Levosimendan Relaxes Pulmonary Arteries and Veins in Precision-Cut Lung Slices - The Role of KATP-Channels,cAMP and cGMP

Introduction

Levosimendan is approved for left heart failure and is also used in right heart failure to reduce right ventricular afterload. Despite the fact that pulmonary arteries (PAs) and pulmonary veins (PVs) contribute to cardiac load, their responses to levosimendan are largely unknown.

Materials and Methods

Levosimendan-induced vasorelaxation of PAs and PVs was studied in precision-cut lung slices from guinea pigs by videomicroscopy; baseline luminal area was defined as 100%. Intracellular cAMP- and cGMP-levels were measured by ELISA and NO end products were determined by the Griess reaction.

Results

Levosimendan relaxed control PVs (116%) and those pre-constricted with an endothelin_A-receptor agonist (119%). PAs were only relaxed if pre-constricted (115%). Inhibition of K_ATP-channels (glibenclamide), adenyl cyclase (SQ 22536) and protein kinase G (KT 5823) largely attenuated the levosimendan-induced relaxation in control PVs, as well as in pre-constricted PAs and PVs. Inhibition of BK_Ca ²⁺-channels (iberiotoxin) and K_v-channels (4-aminopyridine) only contributed to the relaxant effect of levosimendan in pre-constricted PAs. In both PAs and PVs, levosimendan increased intracellular cAMP- and cGMP-levels, whereas NO end products remained unchanged. Notably, basal NO-levels were higher in PVs. The K_ATP-channel activator levcromakalim relaxed PAs dependent on cAMP/PKA/PKG and increased cAMP-levels in PAs.

Discussion

Levosimendan initiates complex and divergent signaling pathways in PAs and PVs. Levosimendan relaxes PAs and PVs primarily via K_ATP-channels and cAMP/cGMP; in PAs, BK_Ca ²⁺- and K_v-channels are also involved. Our findings with levcromakalim do further suggest that in PAs the activation of K_ATP-channels leads to the production of cAMP/PKA/PKG. In conclusion, these results suggest that levosimendan might reduce right ventricular afterload by relaxation of PAs as well as pulmonary hydrostatic pressure and pulmonary edema by relaxation of PVs.     

Mitochondrial ATP-sensitive K channels as redox signals to liver mitochondria in response to hypertriglyceridemia

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK_ATP). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. Isolated HTG liver mitochondria presented lower rates of H₂O₂ production, which were reversed by mitoK_ATP antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoK_ATP activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote mitoK_ATP activation. The mild uncoupling mediated by mitoK_ATP increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK_ATP as a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess.     

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

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

The role of the ATP-sensitive potassium channel in the activation of the K+ cycle in rat liver mitochondria

It is known that the mitochondrial ATP-sensitive potassium channel (mitoK_ATP) plays a key role in protecting myocardium during ischemia. We have suggested that the mechanism of this protection is associated with the potassium cycle in mitochondria. In this paper, for the first time, a direct proof was obtained of the existence of a cycle of potassium ions in rat liver mitochondria that are associated with the functioning of mitoK_ATP. Activation of the cycle was recorded by optical density changes of mitochondrial suspension in the form of two or three swelling-contraction waves of the organelles. Using activators and inhibitors of mitoK_ATP we showed that a significant role in the potassium cycle belongs to the channel. It was found that in vitro sildenafil has a direct effect on mitoK_ATP, being its activator. The results obtained indicate that the cardioprotective effect of sildenafil observed previously is associated with the activation of mitoK_ATP. In order to study the structure and volume changes of mitochondria in various stages of the cycle in the presence of potassium channel modulators, the electron microscopy studies of mitochondria preparations were carried out. A correlation between the optical density decrease of mitochondrial suspension and the swelling of mitochondria was revealed. The data obtained in this study suggest participation of mitoK_ATP in the protection of tissues from hypoxic damage.     

Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect

In the present study, we describe the existence of mitochondrial ATP-dependent K⁺ channel (mitoK_ATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoK_ATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoK_ATP in insect mitochondria. MitoK_ATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects.     

Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channels Prevent Oxidative Stress,Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) protects the heart against ischemic damage. This study examined the effects of mitoK_ATP and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK_ATP opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK_ATP opening in isolated rat heart mitochondria slightly decreased Ca²⁺ uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca²⁺. In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK_ATP prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca²⁺ uptake, inhibiting mitochondrial permeability transition.     

Mitochondrial energy metabolism and redox responses to hypertriglyceridemia

In this work we review recent findings that explain how mitochondrial bioenergetic functions and redox state respond to a hyperlipidemic in vivo environment and may contribute to the maintenance of a normal metabolic phenotype. The experimental model utilized to evidence these adaptive mechanisms is especially useful for these studies since it exhibits genetic hypertriglyceridemia and avoids complications introduced by high fat diets. Liver from hypertrigliceridemic (HTG) mice have a greater content of glycerolipids together with increased mitochondrial free fatty acid oxidation. HTG liver mitochondria have a higher resting respiration rate but normal oxidative phosphorylation efficiency. This is achieved by higher activity of the mitochondrial potassium channel sensitive to ATP (mitoK_ATP). The mild uncoupling mediated by mitoK_ATP accelerates respiration rates and reduces reactive oxygen species generation. Although this response is not sufficient to inhibit lipid induced extra-mitochondrial oxidative stress in whole liver cells it avoids amplification of this redox imbalance. Furthermore, higher mitoK_ATP activity increases liver, brain and whole body metabolic rates. These mitochondrial adaptations may explain why these HTG mice do not develop insulin resistance and obesity even under a severe hyperlipidemic state. On the contrary, when long term high fat diets are employed, insulin resistance, fatty liver and obesity develop and mitochondrial adaptations are inefficient to counteract energy and redox imbalances.     

Short-term synaptic plasticity in the nociceptive thalamic-anterior cingulate pathway

Background

ATP-sensitive potassium (K_ATP) channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of K_ATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both K_ATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of K_ATP channels in control and axotomized DRG neurons.

Results

Cell-attached and cell-free recordings of K_ATP currents in large DRG neurons from control rats (sham surgery, SS) revealed activation of K_ATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i. This NO-induced K_ATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of K_ATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of K_ATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of K_ATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates K_ATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced K_ATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit.

Conclusion

NO activates K_ATP channels in large DRG neurons via direct S-nitrosylation of cysteine residues in the SUR1 subunit. The capacity of NO to activate K_ATP channels via this mechanism remains intact even after spinal nerve ligation, thus providing opportunities for selective pharmacological enhancement of K_ATP current even after decrease of this current by painful-like nerve injury.     

MitoK<Subscript>ATP</Subscript> activity in healthy and ischemic hearts

In addition to their role in energy transduction, mitochondria play important non-canonical roles in cell pathophysiology, several of which utilize the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP). In the normal heart, mitoK_ATP regulates energy transfer through its regulation of intermembrane space volume and is accordingly essential for the inotropic response during periods of high workload. In the ischemic heart, mitoK_ATP is the point of convergence of protective signaling pathways and mediates inhibition of the mitochondrial permeability transition, and thus necrosis. In this review, we outline the experimental evidence that support these roles for mitoK_ATP in health and disease, as well as our hypothesis for the mechanism by which complex cardioprotective signals that originate at plasma membrane receptors traverse the cytosol to reach mitochondria and activate mitoK_ATP.     

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

Background

5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5?HD + CoA + ATP → 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K_ATP (sarcK_ATP) channel activity.

Methods

Single sarcK_ATP channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK_ATP channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A.

Results

5-HD inhibited (IC₅₀ ∼ 30 μM) K_ATP channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 μM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 μM coenzyme A (CoA) inhibited sarcK_ATP channels. Using giant inside-out patches, which are weakly sensitive to “contaminating” CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK_ATP channel activation by either metabolic inhibition or rilmakalim.

General significance

SarcK_ATP channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at “directly” inhibiting sarcK_ATP and mitoK_ATP channels. However, in intact cells, 5-HD fails to inhibit sarcK_ATP channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.     

One potential new target of cancer therapy,mitochondrial ATP-sensitive potassium channels

Plenty of evidence strongly suggests that mitoK_ATP channels played an important role in cellular death and survival in many tissues, but it is still unknown whether mitoK_ATP channels can treat cancer via inducing cellular apoptosis.     

Opening of Astrocytic Mitochondrial ATP-Sensitive Potassium Channels Upregulates Electrical Coupling between Hippocampal Astrocytes in Rat Brain Slices

Astrocytes form extensive intercellular networks through gap junctions to support both biochemical and electrical coupling between adjacent cells. ATP-sensitive K⁺ (K_ATP) channels couple cell metabolic state to membrane excitability and are enriched in glial cells. Activation of astrocytic mitochondrial K_ATP (mitoK_ATP) channel regulates certain astrocytic functions. However, less is known about its impact on electrical coupling between directly coupled astrocytes ex vivo. By using dual patch clamp recording, we found that activation of mitoK_ATP channel increased the electrical coupling ratio in brain slices. The electrical coupling ratio started to increase 3 min after exposure to Diazoxide, a mitoK_ATP channel activator, peaked at 5 min, and maintained its level with little adaptation until the end of the 10-min treatment. Blocking the mitoK_ATP channel with 5-hydroxydecanoate, inhibited electrical coupling immediately, and by 10-min, the ratio dropped by 71% of the initial level. Activation of mitoK_ATP channel also decreased the latency time of the transjunctional currents by 50%. The increase in the coupling ratio resulting from the activation of the mitoK_ATP channel in a single astrocyte was further potentiated by the concurrent inhibiting of the channel on the recipient astrocyte. Furthermore, Meclofenamic acid, a gap-junction inhibitor which completely blocked the tracer coupling, hardly reversed the impact of mitoK_ATP channel''s activation on electrical coupling (by 7%). The level of mitochondrial Connexin43, a gap junctional subunit, significantly increased by 70% in astrocytes after 10-min Diazoxide treatment. Phospho-ERK signals were detected in Connexin43 immunoprecipitates in the Diazoxide-treated astrocytes, but not untreated control samples. Finally, inhibiting ERK could attenuate the effects of Diazoxide on electrical coupling by 61%. These findings demonstrate that activation of astrocytic mitoK_ATP channel upregulates electrical coupling between hippocampal astrocytes ex vivo. In addition, this effect is mainly via up-regulation of the Connexin43-constituted gap junction coupling by an ERK-dependent mechanism in the mitochondria.     

A role of opening of mitochondrial ATP-sensitive potassium channels in the infarct size-limiting effect of ischemic preconditioning via activation of protein kinase C in the canine heart

The opening of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels triggers or mediates the infarct size (IS)-limiting effect of ischemic preconditioning (IP). Because ecto-5′-nucleotidase related to IP is activated by PKC, we tested whether the opening of mitoK_ATP channels activates PKC and contributes to either activation of ecto-5′-nucleotidase or IS-limiting effect. In dogs, IP procedure decreased IS and activated ecto-5′-nucleotidase, both of which were mimicked by transient exposure to either cromakalim or diazoxide, and these effects were blunted by either GF109203X (a PKC inhibitor) or 5-hydroxydecanoate (a mitoK_ATP channel blocker), but not by HMR-1098 (a surface sarcolenmal K_ATP channel blocker). Either cromakalim or diazoxide activated both PKC and ecto-5′-nucleotidase, which was blunted by either GF109203X or 5-hydroxydecanoate, but not by HMR-1098. We concluded that the opening of mitoK_ATP channels contributes to either activation of ecto-5′-nucleotidase or the infarct size-limiting effect via activation of PKC in canine hearts.     

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

The role of mitochondrial K_ATP (mitoK_ATP) channels on muscle fatigue was assessed in adult mouse skeletal muscle bundles. Muscle fatigue was produced by eliciting short repetitive tetani. Isometric tension and the rate of production of reactive oxygen species (ROS) were measured at room temperature (20-22 °C) using a force transducer and the fluorescent indicator CM-H₂DCFDA. We found that opening mitoK_ATP channels with diazoxide (100 μM) significantly reduced muscle fatigue. Fatigue tension was 34% higher in diazoxide-treated fibers relative to controls. This effect was blocked by the mitoK_ATP channel blocker 5-hydroxydecanoate (5-HD), by the protein kinase C (PKC) inhibitor chelerythrine, and by the nitric oxide (NO) synthase inhibitor N^G-nitro-l-arginine methyl ester hydrochloride (l-NAME) but was not accompanied by a change in the rate of ROS production during fatigue. A physiological role of mitoK_ATP channels on muscle fatigue is proposed.     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.     

The role of AKT1 and autophagy in the protective effect of hydrogen sulphide against hepatic ischemia/reperfusion injury in mice

Hydrogen sulphide (H₂S) exerts a protective effect in hepatic ischemia-reperfusion (I/R) injury. However, the exact mechanism of H₂S action remains largely unknown. This study was designed to investigate the role of the PtdIns3K-AKT1 pathways and autophagy in the protective effect of H₂S against hepatic I/R injury. Primary cultured mouse hepatocytes and livers with or without NaHS (a donor of H₂S) preconditioning were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. In certain groups, they were also pretreated with LY294002 (AKT1-specific inhibitor), 3-methyladenine (3MA, autophagy inhibitor) or rapamycin (autophagy enhancer), alone or simultaneously. Cell viability, expression of P-AKT1, T-AKT1, LC3 and BECN1 were examined. The severity of liver injury was measured by the levels of serum aminotransferase and inflammatory cytokine, apoptosis and histological examination. GFP-LC3 redistribution and transmission electron microscopy were used to test the activity of autophagy. H₂S preconditioning activated PtdIns3K-AKT1 signaling in hepatocytes. LY294002 could abolish the AKT1 activation and attenuate the protective effect of H₂S on hepatocytes A/R and hepatic I/R injuries. H₂S suppressed hepatic autophagy in vitro and in vivo. Further reducing autophagy by 3MA also diminished the protective effect of H₂S, while rapamycin could reverse the autophagy inhibitory effect and enhance the protective effect of H₂S against hepatocytes A/R and hepatic I/R injuries, consequently. Taken together, H₂S protects against hepatocytic A/R and hepatic I/R injuries, at least in part, through AKT1 activation but not autophagy. An autophagy agonist could be applied to potentiate this hepatoprotective effect by reversing the autophagy inhibition of H₂S.     

Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4

Background

Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis.

Objectives

In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.

Methods

We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays.

Results

After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.

Conclusions

Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.     

Pharmacological and ischemic preconditioning of the human myocardium: mitoKATP channels are upstream and p38MAPK is downstream of PKC

Background  

These studies investigate the role of mitoK_ATP channels, protein kinase C (PKC) and Mitogen activated protein kinase (p38MAPK) on the cardioprotection of ischemic (IP) and pharmacological preconditioning (PP) of the human myocardium and their sequence of activation.     

Determination of the antioxidant properties of activators of mitochondrial ATP-dependent potassium channels with the Amplex Red fluorescent indicator

The effect of adaptogens-antihypoxants that participate in the activation of mitochondrial ATP-dependent potassium channels (mitoK_ATP) at the oxidation of the Amplex Red (AR) fluorescent indicator in a peroxidase system was tested. It was shown that Extralife, Hypoxen, taurine, and synthetic antioxidant ionol can be arranged in the following row, according to the fluorescence inhibition activity: Extralife > Hypoxen > ionol > taurine; their effect was shown to be concentration-dependent. The calculated K _i value of fluorescence indicators demonstrate fast and slow phases of inhibition of the AR oxidation by Extralife and Hypoxen. The fast phase occurs in the presence of microdoses (0.05–3 μg/mL) of adaptogens and is related to the competition for H₂O₂, which is in agreement with our previous data on the mitoK_ATP activation by doses of adaptogens related to the H₂O₂ consumption. The slow phase is characteristic of high adaptogen and ionol concentrations and is related to the competition for phenoxyl radicals of resorufin formed during AR oxidation. The obtained results allow one to suggest the application of a highly sensitive model peroxidase system with AR for the preliminary testing of compounds activating mitoK_ATP channels.