Upregulation of ATP-Sensitive Potassium Channels as the Potential Mechanism of Cardioprotection and Vasorelaxation Under the Action of Pyridoxal-5-Phosphate in Old Rats.

Background: The aging process is accompanied by the weakening of the protective systems of the organism, in particular by the decrease in the expression of ATP-sensitive potassium (KATP) channels and in the synthesis of H2S. The aim of our work was to investigate the role of KATP channels in the cardioprotection induced by pyridoxal-5-phosphate (PLP) in aging. Methods: Experiments were performed on adult and old (aged 24 months) male Wistar rats, which were divided into 3 groups: adults, old, and old PLP-treated rats. PLP was administered orally once a day for 14 days at a dose of 0.7 mg/kg. The levels of mRNA expression of subunits KATP channels were determined by reverse transcription and real-time polymerase chain reaction analysis. Protein expression levels were determined by the Western blot. Cardiac tissue morphology was determined using transverse 6 µm deparaffinized sections stained with picrosirius red staining. Vasorelaxation responses of isolated aortic rings and the function of Langendorff-perfused isolated hearts during ischemia-reperfusion, H2S levels, and markers of oxidative stress were also studied. Results: Administration of PLP to old rats reduces cardiac fibrosis and improves cardiac function during ischemia-reperfusion and vasorelaxation responses to KATP channels opening. At the same time, there was a significant increase in mRNA and protein expression of SUR2 and Kir6.1 subunits of KATP channels, H2S production, and reduced markers of oxidative stress. The specific KATP channel inhibitor-glibenclamide prevented the enhancement of vasodilator responses and anti-ischemic protection in PLP-treated animals. Conclusions: We suggest that this potential therapeutic effect of PLP in old animals may be a result of increased expression of KATP channels and H2S production.

Glutathione Upregulates the Expression of KATP Channels and Vasorelaxation Responses and Inhibits mPTP Opening and Oxidative Stress in the Heart Mitochondria of Old Rats.

Background: In the present work, we investigated the effect of exogenous glutathione in old rats on the expression of ATP-sensitive potassium (KATP) channels, the mitochondrial permeability transition pore (mPTP) opening in the heart, and the vasorelaxation responses of isolated aortic rings to activation of KATP channels. Methods: Experiments were performed on adult (6 months) and old (24 months) male Wistar rats, which were divided into three groups: adult, old, and glutathione-treated old rats. Glutathione was injected intraperitoneally at a dose of 52 mg/kg 1 hour before the studies. The mRNA expression of KATP channels was determined using reverse transcription and real-time polymerase chain reaction analysis. The effect of glutathione administration on mPTP opening, relaxation responses of isolated aortic rings, and oxidative stress markers was studied. Results: It was shown that the expression levels of Kir6.1, Kir6.2, and SUR1 subunits of KATP channels and levels of reduced glutathione were significantly increased in glutathione-treated old rats (by 8.3, 2.8, 13.1, and 1.5-fold, respectively), whereas the levels of oxidative stress markers (hydrogen peroxide, diene conjugates, malondialdehyde, and rate of superoxide generation) in heart mitochondria and mPTP opening were significantly reduced. Relaxation of aortic rings was significantly increased in response to the actions of KATP channel openers flocalin and pinacidil in glutathione-treated animals, which was prevented by glibenclamide. Conclusions: Thus, the administration of exogenous glutathione to old rats resulted in a significant increase in the expression levels of the Kir6.1, Kir6.2, and SUR1 subunits of KATP channels and a decrease in oxidative stress. This was accompanied by inhibition of mPTP opening and enhancement of vasorelaxation responses to activation of KATP channels.

Exercise restores endogenous H2 S synthesis and mitochondrial function in the heart of old rats.

BACKGROUND: Ageing is accompanied by a decrease in endogenous hydrogen sulphide (H2 S) synthesis and the development of mitochondrial dysfunction. The aim of our work was to study the possible participation of exercise training-induced regulation of endogenous H2 S production in the restoration of mitochondrial function in old rats. MATERIALS AND METHODS: Male rats were divided into three groups: adult, old and exercise-trained old. Exercise training of old rats was performed for 4 weeks. The mRNA expression cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) were determined using reverse transcription and real-time polymerase chain reaction analysis. Mitochondrial dysfunction was determined by mPTP opening, which was investigated by spectrophotometric registration of the swelling of mitochondria isolated from the rat heart. We also studied the effect of exercise on H2 S content, oxidative stress and mtNOS activity. RESULTS: Exercise training in old animals significantly increased the expression of H2 S-synthesizing enzymes CSE and 3-MST and restored endogenous H2 S production in cardiac tissue and cardiac mitochondria to levels of adult animals. In addition, the training significantly reduced oxidative stress in old rats, in particular the rate of formation of •O2 - and H2 O2 , diene conjugates and malondialdehyde levels in the mitochondria of the heart. Simultaneously, in the hearts of these animals, resistance of mPTP to the inducer of its opening of calcium ions was increased. CONCLUSIONS: Thus, exercise training restores endogenous H2 S production, and significantly reduces oxidative stress in cardiac mitochondria of old rats that are associated with the inhibition of calcium-induced mPTP opening as an indicator of mitochondrial dysfunction.

Pyridoxal-5-phosphate induced cardioprotection in aging associated with up-expression of cystathionine-γ-lyase, 3-mercaptopyruvate sulfurtransferase, and ATP-sensitive potassium channels.

BACKGROUND: In the present work, we investigated the cardioprotective potential of pyridoxal-5-phosphate (PLP) in old rats as a cofactor of enzymes that synthesize hydrogen sulphide (H2 S). MATERIALS AND METHODS: PLP was administered per os in a dose of 0.7 mg per kg daily for 2 weeks. Rats were divided into three groups (adult, old and old +PLP) of 20 animals. The cardiac mRNA levels of genes encoding H2 S-synthesizing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), uncoupling proteins (UCP3), subunits of ATP-sensitive potassium (KATP ) channels were determined using real-time polymerase chain reaction analysis. We also studied the effect of PLP-administration on the content of H2 S, oxidative stress, the activities of inducible and constitutive NO-synthase (iNOS, cNOS), arginase and nitrate reductase in the heart homogenates as well as cardiac resistance to ischemia-reperfusion in Langendorff-isolated heart model. RESULTS: It was shown that PLP restored mRNA levels of CSE, 3-MST and UCP3 genes, and H2 S content and also significantly increased the expression of SUR2 and Kir6.1 (2.2 and 3.3 times, respectively) in the heart of old rats. PLP significantly reduced the formation of superoxide, malondialdehyde, diene conjugates as well as the activity of iNOS and arginase. PLP significantly increased constitutive synthesis of NO and prevented reperfusion disturbances of the heart function after ischemia. CONCLUSIONS: Thus, PLP-administration in old rats was associated with up-expression of CSE, 3-MST, UCP3 and SUR2 and Kir6.1 subunits of KATP channels, and also increased cNOS activity and reduced oxidative stress and prevented reperfusion dysfunction of the heart in ischemia-reperfusion.

Glutathione restores the mitochondrial redox status and improves the function of the cardiovascular system in old rats.

Introduction: Aging is accompanied by cardiovascular disorders which is associated with an imbalance of pro- and antioxidant systems, the mitochondrial dysfunction, etc. Glutathione (GSH) plays a critical role in protecting cells from oxidative damage. The aim of the work was to study the effect of exogenous glutathione on the redox status of mitochondria, the content of H2S and the function of the cardiovascular system in old rats. Methods: Experiments were performed on adult (6 months) and old (24 months) Wistar rats divided into three groups: adult, old and glutathionetreated old rats. Glutathione was injected intraperitoneally at a dose of 52 mg/kg. We investigated glutathione redox balance, H2S levels, oxidative stress, the opening of the mitochondrial permeability transition pore (mPTP), the resistance of isolated heart to ischemia/reperfusion in Langendorff model, endothelium-dependent vasorelaxation of isolated aortic rings, and cardiac levels of 3-MST, CSE, and UCP3 mRNA were determined using real-time PCR analysis. Results: Our data shows that in old rats treated with glutathione, the balance of its oxidized and reduced form changes in the direction of a significant increase (by 53.6%) of the reduced form. Glutathione pretreatment significantly increased the H2S levels, mtNOS activity, and UCP3 expression which considered as protective protein, and conversely, significantly decreased oxidative stress markers (the rate of O2•- generation, the levels of H2O2, diene conjugates and malone dialdehyde, in 2.5, 2.3, 2, and 1.6 times, respectively) in heart mitochondria. This was associated with the inhibition mitochondrial permeability transition pore opening and increased resistance of the isolated heart to ischemia/reperfusion in these animals. At the same time, in glutathione-treated old rats, we also observed restoration of endothelium-dependent vasorelaxation responses to acetylcholine, which were almost completely abolished by the NO-synthase inhibitor L-NAME. Conclusion: Thus, the pretreatment of old rats with glutathione restores the mitochondrial redox status and improves the function of the cardiovascular system.

Cardiac dysfunction in spontaneously hypertensive old rats is associated with a significant decrease of SUR2 expression.

ATP-sensitive potassium (KATP) channels are participants of mechanisms of pathological myocardial remodeling containment. The aim of our work was to find the association of changes in the expression of Kir6.1, Kir6.2, SUR1, and SUR2 subunits of KATP channels with changes in heart function and structure during aging under conditions of the constant increase of vascular pressure. The experiments were carried out on young and old spontaneously hypertensive rats (SHR) and Wistar rats. The expression levels of KATP channels subunits were determined using reverse transcription and quantitative PCR. It is shown that the mRNA expression level of Kir6.1 in young SHR rats is significantly lower (6.3-fold, p = 0.035) than that of young Wistar rats that may be one of the causes of arterial hypertension in SHR. At the same time, mRNA expression of both Kir6.1 and Kir6.2 in old SHR rats was significantly higher (6.8-fold, p = 0.003, and 5.9-fold, p = 0.006, respectively) than in young hypertensive animals. In both groups of old animals, SUR2 expression was significantly reduced compared to young animals, in Wistar rats at 3.87-fold (p = 0.028) and in SHR rats at 48.2-fold (p = 0.033). Changes in SUR1 expression were not significant. Thus, significant changes in the cardiovascular system, including impaired function and structure of the heart in old SHR rats, were associated with a significant decrease in SUR2 expression that may be one of the mechanisms of heart failure decompensation. Therefore, it can be assumed that increased expression of SUR2 may be one of the protective mechanisms against pathological myocardial remodeling.

Effects of fluorine-containing opener of ATP-sensitive potassium channels, pinacidil-derivative flocalin, on cardiac voltage-gated sodium and calcium channels.

Fluorine-containing pinacidil-derivative flocalin is an effective adenosine triphosphate-sensitive potassium (K(ATP))-channel opener with pronounced vasodilatory, cardioprotective effects and low general toxicity. By activating cardiac K(ATP) channels, flocalin hyperpolarizes cardiac myocytes, decreases their excitability, reduces Ca(2+) entry, and inhibits Ca(2+)-dependent signalling processes. Since our previous studies indicated that the drug also influences the rate of rise and amplitude of the cardiomyocyte's action potential, here we have investigated its possible actions on depolarizing inward currents through voltage-gated sodium (VGSC) and L-type calcium (VGCC) channels. Experiments were conducted on cultured cardiac myocytes prepared from the whole hearts of neonatal rats and maintained in culture for 1-3 days using whole-cell patch-clamp technique with no distinction of myocyte's type. Flocalin concentration dependently inhibited the Na(+) inward current through VGSCs with IC(50) = 17.4 µM and a maximal extent of 0.54, slowed down its inactivation kinetics, and hyperpolarized steady-state inactivation by 5.6 mV. The drug also inhibited calcium current through L-type VGCCs with IC(50) = 24.1 µM and a maximal block of 0.38, without affecting its inactivation but producing 5.3-mV hyperpolarization shifting of steady-state activation. Inhibition of both depolarizing currents by flocalin in addition to its ability to open K(ATP) channels enhances the suppressive action of the drug on cardiac excitability and broadens its pharmacological effects. Since, according to our previous data, cardiac K(ATP)-channel opening by flocalin occurs with ЕC(50) = 8 µM, the possibility of partial blockade of VGSC and L-type VGCCs should be considered when determining the therapeutic concentrations of the compound during its use as a cardioprotector.

Sarcolemmal cardiac K(ATP) channels as a target for the cardioprotective effects of the fluorine-containing pinacidil analogue, flocalin.

BACKGROUND AND PURPOSE: A class of drugs known as K(ATP) -channel openers induce cardioprotection. This study examined the effects of the novel K(ATP) -channel opener, the fluorine-containing pinacidil derivative, flocalin, on cardiac-specific K(ATP) -channels, excitability of native cardiac myocytes and on the ischaemic heart. EXPERIMENTAL APPROACH: The action of flocalin was investigated on: (i) membrane currents through cardiac-specific K(ATP) -channels (I(KATP) ) formed by K(IR) 6.2/SUR2A heterologously expressed in HEK-293 cells (HEK-293(6.2/2A) ); (ii) excitability and intracellular Ca²(+) ([Ca²(+) ](i) ) transients of cultured rat neonatal cardiac myocytes; and (iii) functional and ultrastructural characteristics of isolated guinea-pig hearts subjected to ischaemia-reperfusion. KEY RESULTS: Flocalin concentration-dependently activated a glibenclamide-sensitive I(KATP) in HEK-293(6.2/2A) cells with an EC50= 8.1 ± 0.4 µM. In cardiac myocytes, flocalin (5 µM) hyperpolarized resting potential by 3-5 mV, markedly shortened action potential duration, reduced the amplitude of [Ca²(+) ](i) transients by 2-3-fold and suppressed contraction. The magnitude and extent of reversibility of these effects depended on the type of cardiac myocytes. In isolated hearts, perfusion with 5 µmol·L⁻¹ flocalin, before inducing ischaemia, facilitated restoration of contraction during reperfusion, decreased the number of extrasystoles, prevented the appearance of coronary vasoconstriction and reduced damage to the cardiac tissue at the ultrastructural level (state of myofibrils, membrane integrity, mitochondrial cristae structure). CONCLUSION AND IMPLICATIONS: Flocalin induced potent cardioprotection by activating cardiac-type K(ATP) -channels with all the benefits of the presence of fluorine group in the drug structure: higher lipophilicity, decreased toxicity, resistance to oxidation and thermal degradation, decreased metabolism in the organism and prolonged therapeutic action.