Apelin C-Terminal Fragments: Biological Properties and Therapeutic Potential.

Creation of bioactive molecules for treatment of cardiovascular diseases based on natural peptides is the focus of intensive experimental research. In the recent years, it has been established that C-terminal fragments of apelin, an endogenous ligand of the APJ receptor, reduce metabolic and functional disorders in experimental heart damage. The review presents literature data and generalized results of our own experiments on the effect of apelin-13, [Pyr]apelin-13, apelin-12, and their chemically modified analogues on the heart under normal and pathophysiological conditions in vitro and in vivo. It has been shown that the spectrum of action of apelin peptides on the damaged myocardium includes decrease in the death of cardiomyocytes from necrosis, reduction of damage to cardiomyocyte membranes, improvement in myocardial metabolic state, and decrease in formation of reactive oxygen species and lipid peroxidation products. The mechanisms of protective action of these peptides associated with activation of the APJ receptor and manifestation of antioxidant properties are discussed. The data presented in the review show promise of the molecular design of APJ receptor peptide agonists, which can serve as the basis for the development of cardioprotectors that affect the processes of free radical oxidation and metabolic adaptation.

Chimeric Agonist of Galanin Receptor GALR2 Reduces Heart Damage in Rats with Streptozotocin-Induced Diabetes.

Neuropeptide galanin and its N-terminal fragments reduce the generation of reactive oxygen species and normalize metabolic and antioxidant states of myocardium in experimental cardiomyopathy and ischemia/reperfusion injury. The aim of this study was to elucidate the effect of WTLNSAGYLLGPßAH-OH (peptide G), a pharmacological agonist of the galanin receptor GalR2, on the cardiac injury induced by administration of streptozotocin (STZ) in rats. Peptide G was prepared by solid phase peptide synthesis using the Fmoc strategy and purified by preparative HPLC; its structure was confirmed by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. Experimental animals were randomly distributed into five groups: C, control; S, STZ-treated; SG10, STZ + peptide G (10 nmol/kg/day); SG50, STZ + peptide G (50 nmol/kg/day); G, peptide G (50 nmol/kg/day). Administration of peptide G prevented hyperglycemia in SG50 rats. By the end of the experiment, the ATP content, total pool of adenine nucleotides, phosphocreatine (PCr) content, and PCr/ATP ratio in the myocardium of animals of the SG50 group were significantly higher than in rats of the S group. In the SG50 and SG10 groups, the content of lactate and lactate/pyruvate ratio in the myocardium were reduced, while the glucose content was increased vs. the S group. Both doses of peptide G reduced the activation of creatine kinase-MB and lactate dehydrogenase, as well as the concentration of thiobarbituric acid reactive products in the blood plasma of STZ-treated rats to the control values. Taken together, these results suggest that peptide G has cardioprotective properties in type 1 diabetes mellitus. Possible mechanisms of peptide G action in the STZ-induced diabetes are discussed.

Modified N-Terminal Fragments of Galanin: Cardioprotective Properties and Mechanisms of Action.

The design of new drugs for treatment of cardiovascular diseases based on endogenous peptide hormones is of undoubted interest and stimulates intensive experimental research. One of the approaches for development in this area is synthesis of the short bioactive peptides that mimic effects of the larger peptide molecules and have improved physicochemical characteristics. In recent years, it has been found that the N-terminal fragments of the neuropeptide galanin reduce metabolic and functional disorders in the experimental heart damage. The review presents literature data and generalized results of our own experiments on the effects of the full-size galanin and its chemically modified N-terminal fragments (2-11) and (2-15) on the heart in normal conditions and in modeling pathophysiological conditions in vitro and in vivo. It has been shown that the spectrum of the peptide actions on the damaged myocardium includes decrease in the necrotic death of cardiomyocytes, decrease in the damage of sarcolemma, improvement in the metabolic state of myocardium, decrease in the formation of reactive oxygen species (ROS) and lipid peroxidation (LPO) products. Mechanisms of the protective action of the modified galanin fragments associated with activation of the GalR2 receptor subtype and manifestation of antioxidant properties are discussed. The data summarized in the review indicate that the molecular design of pharmacological agonists of the GalR2 receptor is a promising approach, because they can serve as a basis for the development of cardioprotectors influencing processes of free radical oxidation and metabolic adaptation.

Antioxidant Properties of Galanin and Its N-Terminal Fragments in in vitro and in vivo Oxidative Stress Modeling.

Antioxidant properties of rat galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2 (Gal), N-terminal fragment of galanin (2-15 aa) WTLNSAGYLLGPHA (G1), and its modified analogue WTLNSAGYLLGPßAH (G2) were studied in vivo in the rat model of regional myocardial ischemia and reperfusion and in vitro in the process of Cu2+-induced free radical oxidation of human blood plasma low-density lipoproteins. Intravenous administration of G1, G2, and Gal to rats after ischemia induction reduced the infarction size and activities of the necrosis markers, creatine kinase-MB and lactate dehydrogenase, in blood plasma at the end of reperfusion. G1, G2, and Gal reduced formation of the spin adducts of hydroxyl radicals in the interstitium of the area at risk during reperfusion, moreover, G2 and Gal also reduced formation of the secondary products of lipid peroxidation in the reperfused myocardium. It was shown in the in vivo experiments and in the in vitro model system that the ability of galanin peptides to reduce formation of ROS and attenuate lipid peroxidation during myocardial reperfusion injury was not associated directly with their effects on activities of the antioxidant enzymes of the heart: Cu,Zn-superoxide dismutase, catalase, and glutathione peroxidase. The peptides G1, G2, and Gal at concentrations of 0.01 and 0.1 mM inhibited Cu2+-induced free radical oxidation of human low-density lipoproteins in vitro. The results of oxidative stress modeling demonstrated that the natural and synthetic agonists of galanin receptors reduced formation of the short-lived ROS in the reperfused myocardium, as well as of lipid radicals in blood plasma. Thus, galanin receptors could be a promising therapeutic target for cardiovascular diseases.

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(+)/Ca(2+) 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(+)/Ca(2+) exchangers.

Enhancement of crystalloid cardioplegic protection by structural analogs of apelin-12.

BACKGROUND: C-terminal fragments of adipokine apelin are able to attenuate myocardial ischemia-reperfusion (I/R) injury, but whether their effects are manifested during cardioplegic arrest remain obscure. This study was designed to evaluate the efficacy of natural apelin-12 (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A12) and its novel structural analogs (H-(N(α)Me)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, AI, and N(G)-Arg(N(G)NO2)-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-NH2, AII) as additives to crystalloid cardioplegia and explore benefits of early reperfusion with these peptides. METHODS: Isolated working rat hearts subjected to normothermic global ischemia and further reperfusion were used. St. Thomas' Hospital cardioplegic solution No.2 (STH2) containing 140 µM A12, AI, or AII was infused for 5 min at 25 °C before ischemia. In separate series, peptide administration was used for 5 min after ischemia. Metabolic state of the hearts was evaluated by myocardial content of high energy phosphates and lactate. Lactate dehydrogenase (LDH) leakage was assessed in myocardial effluent on early reperfusion. RESULTS: Addition of the peptides to STH2 enhanced functional and metabolic recovery of reperfused hearts compared with those of control (STH2 without additives). Cardioplegia with analog AII was the most effective and accompanied by a reduction of postischemic LDH leakage. Infusion of A12, AI, or AII after ischemia improved the majority indices of cardiac function and metabolic state of the heart by the end of reperfusion. However, the overall protective effect of the peptides was less than when they were added to STH2. CONCLUSIONS: Enhancement of apelin bioavailability may minimize myocardial I/R damage during cardiac surgery. Structural analogs of A12 are promising components of clinical cardioplegic solutions.

Effects of structural analogues of apelin-12 in acute myocardial infarction in rats.

OBJECTIVE: To examine cardioprotective effects of Ρ-terminal fragment of adipokine apelin-12 (A12), its novel structural analogue [MeArg(1), NLe(10)]-A12 (I), and [d-Ala(12)]-A12 (II), a putative antagonist of APJ receptor, employing in vivo model of ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: Peptides were synthesized by the automatic solid phase method using Fmoc technology. Anesthetized open-chest male Wistar rats were subjected to left anterior descending (LAD) coronary artery occlusion and coronary reperfusion. Hemodynamic variables and electrocardiogram (ECG) were monitored throughout the experiment. Myocardial injury was assessed by infarct size (IS), activity of necrosis markers in plasma, and metabolic state of the area at risk (AAR). RESULTS: Intravenous injection of A12, I, or II at the onset of reperfusion led to a transient reduction of the mean arterial pressure. A12 or I administration decreased the percent ratio of IS/AAR by 40% and 30%, respectively, compared with control animals which received saline. Both peptides improved preservation of high-energy phosphates, reduced lactate accumulation in the AAR, and lowered CK-MB and LDH activities in plasma at the end of reperfusion compared with these indices in control. Treatment with II did not significantly affect either the IS/AAR, % ratio, or activities of both markers of necrosis compared with control. The overall metabolic protection of the AAR in the treated groups increased in the following rank: II < A12 < I. CONCLUSIONS: The structural analogue of apelin-12 [MeArg(1), NLe(10)]-A12 may be a promising basis to create a new drug for the treatment of acute coronary syndrome.

An attempt to prevent senescence: a mitochondrial approach.

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1=SkQR1>SkQ3>MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H(2)O(2)-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H(2)O(2) or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

Antioxidants decreases the intensification of low density lipoprotein in vivo peroxidation during therapy with statins.

The oxidative modification of low density lipoprotein (LDL) is thought to play an important role in atherogenesis. Drugs of beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) family are usually used as a very effective lipid-lowering preparations but they simultaneously block biosynthesis of both cholesterol and ubiquinone Q10 (coenzyme Q), which is an intermediate electron carrier in the mitochondrial respiratory chain. It is known that reduced form of ubiquinone Q10 acts in the human LDL as very effective natural antioxidant. Daily per os administration of HMG-CoA reductase inhibitor simvastatin to rats for 30 day had no effect on high-energy phosphates (adenosin triphosphate, creatine phosphate) content in liver but decreased a level of these substances in myocardium. We study the Cu2+-mediated susceptibility of human LDL to oxidation and the levels of free radical products of LDL lipoperoxidation in LDL particles from patients with atherosclerosis after 3 months treatment with natural antioxidants vitamin E as well as during 6 months administration of HMG-CoA reductase inhibitors such as pravastatin and cerivastatin in monotherapy and in combination with natural antioxidant ubiquinone Q10 or synthetic antioxidant probucol in a double-blind placebo-controlled trials. The 3 months of natural antioxidant vitamin E administration (400 mg daily) to patients did not increase the susceptibility of LDL to oxidation. On the other hand, synthetic antioxidant probucol during long-time period of treatment (3-6 months) in low-dose (250 mg daily) doesn't change the lipid metabolism parameters in the blood of patients but their high antioxidant activity was observed. Really, after oxidation of probucol-contained LDL by C-15 animal lipoxygenase in these particles we identified the electron spin resonance signal of probucol phenoxyl radical that suggests the interaction of LDL-associated probucol with lipid radicals in vivo. We observed that 6 months treatment of patients with pravastatine (40 mg daily) or cerivastatin (0.4 mg daily) was followed by sufficiently accumulation of LDL lipoperoxides in vivo. In contrast, the 6 months therapy with pravastatin in combination with ubiquinone Q10 (60 mg daily) sharply decreased the LDL initial lipoperoxides level whereas during treatment with cerivastatin in combination with probucol (250 mg daily) the LDL lipoperoxides concentration was maintained on an invariable level. Therefore, antioxidants may be very effective in the prevention of atherogenic oxidative modification of LDL during HMG-CoA reductase inhibitors therapy.

Altered energy supply to the pump function of the isolated heart of spontaneously hypertensive rats.

OBJECTIVE: The reasons for the development of cardiac insufficiency after a prolonged period of compensation accompanying myocardial hypertrophy are still uncertain and a disturbance in the energy metabolism of cardiomyocytes may serve as an underlying cause. The goal of the present work was to study functional and energetic correlates of the isolated heart of spontaneously hypertensive rats (SHR) at the stage of compensation. METHODS: Isolated hearts of SHR and normotensive age-matched Wistar-Kyoto (WKY) rats were subjected to volume and resistance loads. The myocardial content of high-energy phosphates and creatine was determined both before and after the functional loads. RESULTS: The contractile performances of the SHR hearts was significantly higher than those of the WKY hearts, the maximal cardiac output during volume load was higher by 36% and the maximal cardiac work index at complete aortic clamping was 68% higher. However, because the dry weight of SHR hearts was 48% higher, the normalized functional indices did not differ significantly between the groups. The ATP-to-ADP ratio and the total creatine level were significantly lower by 10% to 13% in the SHR group before and after the functional loads. In addition, the total adenine nucleotide pool and ATP content were 17% to 20% lower in SHR hearts after the functional loads. The content of high-energy phosphates correlated with contractile indices in the WKY group but not in the SHR group. CONCLUSIONS: The results showed that the SHR hearts were better adapted to increased loads than the WKY hearts; however, this advantage combines with an altered interrelation between myocardial energy state and its function.