and phosphocreatine (PCr) content, intensification of lipid peroxidation (LPO), and inhibition of the antioxidant system (AOS) in cardiomyocytes. Intravenous administration of uridine (30 mg/kg) had a protective effect on the myocardial metabolism in the zone of I/RP. It prevented the I/RP-induced decrease of ATP and PCr, limited LPO processes, evaluated by the content of lipid hydroperoxides (LHP) and conjugated dienes (CD), and improved the AOS state by, preventing a decrease of superoxide dismutase (SOD) activity and increasing the content of reduced glutathione (GSH). The intravenous injection of mitoKATP-channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg, 5 min before uridine) eliminated the ability of uridine to maintain the ATP level and to exhibit its positive effect on the intensity of the LPO and activity of AOS. The data obtained suggest that the activation of mitoKATP-channels plays an important role in the mechanism of the cardioprotective effect of uridine in the myocardial I/RP.

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.

Protective effect of uridine on metabolic processes in rat myocardum during its ischemia/reperfusion damage

The experimental study of the cardioprotective effect of uridine, the metabolic precursor of the endogenous activator of mitochondrial ATP-dependent K+-channels (mitoKATP-channels), was performed using the model of myocardial ischemia/reperfusion (I/RP) in rats. Ischemia for 30 min followed by reperfusion for 120 min resulted in a significant decrease in ATP and phosphocreatine (PC) content, intensification of lipid peroxidation (LPO), and inhibition of the antioxidant system (AOS) in cardiomyocytes. Uridine in a dose of 30 mg/kg, administered intravenously prior to reperfusion, had a protective effect on myocardial metabolism in the I/RP zone. It prevented the decrease of ATP and PC, limited the LPO processes, evaluated by the content of lipid hydroperoxides and conjugated dienes, and improved the AOS state by, preventing the decrease of superoxide dismutase (SOD) activity and increasing the content of reduced glutathione (GSH). The mitoKATP-channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg) eliminated the ability of uridine to maintain the ATP level and to exhibit its positive effect on the intensity of the LPO and activity of AOS. The obtained data allow us to conclude that activation of mitoKATP-channels play an important role in the mechanism of the cardioprotective effect of uridine in I/RP damage of myocardium.

Cardioprotection of Ischemic Myocardium

Aim. Cardioprotective effect of precursors in the synthesis of the uridine-5ʼ-diphosphate (UDP) - the mitochondrial ATP-dependent potassium channels (mitoKATP channels) endogenous activator - uridine and uridine-5ʼ-monophosphate (UMP) and the relation between there mechanism of action and activity of mitoKATP channels were studied. Methods. The experiments were performed on the male Wistar rats weighing 300-350 g. Acute myocardial ischemia (MI) lasting 60 min was produced by occlusion of the descending branch of the left coronary artery (LCA) under artificial pulmonary ventilation. Animals were anesthetized with sodium ethaminal (50 mg/kg). Uridine or UMP in the dose of 30 mg/kg was injected intravenously 5 min prior to LCA occlusion. A selective blocker of these channels 5-hydroxydecanoate (5-HD, 5 mg/kg intravenously 5 min prior to injection of uridine or UMP) was used to detect the involvement of mitoKATP channels in the effects of drugs. ATP and creatine phosphate (CP) was determined in the heart homogenates. The intensity of lipid peroxidation (LPO) was estimated by the content of lipid hydroperoxides (LHP) and the state of the antioxidant system (AOS) by superoxidedismutase (SOD) activity and the reduced glutathione (GH) content. Results. Occlusion of the LCA during 60 min led to the decrease of ATP and CP content in the myocardium by 35% and 59% respectively. At the same time changes in LPO and AOS were observed. The amount of LHP increased by 97%, the activity of SOD was reduced by 28% and the content of GH decreased by 30%. Uridine and UMP given 5 minutes prior to LCA occlusion prevented the development of these metabolic disorders in the ischemic myocardium. Selective blocker of mitoKATP channels 5-HD eliminated the protective effect of both drugs. Conclusion. Uridin and UMP have the evident cardioprotective effect in the acute MI, stabilizing the miocardium energy metabolism, preventing the AOS function depression and excessive activation of LPO. The mechanism of protective action of the drugs is associated with the activation of mitoKATP channels. (For citation: Bulion VV, Krylova IB, Selina EN. Cardioprotection of ischemic myocardium. Reviews on Clinical Pharmacology and Drug Therapy. 2018;16(2):13-17. doi: 10.17816/RCF16213-17).

Correction of hypoxic state by metabolic precursors of endogenous activator of mitochondrial ATP-dependent K+channels

Aim. The antihypoxic properties of uridine and uridine-5'-monophosphate (UMP), which are the metabolic precursors of the natural activator of mitochondrial ATP-dependent K+ channels (mitoKATP channels) uridine diphosphat were investigated on the models of hypoxic hypoxia with hypercapnia (HHH), hemic hypoxia and local circulatory hypoxia.&#x0D; Methods. HHH was created in males and females white mice weighing 28-30 g. The animals were placed one by one in hermetically closed container and the duration of their life was determined. The antihypoxic activity of the substances was compared with the reference anthypoxant amtizole (50 mg/kg). Hemic hypoxia was caused in Wistar rats weighing 350-370 g by the injection of sodium nitrite (intramuscularly, 100 mg/kg). Uridine or UMP 30 mg/kg was injected intraperitoneally 30 minutes before the onset of HHH and hemic hypoxia. Local circulatory hypoxia was modeled in male Wistar rats weighing 250-300 g. Acute coronary occlusion lasting 60 min was reproduced by legation of descending branch of the left coronary artery (LCA). Uridine or UMP (30 mg/kg) was administered intravenously 5 minutes prior to LCA occlusion. Selective blocker of mitoKATP channels 5-hydroxydecanoate (5 mg/kg, intravenously, 5 minutes prior uridine or UMP) was used to determine the role of these channels in the mechanism of antihypoxic action of the studied drugs. The volume of the damaged myocardium was used as the marker of antihypoxic activity of uridine and UMP.&#x0D; Results. Different resistance to hypoxia in female and male mice was observed in HHH. The female mice were more resistant, their life duration was 43% more than the males. Uridine and UMP displayed antihypoxic activity only in male mice, increasing their life duration by 25% and 20% respectively. This effect was 2 times less than that of amtisol. In similar conditions in females mice the preparations did not show a protective effect. In hemic hypoxia the life duration of rats treated with uridine and UMP did not differ from the control values. Circulatory hypoxia, caused by occlusion of the LCA, led to the formation of a local zone of myocardial damage. Uridine or UMP decreased the damage zone in 2 and 3,5 times respectively. The inhibitor of mitoKATP channels blocked the protective effect of these compounds.&#x0D; Conclusion. Uridine and UMP have a distinct antihypoxic effect in HHH and a marked protective effect in local circulatory hypoxia. The antihypoxic activity of druges in HHH is manifested differently in female and male mice. It may be due to sexual differences in the resistance to hypoxia. The maximum effect is observed in male who have initially low resistance to oxygen deficiency. The mechanism of the protective action of uridine and UMP in the circulatory hypoxia is associated with the activation of mitoKATP channels.

Transgenic overexpression of the SUR2A-55 splice variant in mouse heart reduces infract size and promotes protective mitochondrial function

ATP-sensitive potassium channels found in both the sarcolemma (sarcKATP) and mitochondria (mitoKATP) of cardiomyocytes are important mediators of cardioprotection during ischemic heart disease. Sulfonylurea receptor isoforms (SUR2), encoded by Abcc9, an ATP-binding cassette family member, form regulatory subunits of the sarcKATP channel and are also thought to regulate mitoKATP channel activity. A short-form splice variant of SUR2 (SUR2A-55) was previously shown to target mitochondria and display diaxoxide and ATP insensitive KATP activity when co-expressed with the inward rectifier channels Kir6.2 and Kir6.1. We hypothesized that mice with cardiac specific overexpression of SUR2A-55 would mediate cardioprotection from ischemia by altering mitoKATP properties. Mice overexpressing SUR2A-55 (TGSUR2A-55) in cardiomyocytes were generated and showed no significant difference in echocardiographic measured chamber dimension, percent fractional shortening, heart to body weight ratio, or gross histologic features compared to normal mice at 11–14 weeks of age. TGSUR2A-55 had improved hemodynamic functional recovery and smaller infarct size after ischemia reperfusion injury compared to WT mice in an isolated hanging heart model. The mitochondrial membrane potential of TGSUR2A-55 mice was less sensitive to ATP, diazoxide, and Ca2+ loading. These data suggest that the SUR2A-55 splice variant favorably affects mitochondrial function leading to cardioprotection. These data support a role for the regulation of mitoKATP activity by SUR2A-55.

Mitochondrial ATP-sensitive potassium channel regulates mitochondrial dynamics to participate in neurodegeneration of Parkinson's disease

Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. Mitochondrial dysfunction has been the focus of the pathogenesis of PD. The mitochondrial ATP-sensitive potassium channel (mitoKATP) plays a significant role in mitochondrial physiology and has been extensively shown to protect against ischemic and brain reperfusion injury. However, there have long been controversies regarding its role in Parkinson's disease. We investigated the role of mitoKATP channels in rotenone-induced PD model in vivo and vitro and the interactions of mitoKATP channels, mitochondrial dynamics and PD. The results indicated that the use of diazoxide to activate mitoKATP channels resulted in the aggravation of rotenone-induced dopamine neurodegeneration in PC12 cells and SD rats. In contrast, the use of 5-hydroxydecanoate (5-HD) to inhibit mitoKATP channels improved rotenone-induced dopamine neurodegeneration, which was not consistent with mitoKATP channels in ischemic and brain reperfusion injury. Further analysis determined that the mitoKATP channel was involved in PD mainly via the regulation of mitochondrial biogenesis and fission/fusion. And the pore subunits of Kir6.1, the major component of mitoKATP channels, was the key contributor in its interaction with mitochondrial dynamics in rotenone-induced dopamine neurodegeneration. Therefore, it can be concluded that mitoKATP channels regulate mitochondrial dynamics to participate in rotenone-induced PD mainly attributes to the pore subunits of Kir6.1. And additionally, though mitoKATP channels may represent a direction of one potential target for neuroprotection, it should be noted that the effects are different in the activation or inhibition of mitoKATP channels in different models.

Abstract 453: FADS2 Overexpression Exacerbates Myocardial Ischemia-reperfusion Injury in Mice: Role of Mitochondria

Common FADS2 haplotypes associated with hyperactivity of its gene product, delta-6-desaturase (D6D), predict cardiovascular morbidity and mortality in humans. D6D is the rate-limiting enzyme in essential polyunsaturated fatty acid (PUFA) metabolism, but its role in the pathogenesis of cardiovascular disease is unclear. To investigate this, we generated mice with global (CMV promoter) transgenic overexpression of FADS2 and evaluated their cardiometabolic phenotype. FADS2 -tg mice exhibit mild glucose intolerance and aortic stiffening with advancing age, but no overt cardiac pathology. However, when challenged with ischemia-reperfusion (I/R) ex vivo , FADS2 -tg mouse hearts have greater infarct sizes compared to their wild-type (WT; FVB) counterparts. Mitochondrial phospholipid analyses revealed a 65% increase in the membrane arachidonic/linoleic acid (AA/LA) ratio of FADS2- tg versus WT hearts, consistent with previous work in our lab linking D6D hyperactivity with mitochondrial phospholipid remodeling and disease progression in heart failure. Thus, we hypothesized that FADS2 might exacerbate myocardial injury by altering mitochondrial responses to ischemia-reperfusion. To examine this further, mitochondria were isolated from FADS2 -tg or WT mouse hearts for functional studies following 45/90 minutes of global I/R ex vivo . FADS2- tg - I/R mitochondria exhibited lower ADP-stimulated respiration and higher H 2 O 2 emission per O 2 consumed compared to WT-I/R and non-ischemic controls , suggesting impaired cardiac energetics and greater oxidant burden following ischemia. I/R augmented Ca 2+ -induced swelling in both WT and FADS2 -tg mitochondria. This was attenuated in the presence of K + in WT, but not FADS2 -tg, implicating FADS2 -dependent alterations post-ischemic in mitochondrial K + handling. Interestingly, ischemia led to a 38% depletion of mitochondrial membrane AA in FADS2 -tg, but not WT hearts, suggesting an interaction of FADS2 and mitochondrial membrane AA liberation following ischemia. Ongoing studies in our laboratory are investigating the potentially novel links between FADS2 expression with mitochondrial phospholipases, AA signaling, and mitoK ATP channel activity in the pathogenesis of myocardial I/R injury.

The novel H2S-donor 4-carboxyphenyl isothiocyanate promotes cardioprotective effects against ischemia/reperfusion injury through activation of mitoKATP channels and reduction of oxidative stress

The endogenous gasotransmitter hydrogen sulphide (H2S) is an important regulator of the cardiovascular system, particularly of myocardial function. Moreover, H2S exhibits cardioprotective activity against ischemia/reperfusion (I/R) or hypoxic injury, and is considered an important mediator of “ischemic preconditioning”, through activation of mitochondrial potassium channels, reduction of oxidative stress, activation of the endogenous “anti-oxidant machinery” and limitation of inflammatory responses. Accordingly, H2S-donors, i.e. pro-drugs able to generate exogenous H2S, are viewed as promising therapeutic agents for a number of cardiovascular diseases. The novel H2S-donor 4-carboxy phenyl-isothiocyanate (4CPI), whose vasorelaxing effects were recently reported, was tested here in different experimental models of myocardial I/R.In Langendorff-perfused rat hearts subjected to I/R, 4CPI significantly improved the post-ischemic recovery of myocardial functional parameters and limited tissue injury. These effects were antagonized by 5-hydroxydecanoic acid (a blocker of mitoKATP channels). Moreover, 4CPI inhibited the formation of reactive oxygen species. We found the whole battery of H2S-producing enzymes to be present in myocardial tissue: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST). Notably, 4CPI down-regulated the post-ischemic expression of CSE.In Langendorff-perfused mouse hearts, 4CPI reduced the post-ischemic release of norepinephrine and the incidence of ventricular arrhythmias. In both rat and mouse hearts, 4CPI did not affect the degranulation of resident mast cells.In isolated rat cardiac mitochondria, 4CPI partially depolarized the mitochondrial membrane potential; this effect was antagonized by ATP (i.e., the physiological inhibitor of KATP channels). Moreover, 4CPI abrogated calcium uptake in the mitochondrial matrix.Finally, in an in vivo model of acute myocardial infarction in rats, 4CPI significantly decreased I/R-induced tissue injury.In conclusion, H2S-donors, and in particular isothiocyanate-based H2S-releasing drugs like 4CPI, can actually be considered a suitable pharmacological option in anti-ischemic therapy.

Role of Vitamin C in Cardioprotection of Ischemia/Reperfusion Injury by Activation of Mitochondrial KATP Channel.

How to provide effective prevention and treatment of myocardial ischemia/reperfusion (I/R) injury and study of the mechanism underlying I/R injury are hotspots of current research. This study aimed to elucidate the effect and cardioprotective mechanism of vitamin C (VC) on myocardial I/R injury. Our study introduced two different I/R models: I/R in vitro and oxygen-glucose deprivation/recovery (OGD/R) in primary neonatal rat cardiac myocytes. We used the mitochondrial permeability transition pore (mPTP) opener lonidamine (LND) and the mitochondrial KATP (mitoKATP) channel inhibitor 5-hydroxydecanoate (5-HD) to analyze the underlying mechanisms. We found that post-treatment with VC decreased I/R injury in our models. Post-treatment with VC significantly decreased I/R-induced injury, attenuated apoptosis, and maintained the functional integrity of mitochondria via alleviation of Ca(2+) overload, reactive oxygen species burst, inhibition of the opening of mPTP, and prevention of mitochondrial membrane potential (ΔΨm) depolarization. VC post-treatment increased the phosphorylation of Akt and glycogen synthase kinase (GSK)-3β. The present results demonstrate that VC might protect the myocardium from I/R-induced injury by inhibiting the mPTP opening via activation of mitoKATP channels. VC mediates cardioprotection via activation of the phosphatidyl inositol 3-kinase (PI3K)-Akt signaling pathway. These findings may contribute toward the development of novel strategies for clinical cardioprotection against I/R injury.

Signaling pathways of a structural analogue of apelin-12 involved in myocardial protection against ischemia/reperfusion injury

Exogenously administered chemically modified apelin-12 (MA) has been shown to exhibit protective effects in myocardial ischemia/reperfusion (I/R) injury. They include reduction of ROS formation, cell death and cardiometabolic abnormalities. The aim of the present study was to explore the role of the underlying signaling mechanisms involved in cardioprotection afforded by MA. Isolated perfused working rat hearts subjected to global ischemia and anaesthetized rats in vivo exposed to LAD coronary artery occlusion were used. Myocardial infarct size, cell membrane damage, cardiac dysfunction and metabolic state of the heart were used as indices of I/R injury at the end of reperfusion. Administration of specific inhibitors of MEK1/2, PI3K, NO synthase (NOS) or the mitochondrial ATP-sensitive K+ (mito KATP) channels (UO126, LY294002, L-NAME or 5-hydroxydecanoate, respectively) reduced protective efficacy of MA in both models of I/R injury. This was evidenced by abrogation of infarct size limitation, deterioration of cardiac function recovery, and attenuation of metabolic restoration and sarcolemmal integrity. An enhancement of functional and metabolic recovery in isolated reperfused hearts treated with MA was suppressed by U-73122, chelerythrine, amiloride or KB-R7943 (inhibitors of phospholipase С (PLC), protein kinase C (PKC), Na+/H+ or Na+/Ca2+ exchange, respectively). Additionally, co-infusion of MA with amiloride or L-NAME reduced the integrity of cell membranes at early reperfusion compared with the effect of peptide alone. In conclusion, cardioprotection with MA is mediated by signaling via PLC and survival kinases, PKC, PI3K, and MEK1/2, with activation of downstream targets, NOS and mito KATP channels, and the sarcolemmal Na+/H+ and Na+/Ca2+ exchangers.

Cardio-vascular activity of catestatin: interlocking the puzzle pieces.

Catestatin (CST), the Chromogranin A (CgA)-derived cationic and hydrophobic peptide, firstly recognized as an endogenous inhibitor of catecholamine secretion, functions as a physiological brake of the adreno-sympathetic-chromaffin system. Its wide spectrum of activities includes relevant multilevel cardiovascular and antihypertensive influences. At central systemic level, CST seems to modulate the autonomic cardiovascular control possibly acting on baroreceptor afferent fibers of the nucleus tractus solitarius. This, as well as clinical and experimental (CgA-KO mice) evidences point to an important role of CST in the determinism and prevention of essential hypertension. At organ level, CST exerts myocardial (negative inotropy and lusitropy) effects and potently vasodilates endothelin-1 (ET-1)-preconstricted coronaries through β2-adrenergic receptor (AR)-Gi/o protein-nitric oxide (NO)-cGMP signalling, while counterbalancing β adrenergic (ISO) stimulation. The contractile myocardial effects have been deeply analysed in fish and amphibian hearts, highlighting finely diversified mechanisms of action. CST also acts as cardioprotective agent in both pre- and post-conditioning through NO-dependent mechanisms implicating the Reperfusion Injury Salvage Kinase (RISK) signalling and the activation of mitoKATP channels. The CST-elicited cardiotropic and coronarotropic influences, along with the recently discovered proangiogenic and regulatory effects in glucose and lipid metabolism, contribute to delineate an integrated and updated picture of the peptide which emerges as a pleiotropic hormone with a wide range of cytokine-like characteristics. The aim of this review is to interlock some older and more recent evidences which may help to better perceive the subtle links and differences among the puzzle pieces that still need to be deciphered.

Diversity of mitochondria-dependent dilator mechanisms in vascular smooth muscle of cerebral arteries from normal and insulin-resistant rats.

Mitochondrial depolarization following ATP-sensitive potassium (mitoKATP) channel activation has been shown to induce cerebral vasodilation by generation of mitochondrial reactive oxygen species (ROS), which sequentially promotes frequency of calcium sparks and activation of large conductance calcium-activated potassium channels (BKCa) in vascular smooth muscle (VSM). We previously demonstrated that cerebrovascular insulin resistance accompanies aging and obesity. It is unclear whether mitochondrial depolarization without the ROS generation enhances calcium sparks and vasodilation in phenotypically normal [Sprague Dawley (SD); Zucker lean (ZL)] and insulin-resistant [Zucker obese (ZO)] rats. We compared the mechanisms underlying the vasodilation to ROS-dependent (diazoxide) and ROS-independent [BMS-191095 (BMS)] mitoKATP channel activators in normal and ZO rats. Arterial diameter studies from SD, ZL, and ZO rats showed that BMS as well as diazoxide induced vasodilation in endothelium-denuded cerebral arteries. In normal rats, BMS-induced vasodilation was mediated by mitochondrial depolarization and calcium sparks generation in VSM and was reduced by inhibition of BKCa channels. However, unlike diazoxide-induced vasodilation, scavenging of ROS had no effect on BMS-induced vasodilation. Electron spin resonance spectroscopy confirmed that diazoxide but not BMS promoted vascular ROS generation. BMS- as well as diazoxide-induced vasodilation, mitochondrial depolarization, and calcium spark generation were diminished in cerebral arteries from ZO rats. Thus pharmacological depolarization of VSM mitochondria by BMS promotes ROS-independent vasodilation via generation of calcium sparks and activation of BKCa channels. Diminished generation of calcium sparks and reduced vasodilation in ZO arteries in response to BMS and diazoxide provide new insights into mechanisms of cerebrovascular dysfunction in insulin resistance.