The structural analogue of apelin-12 prevents energy disorders in the heart in experimental type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is the most severe form of diabetes, which is characterized by absolute insulin deficiency induced by the destruction of pancreatic beta cells. The aim of this study was to evaluate the effect of a structural analogue of apelin-12 ((NαMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, metilin) on hyperglycemia, mitochondrial (MCh) respiration in permeabilized cardiac left ventricular (LV) fibers, the myocardial energy state, and cardiomyocyte membranes damage in a model of streptozotocin (STZ) diabetes in rats. Metilin was prepared by solid-phase synthesis using the Fmoc strategy and purified using HPLC. Four groups of animals were used: initial state (IS); control (C), diabetic control (D) and diabetic animals additionally treated with metilin (DM). The following parameters have been studied: blood glucose, MCh respiration in LV fibers, the content of cardiac ATP, ADP, AMP, phosphocreatine (PCr) and creatine (Cr), the activity of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in blood plasma. Administration of metilin to STZ-treated rats decreased blood glucose, increased state 3 oxygen consumption, the respiratory control ratio in MCh of permeabilized LV fibers, and increased the functional coupling of mitochondrial CK (mt-CK) to oxidative phosphorylation compared with these parameters in group D. In STZ-treated animals metilin administration caused an increase in the PCr content and prevention of the loss of total creatine (ΣCr=PCr+Cr) in the diabetic hearts, as well as restoration of the PCr/ATP ratio in the myocardium and a decrease in the activity of CK-MB and LDH in plasma to initial values. Thus, metilin prevented energy disorders disturbances in cardiomyocytes of animals with experimental T1DM.

[The anti-ischemic and antioxidant activity of the pharmacological agonist of galanin receptor GalR2 and carnosine in in vitro and in vivo model systems]. / Protivoishemicheskaia i antioksidantnaia aktivnost' farmakologicheskogo agonista retseptora galanina GalR2 i karnozina v model'nykh sistemakh in vitro i in vivo.

Antioxidant and anti-ischemic properties of the pharmacological agonist of galanin receptor GalR2 WTLNSAGYLLGPßAH (Gal) and its C-terminal fragment, dipeptide carnosine (ßAH), were studied in the model of regional ischemia and reperfusion of the rat heart in vivo in the dose range of 0.5-5.0 mg/kg and Cu²âº-induced free radical oxidation of low density lipoproteins (LDL) of human plasma in vitro for peptide concentrations of 0.01 mM and 0.1 mM. Gal was obtained by automatic solid phase synthesis using the Fmoc methodology; its structure was characterized by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. Intravenous administration of the optimal dose of Gal (1 mg/kg) to rats after ischemia was more effective than carnosine in reducing of the myocardial infarct size and the activity of creatine kinase-MB and lactate dehydrogenase in blood plasma at the end of reperfusion. It also improved the metabolic state of the reperfused myocardium and reduced the formation of peroxidation products during reperfusion. Gal reduced more effectively the formation of adducts of hydroxyl radicals in the interstitium of the area at risk (AAR) of the rat heart than carnosine. Carnosine at a dose of 1 mg/kg more effectively increased the activity of catalase and glutathione peroxidase in the AAR by the end of reperfusion compared to Gal. In a model of Cu²âº-initiated oxidation of human plasma LDL 0.1 mM carnosine demonstrated a significantly more pronounced reduction in the formation of lipid radicals compared to Gal. The results show that Gal can be considered as a promising agent that reduces myocardial injury during reperfusion and oxidative stress.

[Cardiometabolic efficacy and toxicological evaluation of a pharmacological galanin receptor agonist]. / Kardiometabolicheskaia éffektivnost' i toksikologicheskaia kharakteristika farmakologicheskogo agonista retseptorov galanina.

The goal of this study was to examine effects of a novel galanin receptor agonist GalR1-3 [bAla14, His15]-galanine 2-15 (G), obtained by automatic solid-phase synthesis, on the metabolic state of the area at risk and the size of acute myocardial infarction (MI) in rats in vivo and evaluate its toxicity in BALB /c mice. In anesthetized rats, regional ischemia was simulated by coronary artery occlusion and then coronary blood flow was restored. The peptide G was administered intravenously (i.v.) with a bolus after a period of regional ischemia in the dose range of 0.25-3.0 mg/kg. The sizes of MI and the activities of creatine kinase-MB (СK-MB) and lactate dehydrogenase (LDH) in blood plasma were estimated. The effect of administration of the optimal dose of G (1.0 mg/kg) on myocardial content of adenine nucleotides (AN), phosphocreatine (PCr), creatine (Cr) and lactate was studied. I.v. administration of G to rats at a dose of 1.0 mg/kg slightly affected hemodynamic parameters, but reduced MI size by 40% and decreased plasma LDH and CK-MB activity by the end of reperfusion compared to control. These effects were accompanied by a significant improvement in energy state of area at risk (AAR) - an increase in myocardial content of ATP, åAN, PCr and åCr, and combined with a decrease in myocardial lactate level compared with the control. Toxicity of peptide G was studied with a single intraperitoneal injection of 0.5-3.0% solution of the peptide substance to mice. The absence of signs of intoxication and death of animals after G injection in the maximum possible dose did not allow determining the value of the average lethal dose. The results indicate therapeutic potential of the peptide G for preventing myocardial ischemia and reperfusion injury and feasibility for further study of its pharmacological properties and mechanisms of action.

Apelin-12 and its structural analog enhance antioxidant defense in experimental myocardial ischemia and reperfusion.

This study investigated the effects of peptide apelin-12 (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A12) and its novel structural analog (H-(N(α)Me)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, AI) on myocardial antioxidant enzyme activities, lipid peroxidation, and reactive oxygen species formation in ex vivo and in vivo models of myocardial ischemia/reperfusion (I/R) injury. Isolated working rat hearts were subjected to global ischemia and reperfusion. Infusion of 140 µM A12 or AI before global ischemia improved cardiac function recovery; increased the activity of Cu,Zn superoxide dismutase (Cu,Zn SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); decreased malondialdehyde (MDA) content in reperfused heart; and reduced the formation of hydroxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide in the myocardial effluent during early reperfusion compared with these indices in control. Anesthetized open-chest rats were subjected to the left anterior descending coronary artery occlusion and coronary reperfusion. Peptide A12 or its analog AI was injected intravenously at the onset of reperfusion at a dose of 0.35 µmol/kg. Treatment with A12 or AI significantly limited infarct size and reduced the activity of lactate dehydrogenase and creatine kinase MB isoenzyme in blood plasma at the end of reperfusion compared with control. These effects were accompanied by complete recovery of Cu,Zn SOD, CAT, and GSH-Px activities; and decrease in MDA content in the area at risk by the end of reperfusion. The study concluded that C-terminal fragment of native peptide apelin-12 and its synthesized analog is involved in the upregulation of cardiac antioxidant defense systems and attenuation of lipid peroxidation in myocardial I/R injury.

In vivo reduction of reperfusion injury to the heart with apelin-12 peptide in rats.

Apelin-12 (A-12) peptide was synthesized by automated solid phase method and purified by reverse phase HPLC. Its homogeneity and structure were confirmed by HPLC, (1)H-NMR spectroscopy, and mass spectroscopy. Acute myocardial infarction was induced by 40-min occlusion of the left coronary artery with subsequent 60-min reperfusion in narcotized Wistar rats. Peptide A-12 was injected (intravenous bolus, 0.07 or 0.35 µmol/kg) to experimental animals simultaneously with the beginning of reperfusion. Injections of A-12 in these doses led to reduction of systolic BP to 67 and 85% of the initial level, respectively, which was virtually restored completely by the end of reperfusion, and to a significant reduction of the infarction focus in the myocardium (by 21 and 34% in comparison with the control, respectively). Injection of A-12 in a dose of 0.35 µmol/kg led to reduction of plasma concentrations of necrosis markers in comparison with the control by the end of reperfusion: MB-creatine kinase by 56%, lactate dehydrogenase by 30%. The results attest to vasodilatory effects of A-12 under conditions of heart reperfusion in vivo; the peptide injected after local ischemia limits the myocardial infarction size and reduces damage to cardiomyocyte membrane.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke).

Effects of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6'-plastoquinonyl) decylrhodamine 19 (SkQR1) on rat models of H2O2- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H2O2 or by ischemia/reperfusion. Higher SkQ1 (125-250 nmol/kg per day for 2-3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2-4 days, whereas one injection of SkQ1 or SkQR1 (1 micromol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 micromol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound.

Metabolic correction reduces the area of acute ischemic myocardial infarction in rats.

The possibility of decreasing the degree of irreversible alterations in cardiomyocytes with original saline reperfusion solution enriched with L-aspartic acid, D-glucose, and Dmannitol was studied on experimental rats with regional ischemia and reperfusion. Infusion of the test solution into the left ventricle during the early reperfusion stage significantly reduced the area of myocardial infraction. This effect was accompanied by improvement of energy metabolism and decrease in damage to cell membranes in the risk zone. Our results indicate that metabolic protection during reperfusion increases myocardial resistance to ischemic and reperfusion stress.

Metabolic and antioxidant effects of R(+/-)-N6-(2-phenylisopropyl)-adenosine following regional ischemia and reperfusion in canine myocardium.

Recent studies have indicated that activation of A1/A2-receptors may mediate metabolic adaptation of the heart to ischemia/reperfusion stress. This study tests whether pretreatment with A1-selective agonist R(-)-N6-(2-phenylisopropyl) adenosine (R-PIA) might mimic effects of a brief period of coronary occlusion (ischemic preconditioning, IP) on energy metabolism and hydroxyl radical (OH.) formation in canine myocardium following subsequent prolonged ischemia and reperfusion. Anaesthetized dogs were randomized to a control group subjected to 40-min occlusion of a diagonal branch of left anterior descending coronary artery (LAD) followed by 1-h reperfusion, or a preconditioned group (PC) in which the same period of sustained ischemia and reperfusion was preceded by a single cycle of IP (5-min occlusion of the same LAD branch and 10-min reperfusion), or to PIA group in which R-PIA infusion into the same branch of LAD (0.4 microg/kg per min during 5 min) was followed by 10 min of perfusion prior to sustained ischemia-reperfusion. Pretreatment with R-PIA similarly to IP reduced lactate (Lac), creatine (Cr) and inorganic phosphate (Pi) release from myocytes into the interstitial fluid during sustained ischemia compared to these indices in control. By the end of reperfusion, both IP and R-PIA infusion enhanced recovery of myocardial ATP and phosphocreatine (PCr) and attenuated the total creatine (sigmaCr = PCr + Cr) loss, an index of cell membrane damage. A1-receptor activation by R-PIA, as IP, led to a significant reduction in OH. radical generation following reperfusion assessed by a spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) using cardiac microdialysis. R-PIA pretreatment did not affect systemic and cardiac hemodynamic parameters. We conclude that (1) adaptive mechanisms of IP involve A1-receptor activation that contributes to the overall metabolic response and (2) R-PIA acts as a useful preconditioning-mimetic and anti-ischemic agent in dogs.

Allopurinol: kinetics, inhibition of xanthine oxidase activity, and protective effect in ischemic-reperfused canine heart as studied by cardiac microdialysis.

With microdialysis, we monitored cardiac interstitial fluid (ISF) levels of allopurinol, its metabolites, and the adenine nucleotide breakdown products (ANBP), inosine, hypoxanthine (HYP), xanthine (Xa), uric acid (UA) in dogs that received 1 and 10 mg/kg allopurinol intravenously (i.v.). Half-life (t1/2) of drug penetration into the heart was dose independent (1.8 min), whereas for the 10-mg/kg dose terminal elimination t1/2 (96 min) was much prolonged and ISF clearance (9.6 l/min kg) was reduced as compared with that induced by 1 mg/kg (28 min and 30.4 l/min kg) probably due to capacity limitation of allopurinol conversion to oxypurinol by Xa dehydrogenase/oxydase (Xa D/O). Inhibition of Xa D/O activity by allopurinol resulted in a dose-dependent increase in ISF HYP and Xa levels and a decrease in UA level. For a 10-mg/kg dose, maximal effect was attained approximately 40 min after drug injection. Allopurinol (1 mg/kg) given 30 min after the start of 40-min coronary artery occlusion during ischemia entered the ischemic zone ISF very slowly as compared with that of the control zone; the no-reflow phenomenon was evident because the levels became similar in both zones only 15 min after initiation of reperfusion. To examine cardioprotective efficiency, we administered allopurinol (10 mg/kg) 40 min before 40-min occlusion; it had little effect on total ANBP release during ischemia but facilitated washout of ANBP from the ischemic zone during reperfusion, thus manifesting protective efficacy against reperfusion injury and no-reflow. As shown by the lack of ischemia-induced increase in ISF Xa, myocardial Xa D/O activity was completely blocked by allopurinol.

Microdialysis study of ischemia-induced hydroxyl radicals in the canine heart.

A new experimental approach for spin-trapping of oxygen radicals in a selected region of the heart in situ is described. This approach is based on microdialysis, and it permits the detection of oxygen radicals in conditions of local ischemia and restoration of normal blood flow. Increased hydroxyl radical generation in an ischemic area of canine myocardium, as a result of 40 min local occlusion, has been studied.

Cardiac microdialysis measurement of extracellular adenine nucleotide breakdown products during regional ischemia and reperfusion in canine heart: protective effect of propranolol against reperfusion injury.

Using cardiac microdialysis, we studied release of the adenine nucleotide breakdown products (ANBP) adenosine (ADS), inosine (INS), and hypoxanthine (HYP) into the interstitium of canine myocardium during 20- and 40-min occlusion of the anterior descending coronary artery and reperfusion. Dialysate ANBP concentrations reached maximum values not at the end of ischemia but in the first 10 min of reperfusion. The effect was more pronounced after 20-min ischemia. Further reperfusion led to an ANBP decrease that was more prolonged after 40-min ischemia. Pretreatment with DL-propranolol (0.5 mg/kg, intravenously, i.v.) given 40 min before coronary occlusion had no effect on adenine nucleotide catabolism rate during 20- and 40-min ischemia, but it facilitated washout of ANBP from ischemic zone immediately after the start of reperfusion. A similar effect was elicited by a D-stereoisomer of propranolol with no beta-adrenoceptor blocking activity. Results suggest that the reperfusion injury and probably the no-reflow phenomenon were the cause of enhanced adenine nucleotide catabolism at the beginning of reperfusion and prolonged ANBP washout from the ischemic zone. Reduction of reperfusion injury by propranolol could be related to the membrane stabilizing and antioxidant activity of this agent. Examination of DL-propranolol kinetics in arterial and coronary venous blood plasma showed that drug accumulation in the myocardium was almost maximum at the start of ischemia; therefore, the efficiency of cardio-protection with DL-propranolol was not limited by pharmacokinetic causes. Insertion of an additional microdialysis probe in the myocardium allowed monitoring of extracellular propranolol concentrations.

Function and metabolism of dog heart in ischemia and in subsequent reperfusion: effect of exogenous glutamic acid.

The effect of intravenous infusion of glutamic acid on cardiac contractile function during short-term ischemia and subsequent reperfusion was studied in anaesthetized dogs. Left ventricular ischemia was induced by underperfusion of the anterior descending and circumflex coronary arteries. Infusion of glutamic acid at 3 mg/kg/min resulted in less depression of cardiac function when given after a 2 min period of 60% coronary blood flow reduction: left ventricular systolic pressure decreased by 9% vs. 22%, dP/dt decreased by 16% vs. 29%, the double product (left ventricular systolic pressure by heart rate) was reduced by 16% vs. 31%. When reperfusion was carried out during glutamic acid infusion there was a significantly enhanced recovery in cardiac function. The augmentation of cardiac performance in ischemia and reperfusion caused by glutamic acid was not accompanied by changes in myocardial oxygen consumption. Glutamic acid uptake by the ischemic myocardium increased 2-fold during infusion. This led to cessation of ammonia release from the heart due to stimulation of glutamine synthesis, and an enhancement of alanine formation coupled with pyruvate uptake but it did not effect lactate production. However, glutamic acid infusion did not influence cardiac performance and metabolism under conditions of normal coronary flow. The results suggest that elevation of glutamate arterial concentration exerts a beneficial effect on ischemic heart. The mechanisms of the protective action are discussed.