Effect of Resistance Training Combined with Beta-Hydroxy-Beta-Methylbutyric Acid Supplements in Elderly Patients with Sarcopenia after Hip Replacement.

OBJECTIVES: To evaluate the efficacy of resistance training (RT) combined with beta-hydroxy-beta-methylbutyric acid (HMB) in the treatment of elderly patients with sarcopenia after hip replacement. METHODS: From January 1, 2018 to December 31, 2018, 200 elderly patients (68 men, mean age 76.3 years and 137 women, mean age 79.1 years) who experienced femoral neck fracture with sarcopenia after hip arthroplasty were assigned to four groups: RT + HMB group, RT group, HMB group, and negative control group. Baseline data, body composition, grip strength, Barthel index (BI), Harris hip score (HHS), and visual analog scale score (VAS) were compared among the four groups before and 3 months after surgery. RESULTS: A total of 177 participants completed the trial, including 43 in the HMB + RT group, 44 in the HMB group, 45 in the RT group, and 45 in the negative control group. At the 3-month follow-up, the body composition and grip strength of the HMB + RT group and RT group were significantly improved compared with those before operation. The HMB group had no significant change, while the measures in the negative control group significantly decreased. Postoperative BI and HSS did not reach pre-injury levels in any of the four groups, but postoperative VAS score was significantly improved. However, there was no significant difference in BI, HSS, or VAS among the four groups. CONCLUSION: RT, with or without HMB supplementation, can effectively improve body composition and grip strength in elderly patients with sarcopenia after hip replacement at short-term follow-up. Simultaneously, use of exclusive HMB supplementation alone may also help to prevent decreases in muscle mass and grip strength in these patients.

Cardioprotective effect of anisodamine against ischemia/reperfusion injury through the mitochondrial ATP-sensitive potassium channel.

Previous clinical studies have shown that anisodamine could improve no-reflow phenomenon and prevent reperfusion arrhythmias, but whether this protective effect is related to the antagonism of the M-type cholinergic receptor or other potential mechanisms is uncertain. The aim of the present study was to investigate the role of the mitochondrial ATP-sensitive potassium channel (mitoK ATP ) in cardioprotective effect of anisodamine against ischemia/reperfusion injury. Anisodamine and 5- hydroxydecanoic acid were used to explore the relationship between anisodamine and mitoK ATP . Using a Langendorff isolated heart ischemia/reperfusion injury model, hemodynamic parameters and reperfusion ventricular arrhythmia were evaluated; in addition, changes in myocardial infarct size, cTnI from coronary effluent and myocardial ultrastructure, as well as ATP, MDA and SOD in myocardial tissues, were detected. In the hypoxia/reoxygenation injury model of neonatal rat cardiomyocyte, cTnI release in the culture medium and levels of ATP, MDA and SOD in cardiomyocytes and mitochondrial membrane potential, were analyzed. Overall, anisodamine could significantly improve the hemodynamic indexes of isolated rat heart injured by ischemia/reperfusion, reduce the occurrence of ventricular reperfusion arrhythmia and myocardial infarction area, and improve the ultrastructural damage of myocardium and mitochondria. The in vitro results demonstrated that anisodamine could improve mitochondrial energy metabolism, reduce oxidative stress and stabilize mitochondrial membrane potential. The cardioprotective effects were significantly inhibited by 5-hydroxydecanoic acid. In conclusion, this study suggests that the opening of mitoK ATP could play an important role in the protective effect of anisodamine against myocardial ischemia/reperfusion injury.

ATP-Sensitive Potassium Channels Mediate the Cardioprotective Effect of Panax notoginseng Saponins against Myocardial Ischaemia-Reperfusion Injury and Inflammatory Reaction.

Inflammatory response during myocardial ischemia reperfusion injury (MIRI) is essential for cardiac healing, while excessive inflammation extends the infarction and promotes adverse cardiac remodeling. Understanding the mechanism of these uncontrolled inflammatory processes has a significant impact during the MIRI therapy. Here, we found a critical role of ATP-sensitive potassium channels (KATP) in the inflammatory response of MIRI and its potential mechanism and explored the effects of Panax Notoginseng Saponins (PNS) during this possess. Rats underwent 40 min ischemia by occlusion of the left anterior descending (LAD) coronary artery and 60 min of reperfusion. PNS was treated at the corresponding time point before operation; 5-hydroxydecanoate (5-HD) and glybenclamide (Gly) (or Nicorandil (Nic)) were used as pharmacological blocker (or nonselective opener) of KATP. Cardiac function and pathomorphology were evaluated and a set of molecular signaling experiments was tested. KATP current density was measured by patch-clamp. Results revealed that in MIRI, PNS pretreatment restored cardiac function, reduced infarct size, and ameliorated inflammation through KATP. However, inhibiting KATP by 5-HD and Gly significantly reversed the effects, including NLRP3 inflammasome and inflammatory mediators IL-6, MPO, TNF-α, and MCP-1. Moreover, PNS inhibited the phosphorylation and nuclear translocation of NF-κB in I/R myocardium when the KATP was activated. Importantly, PNS promoted the expression of subunits and activation of KATP. The study uncovered KATP served as a new potential mechanism during PNS modulating MIRI-induced inflammation and promoting injured heart recovery. The manipulation of KATP could be a potential therapeutic approach for MIRI and other inflammatory diseases.

Biological function and molecular properties of Pyrenaican SF-1 as biological macromolecule extracted from Daldinia pyrenaica.

Molecular properties and biological functions of Pyrenaican SF-1 as a novel biological macromolecule extracted from a fungal isolate were studied. The isolate was identified as Daldinia pyrenaica on the basis of 5.8S rDNA sequencing. Pyrenaican SF-1 was obtained from the culture filtrate of the fungal isolate. The partial characterization of biochemical structure of Pyrenaican SF-1 was conducted. The fungal extract was also tested for the treatment of AGS, MDA and HeLa cell lines to assess cells proliferation, cells cycle and apoptosis. Furthermore, Pyrenaican SF-1 extract was tested for its antibacterial and antioxidant activity. Initial chemical analysis revealed that Pyrenaican SF-1 extract was composed of various monosaccharides such as d-glucose, D- mannitol, D-arabinose and ß-D-ribopyranose. In vitro study indicated that Pyrenaican SF-1 could effectively elevate percentage of apoptosis and necrosis of cancer cells and block cell cycle phase of the control group. The fungal extract could inhibit proliferation of Hela and MDA cell up to 67% and 56%, respectively. Moreover, Pyrenaican SF-1 represented a strong antioxidant activity compared to that one obtained from vitamin C. On the other hand, Pyrenaican SF-1 exhibited growth inhibitory effects against different Gram-negative and Gram-positive bacterial strains. Pyrenaican SF-1 can be considered as a bioactive macromolecule with promising application in pharmaceutical and medical sectors.

Diazoxide affects mitochondrial bioenergetics by the opening of mKATP channel on submicromolar scale.

BACKGROUND: Cytoprotection afforded by mitochondrial ATP-sensitive K+-channel (mKATP-channel) opener diazoxide (DZ) largely depends on the activation of potassium cycle with eventual modulation of mitochondrial functions and ROS production. However, generally these effects were studied in the presence of Mg∙ATP known to block K+ transport. Thus, the purpose of our work was the estimation of DZ effects on K+ transport, K+ cycle and ROS production in rat liver mitochondria in the absence of Mg∙ATP. RESULTS: Without Mg·ATP, full activation of native mKATP-channel, accompanied by the increase in ATP-insensitive K+ uptake, activation of K+-cycle and respiratory uncoupling, was reached at ≤0.5 µM of DZ,. Higher diazoxide concentrations augmented ATP-insensitive K+ uptake, but not mKATP-channel activity. mKATP-channel was blocked by Mg·ATP, reactivated by DZ, and repeatedly blocked by mKATP-channel blockers glibenclamide and 5-hydroxydecanoate, whereas ATP-insensitive potassium transport was blocked by Mg2+ and was not restored by DZ. High sensitivity of potassium transport to DZ in native mitochondria resulted in suppression of mitochondrial ROS production caused by the activation of K+-cycle on sub-micromolar scale. Based on the oxygen consumption study, the share of mKATP-channel in respiratory uncoupling by DZ was found. CONCLUSIONS: The study of mKATP-channel activation by diazoxide in the absence of MgATP discloses novel, not described earlier, aspects of mKATP-channel interaction with this drug. High sensitivity of mKATP-channel to DZ results in the modulation of mitochondrial functions and ROS production by DZ on sub-micromolar concentration scale. Our experiments led us to the hypothesis that under the conditions marked by ATP deficiency affinity of mKATP-channel to DZ can increase, which might contribute to the high effectiveness of this drug in cardio- and neuroprotection.

New Hydroxydecanoic Acid Derivatives Produced by an Dndophytic Yeast Aureobasidium pullulans AJF1 from Flowers of Aconitum carmichaeli.

Endophytes have been recognized as a source for structurally novel and biologically active secondary metabolites. Among the host plants for endophytes, some medicinal plants that produce pharmaceuticals have been reported to carry endophytes, which could also produce bioactive secondary metabolites. In this study, the medicinal plant Aconitum carmichaeli was selected as a potential source for endophytes. An endophytic microorganism, Aureobasidium pullulans AJF1, harbored in the flower of Aconitum carmichaeli, was cultured on a large scale and extracted with an organic solvent. Extensive chemical investigation of the extracts resulted in isolation of three lipid type compounds (1-3), which were identified to be (3R,5R)-3,5-dihydroxydecanoic acid (1), (3R,5R)-3-(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-5-hydroxydecanoic acid (2), and (3R,5R)-3-(((3R,5R)-5-(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-3-hydroxydecanoyl)oxy)-5-hydroxydecanoic acid (3) by chemical methods in combination with spectral analysis. Compounds 2 and 3 had new structures. Absolute configurations of the isolated compounds (1-3) were established using modified Mosher's method together with analysis of NMR data for their acetonide derivatives. All the isolates (1-3) were evaluated for antibiotic activities against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and their cytotoxicities against MCF-7 cancer cells. Unfortunately, they showed low antibiotic activities and cytotoxic activities.

Uridine as a protector against hypoxia-induced lung injury.

The effect of the activation of the mitochondrial ATP-dependent potassium channel (mitoKATP) on the ultrastructure of rat lung in acute hypoxic hypoxia (7% of oxygen in nitrogen, exposure 30 min) was studied. It was shown that uridine, a precursor of the mitoKATP activator UDP, exerted a protective effect against hypoxic damage to the lung. The administration of uridine to animals prior to hypoxia decreased the number of mitochondria with altered ultrastructure and prevented the hypoxia-induced mitochondrial swelling. Uridine also protected the epithelial, interstitial and endothelial layers of the air-blood barrier from the hypoxia-induced hyperhydration. The protective action of uridine against hypoxia-induced lung injury was eliminated by the selective blocker of mitoKATP 5-hydroxydecanoate. These data suggest that one of the mechanisms of the positive effect of uridine is related to the activation of the mitoKATP channel, which, according to the literature and our data, is involved in the protection of tissues from hypoxia and leads to adaptation to it. A possible role of uridine in the maintenance of the mitochondrial structure upon hypoxia-induced lung injury and the optimization of oxygen supply of the organism is discussed.

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.

PI3K-AKT Pathway Protects Cardiomyocytes Against Hypoxia-Induced Apoptosis by MitoKATP-Mediated Mitochondrial Translocation of pAKT.

BACKGROUND/AIMS: The phosphatidylinositol-3-kinase -AKT (PI3K-AKT) is an important intracellular signal pathway in regulating cell proliferation, differentiation and apoptosis. In previous studies, we've demonstrated that PI3K-AKT pathway protects cardiomyocytes from ischemic and hypoxic apoptosis through mitochondrial function. However, the molecular mechanisms underlying hypoxia-induced cardiomyocyte apoptosis via PI3K-AKT pathway remain ill-defined. Here, we addressed this question. METHODS: Cardiomyocytes were exposed to hypoxia, with/without different inhibitors and then protein levels were assessed by Western blotting. RESULTS: We found that the PI3K-AKT pathway was activated in cardiomyocytes that were exposed to hypoxia. Moreover, the phospho-AKT (pAKT) translocated from cytosol to mitochondria via mitochondrial adenosine triphosphate-dependent potassium (mitoKATP), leading to an increase in cytochrome c oxidase (CcO) activity to suppress apoptosis. On the other hand, the mitoKATP specific blocker, 5-hydroxydecanote (5-HD), or suppression of CcO using siRNA, inhibited the pAKT mitochondrial translocation to maintain the CcO activity, resulting in mitochondrial dysfunction and cellular apoptosis induced by hypoxia. CONCLUSION: These findings suggest that the anti-apoptotic effect of the PI3K-AKT pathway through pAKT translocation to mitochondrial via mitoKATP may be conducted through modification of CcO activity.

Involvement of brain natriuretic peptide signaling pathway in the cardioprotective action of sitagliptin.

BACKGROUND: The current study is focusing on the role of brain natriuretic peptide (BNP), a substrate of dipeptidyl peptidase-4 (DPP-4) enzyme, and its signaling survival pathway in the cardioprotective mechanism of sitagliptin, a DPP-4 inhibitor. METHODS: Male Wistar rats were randomized into 7 groups, sham, I/R, KT-5823 (selective protein kinase (PK) G inhibitor), 5-HD (selective mito-KATP channel blocker), sitagliptin (300mg/kg, po), sitagliptin+KT-5823, and sitagliptin+5-HD. Sitagliptin was administered for 3 days prior to induction of coronary I/R, while either KT-5823 or 5-HD was administered intravenously 5min before coronary ligation. RESULTS: Pretreatment with sitagliptin provided significant protection against I/R injury as manifested by decreasing, percentage of infarct size, suppressing the elevated ST segment, reducing the increased cardiac enzymes, as well as DPP-4 activity and elevating both heart rate (HR) and left ventricular developed pressure (LVDP). However, the addition of either blocker to sitagliptin regimen reversed partly its cardioprotective effects. Although I/R increased BNP content, it unexpectedly decreased that of cGMP; nevertheless, sitagliptin elevated both parameters, an effect that was not affected by the use of the two blockers. On the molecular level, sitagliptin decreased caspase-3 activity and downregulated the mRNA levels of BNP, Bax, and Cyp D, while upregulated that of Bcl2. The use of either KT-5823 or 5-HD with sitagliptin hindered its effect on the molecular markers tested. CONCLUSIONS: The results of the present study suggest that the cardioprotective effect of sitagliptin is mediated partly, but not solely, through the BNP/cGMP/PKG survival signaling pathway.

DNA Transfection to Mesenchymal Stem Cells Using a Novel Type of Pseudodendrimer Based on 2,2-Bis(hydroxymethyl)propionic Acid.

In the search for effective vehicles to carry genetic material into cells, we present here new pseudodendrimers that consist of a hyperbranched polyester core surrounded by amino-terminated 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) dendrons. The pseudodendrimers are readily synthesized from commercial hyperbranched bis-MPA polyesters of the second, third, and fourth generations and third-generation bis-MPA dendrons, bearing eight peripheral glycine moieties, coupled by the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). This approach provides globular macromolecular structures bearing 128, 256, and 512 terminal amino groups, and these can complex pDNA. The toxicity of the three pseudodendrimers was studied on two cell lines, mesenchymal stem cells, and HeLa, and it was demonstrated that these compounds do not affect negatively cell viability up to 72 h. The complexation with DNA was investigated in terms of N-to-P ratio and dendriplex stability. The three generations were found to promote internalizing of pDNA into mesenchymal stem cells (MSCs), and their transfection capacity was compared with two nonviral commercial transfection agents, Lipofectamine and TransIT-X2. The highest generations were able to transfect these cells at levels comparable to both commercial reagents.

The Opening of ATP-Sensitive K+ Channels Protects H9c2 Cardiac Cells Against the High Glucose-Induced Injury and Inflammation by Inhibiting the ROS-TLR4-Necroptosis Pathway.

BACKGROUND/AIMS: Hyperglycemia activates multiple signaling molecules, including reactive oxygen species (ROS), toll-like receptor 4 (TLR4), receptor-interacting protein 3 (RIP3, a kinase promoting necroptosis), which mediate hyperglycemia-induced cardiac injury. This study explored whether inhibition of ROS-TLR4-necroptosis pathway contributed to the protection of ATP-sensitive K+ (KATP) channel opening against high glucose-induced cardiac injury and inflammation. METHODS: H9c2 cardiac cells were treated with 35 mM glucose (HG) to establish a model of HG-induced insults. The expression of RIP3 and TLR4 were tested by western blot. Generation of ROS, cell viability, mitochondrial membrane potential (MMP) and secretion of inflammatory cytokines were measured as injury indexes. RESULTS: HG increased the expression of TLR4 and RIP3. Necrostatin-1 (Nec-1, an inhibitor of necroptosis) or TAK-242 (an inhibitor of TLR4) co-treatment attenuated HG-induced up-regulation of RIP3. Diazoxide (DZ, a mitochondrial KATP channel opener) or pinacidil (Pin, a non-selective KATP channel opener) or N-acetyl-L-cysteine (NAC, a ROS scavenger) pre-treatment blocked the up-regulation of TLR4 and RIP3. Furthermore, pre-treatment with DZ or Pin or NAC, or co-treatment with TAK-242 or Nec-1 attenuated HG-induced a decrease in cell viability, and increases in ROS generation, MMP loss and inflammatory cytokines secretion. However, 5-hydroxy decanoic acid (5-HD, a mitochondrial KATP channel blocker) or glibenclamide (Gli, a non-selective KATP channel blocker) pre-treatment did not aggravate HG-induced injury and inflammation. CONCLUSION: KATP channel opening protects H9c2 cells against HG-induced injury and inflammation by inhibiting ROS-TLR4-necroptosis pathway.

Effect of dexmedetomidine on cerebral ischemia-reperfusion rats by activating mitochondrial ATP-sensitive potassium channel.

The aim of the study reported here was to evaluate whether the mitochondrial ATP-sensitive potassium (mitoKATP) channel could participate in the effect of dexmedetomidine on cerebral ischemia-reperfusion (I/R) rats. Forty rats were randomly assigned into 5 groups: sham operation (S) group; cerebral I/R group; dexmedetomidine (D) group; 5-hydroxydecanoate (5-HD) group; 5-HD + D group. The cerebral I/R were produced by 2 h right middle cerebral artery occlusion followed by 24 h reperfusion. Dexmedetomidine (50µg/kg) was injected intraperitoneally before ischemia and after the onset of reperfusion. 5-HD (30 mg/kg) was injected intraperitoneally at 1 h before ischemia. The neurological deficit score (NDS) and the levels of super oxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) were evaluated. Compared to group S, NDS and the levels of MDA, MPO, IL-6 and TNF-α were significantly higher, and SOD levels were significantly lower in the other groups (P < 0.05). Compared to group I/R,NDS and the levels of MDA, MPO, IL-6 and TNF-α were significantly lower, and SOD level was significantly higher in group D (P < 0.05). Compared to group D, NDS and the levels of MDA, MPO, IL-6 and TNF-α were significantly higher, and SOD level was significantly lower in group5-HD + D (P < 0.05). The activation of the mitoKATP channel could contribute to the protective effect of dexmedetomidine on rats induced by focal cerebral ischemia-reperfusion injury.

Lidocaine relaxation in isolated rat aortic rings is enhanced by endothelial removal: possible role of Kv, KATP channels and A2a receptor crosstalk.

BACKGROUND: Lidocaine is an approved local anesthetic and Class 1B antiarrhythmic with a number of ancillary properties. Our aim was to investigate lidocaine's vasoreactivity properties in intact versus denuded rat thoracic aortic rings, and the effect of inhibitors of nitric oxide (NO), prostenoids, voltage-dependent Kv and KATP channels, membrane Na+/K+ pump, and A2a and A2b receptors. METHODS: Aortic rings were harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs-Henseleit solution, pH 7.4, 37 °C. The rings were pre-contracted sub-maximally with 0.3 µM norepinephrine (NE), and the effect of increasing lidocaine concentrations was examined. Rings were tested for viability after each experiment with maximally dilating 100 µM papaverine. The drugs 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate, ouabain, 8-(3-chlorostyryl) caffeine and PSB-0788 were examined. RESULTS: All drugs tested had no significant effect on basal tension. Lidocaine relaxation in intact rings was biphasic between 1 and 10 µM (Phase 1) and 10 and 1000 µM (Phase 2). Mechanical removal of the endothelium resulted in further relaxation, and at lower concentrations ring sensitivity (% relaxation per µM lidocaine) significantly increased 3.5 times compared to intact rings. The relaxing factor(s) responsible for enhancing ring relaxation did not appear to be NO- or prostacyclin-dependent, as L-NAME and indomethacin had little or no effect on intact ring relaxation. In denuded rings, lidocaine relaxation was completely abolished by Kv channel inhibition and significantly reduced by antagonists of the MitoKATP channel, and to a lesser extent the SarcKATP channel. Curiously, A2a subtype receptor antagonism significantly inhibited lidocaine relaxation above 100 µM, but not the A2b receptor. CONCLUSIONS: We show that lidocaine relaxation in rat thoracic aorta was biphasic and significantly enhanced by endothelial removal, which did not appear to be NO or prostacyclin dependent. The unknown factor(s) responsible for enhanced relaxation was significantly reduced by Kv inhibition, 5-HD inhibition, and A2a subtype inhibition indicating a potential role for crosstalk in lidocaine's vasoreactivity.

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 ß-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.

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.

The Effects of Fentanyl on Hepatic Mitochondrial Function.

BACKGROUND: Remifentanil interferes with hepatic mitochondrial function. The aim of the present study was to evaluate whether hepatic mitochondrial function is affected by fentanyl, a more widely used opioid than remifentanil. METHODS: Human hepatoma HepG2 cells were exposed to fentanyl or pretreated with naloxone (an opioid receptor antagonist) or 5-hydroxydecanoate (5-HD, an inhibitor of mitochondrial adenosine triphosphate (ATP)-sensitive potassium [mitoKATP] channels), followed by incubation with fentanyl. Mitochondrial function and metabolism were then analyzed. RESULTS: Fentanyl marginally reduced maximal mitochondrial complex-specific respiration rates using exogenous substrates (decrease in medians: 11%-18%; P = 0.003-0.001) but did not affect basal cellular respiration rates (P = 0.834). The effect on stimulated respiration was prevented by preincubation with naloxone or 5-HD. Fentanyl reduced cellular ATP content in a dose-dependent manner (P < 0.001), an effect that was not significantly prevented by 5-HD and not explained by increased total ATPase concentration. However, in vitro ATPase activity of recombinant human permeability glycoprotein (an ATP-dependent drug efflux transporter) was significantly stimulated by fentanyl (P = 0.004). CONCLUSIONS: Our data suggest that fentanyl reduces stimulated mitochondrial respiration of cultured human hepatocytes by a mechanism that is blocked by a mitoKATP channel antagonist. Increased energy requirements for fentanyl efflux transport may offer an explanation for the substantial decrease in cellular ATP concentration.

Myocardial triggers involved in activation of remote ischaemic preconditioning.

NEW FINDINGS: What is the central question of this study? Ischaemia-reperfusion of peripheral tissues protects the heart from subsequent myocardial ischaemia-reperfusion injury, a phenomenon referred to as remote ischaemic preconditioning (rIPC). This study evaluated the possible myocardial triggers of rIPC. What is the main finding and its importance? Remote ischaemic preconditioning reduces infarct size through a vagal pathway and a mechanism involving phosphorylation of Akt and endothelial nitric oxide synthase, opening of mitochondrial ATP-dependent K(+) channels and an increase in mitochondrial H2 O2 production. All these phenomena occur before the myocardial ischaemia; hence, they could act as 'triggers' of rIPC. It has been proposed that remote ischaemic preconditioning (rIPC) activates a parasympathetic neural pathway. However, the myocardial intracellular mechanism of rIPC remains unclear. Here, we characterized some of the intracellular signals participating as rIPC triggers. Isolated rat hearts were subjected to 30 min of global ischaemia and 120 min of reperfusion (Non-rIPC group). In a second group, before the isolation of the heart, an rIPC protocol (three cycles of hindlimb ischaemia-reperfusion) was performed. The infarct size was measured with tetrazolium staining. Expression/phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) and mitochondrial H2 O2 production were evaluated at the end of the rIPC protocol, before myocardial ischaemia-reperfusion. The rIPC significantly decreased the infarct size and induced Akt and eNOS phosphorylation. The protective effect on infarct size was abolished by cervical vagal section, l-NAME (an NO synthesis inhibitor) and 5-hydroxydecanoate (a mitochondrial ATP-dependent K(+) channel blocker). Mitochondrial production of H2 O2 was increased by rIPC, whereas it was abolished by cervical vagal section, l-NAME and 5-hydroxydecanoate. We conclude that rIPC activates a parasympathetic vagal pathway and a mechanism involving the phosphorylation of Akt and eNOS, the opening of mitochondrial ATP-dependent K(+) channels and the release of H2 O2 by the mitochondria. All these phenomena occur before myocardial ischaemia and could act as triggers of rIPC.

Sevoflurane postconditioning affects post-ischaemic myocardial mitochondrial ATP-sensitive potassium channel function and apoptosis in ageing rats.

This study investigated the effect of sevoflurane postconditioning on post-ischaemic cardiac function, infarct size, myocardial mitochondrial ATP-sensitive potassium channel (mitoKATP) function and apoptosis in ageing rats to determine the possible mechanism underlying the cardioprotective property of sevoflurane. Ageing rat hearts were isolated and attached to a Langendorff apparatus. The hearts were then exposed or not to sevoflurane postconditioning in the presence or absence of 100 µmol/L 5-hydroxydecanoate (5-HD), a selective mitoKATP inhibitor. The infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Mitochondrial morphology was observed by electron microscopy and scored using FlaMeng semiquantitative analysis. In addition, the expression levels of Bax, Bcl-2, and cytochrome-C (Cyt-C) were determined by Western blot analysis at the end of reperfusion. Sevoflurane postconditioning increased coronary flow, improved functional recovery, reduced Bax/Bcl-2 and Cyt-C phosphorylation levels, and decreased mitochondrial lesion severity and the extent of apoptosis. The protective effects of sevoflurane postconditioning were prevented by the mitoKATP inhibitor 5-HD. Sevoflurane postconditioning significantly protected the function of ageing hearts that were subjected to ischaemia and reperfusion, and these protective effects were mediated by mitoKATP opening.

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

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