[Antioxidant properties of apelin-12 and its structural analogue in experimental ischemia and reperfusion].

Effects of apelin-12 H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH (A12) and its modified analogue H-(NMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH (I) on activity of antioxidant enzymes, formation of malonic dialdehyde (MDA) and generation of reactive oxygen species (ROS) were studied in ex vivo and in vivo models of myocardial ischemia and reperfusion (I/R) injury in Wistar rats. Preischemic infusion of peptide A12 or AI enhanced cardiac function recovery of isolated perfused heart and was accompanied by a marked attenuation of ROS generation detected by electron paramagnetic resonance (EPR) technique in myocardial effluent at early reperfusion compared with control. Intravenous administration (i.v.) of peptides in narcotized rats with regional myocardial ischemia limited infarct size and reduced activity of lactate dehydrogenase and MB-fraction of creatine kinase in plasma at the end of reperfusion. Treatment with peptide A12 prevented reduction or augmented activity of myocardial u/Zn superoxide dismutase, catalase and glutathione peroxidase by the end of reperfusion in both I/R models compared with control. Increased MDA content in the area at risk of rat heart in situ at the end of reperfusion was reduced to the initial value under the effect of i.v. A12 administration. Therefore, cardioprotective action of natural apelin-12 and its structural analog AI involve reduction of short-lived ROS generation and improvement of the antioxidant state of ischemic heart during reperfusion.

[Synthesis and cardioprotective properties of apelin-12 and its structural analogs].

The apelin-12 and a number of its analogs, resistant to degradation of proteases, were synthesized by Fmoc- method of SPPS. By-products of synthesis were examined. It was found that serine hydroxyl group was sulfating during the final deprotection of apelin-12 (I) and its analogs. Sulfate moiety of Arg-protecting group transfer into hydroxyl group of Ser. Amount of by-product depends on presence of water in cleavage mixture. Furthermore, the final deprotection of amide analogs of apelin-12 (III, IV) is closed with formation of by-product--4-hydroxybenzylamide, its amount range on 20-8% on reaction mixture accordance HPLC data and also depend on composition of cleavage mixture. Effects of the synthesized peptides on recovery of cardiac function after ischemia were examined in a model of isolated perfused rat heart. Infusions of any of the peptides (I-V) before ischemia resulted in a significant improvement of contractile and pump function recovery compared to the control. Cardioptotective efficacy of the peptides increased in the following rank (I) < (II) = (III) < (IV) = (V).

[The influence of inhibiting no formation on metabolic recovery of ischemic rat heart by apelin-12].

Apelin 12 (A-12) was synthesized by the automatic solid phase method with use of Fmoc 1H-NMR spectroscopy and mass spectrometry. Effects of apelin-12 (a peptide comprised of 12 aminoacids, A-12) on recovery of energy metabolism and cardiac function were studied in isolated working rat hearts perfused with Krebs buffer (KB) containing 11 mM glucose that were subjected to global ischemia and reperfusion. A short-term infusion of microM 140 A-12 in KB prior to ischemia enhanced myocardial ATP, the total adenine nucleotide pool (SigmaAN = ATP + ADP + AMP) and the energy charge of cardiomyocites ((ATP + 0.5ADP)/SigmaAN) at the end of reperfusion compared with control (KB infusion) and reduced lactate content and lactate/pyruvate ratio in reperfused myocardium to the initial values. This effect was accompanied by improved recovery of coronary flow and cardiac function. Coadministration of 140 microM A-12 and 100 microM L-NAME (the nonspecific NOS inhibitor) profoundly attenuated the peptide influence on metabolic and functional recovery of reperfused hearts. The results indicate involvement of NO, formed under the peptide action, in mechanisms of cardioprotection that are tightly associated with recovery of energy metabolism in postischemic heart.

[Effects of dinitrosyl iron complex on metabolism and cellular membranes in ischemic rat heart].

Effects of exogenous NO donor--dinitrosyl iron complex with reduced glutathione (DNIC-GS) on functional recovery of isolated perfused rat heart subjected to global ischemia and reperfusion have been studied. DNIC-GS administration after ischemia substantially improved contractile and pump function recovery within a concentration range of 34 nM - 5 uM. In case of DNIC-GS administration before ischemia a two-phase influence was found--cardioprotective action for 67 nM and damaging one for 250 nM. Enhanced recovery of cardiac function after preischemic infusion of 67 nM DNIC-GS was associated with augmented preservation of ATP, phosphocreatine, total adenine nucleotide pool and total creatine content in myocardial tissue, and with reduction of lactate dehydrogenase (LDH) release into myocardial effluent compared with these indices in control. In contrast, infusion of 250 nM DNIC-GS resulted in poor recovery of energy metabolism and increased membrane injury than in control. The results suggest that a worse recovery of myocardial energy state and increased sarcolemma permeability in the 250 nM DNIC-GS group were caused by inhibiting oxidation of glucose, the main energy substrate for isolated perfused heart. Molecular mechanisms of protective and injurious action of DNIC-GS on ischemic heart are discussed.

[The effect of myosin ATPase inhibition on metabolic and functional recovery of isolated rat heart after global ischemia].

The effect of myosin ATPase inhibitor, 2,3-butanedione monoxime (BDM; used in the range of concentrations 1.25-10.0 mM), on recovery of functions of isolated rat heart subjected to normothermic (37 degrees C) total ischemia for 35 min has been investigated. BDM perfusion was performed at a flow rate of 4 ml/min during 5 min before ischemia (BDM-I) or before 25-min reperfusion (BDM-R). Control hearts were perfused with Krebs solution at the same flow rate. The highest functional recovery of heart and coronary vessels was observed during infusion of 2.5 mM BDM before ischemia. At the end of reperfusion ATP and phosphocreatine (PCr) content in hearts of this group was significantly higher whereas the level of lactate was two times lower than in control; total creatine content (sigmaCr) did not differ from the initial level. Similar but less pronounced changes in the improvement of aerobic metabolism and maintenance of sigmaCr after reperfusion were also observed in the case of infusion of 2.5 mM BDM before reperfusion. They were consistent with reduced recovery of functions of heart and coronary flow compared with these parameters observed in the BDM-I group. 2.5 mM BDM caused almost 2-fold decrease in release of cardiac lactate dehydrogenase into myocardial perfusate in the BDM-I and BDM-R groups (compared with control); this suggests lower damage of cell membranes. These results suggest that improvement of energy supply of postischemic cardiomyocytes may be a key factor determining cardioprotector effectiveness of short-term administration of BDM before ischemia.

[Effects of insulin and acidosis on metabolism and function of postischemic rat heart].

Isolated perfused hearts of male Wistar rats were subjected to 25-min normothermic (37 degrees C) global ischemia, then 5-min infusion of a modified perfusate (pH=7.4) and 25-min reperfusion (control). Experimental groups were treated after ischemia with: a perfusate of the same composition (pH=6.0, RA), a perfusate, containing insulin 2 U/l (pH=7.4, RI) or a perfusate with insulin 2 U/l (pH=6.0, RIA). In the RA, RI and RIA groups a 2-4 folds reduction of contracture was combined with an augmented recovery of the aortic outflow, the cardiac and stroke volumes, compared with these indices in the control during reperfusion. At the end of the reperfusion an improved ATP and phosphocreatine (PCr) recovery in the RA and RI groups was accompanied by reduction of intracellular total creatine (SigmaCr = PCr + creatine) compared with corresponding values in control. Lactate content of reperfused hearts in the RA group was 2.3-times lower than in the control one, but it was almost twice higher in the RI group because of glycolysis stimulation. The most effective recovery of high energy phosphates in the RI group corresponded to reduction of glutamate and aspartate catabolism--total myocardial pool of these amino acids was 1.5-times higher than in the control, and individual amino acid contents did not differ significantly from initial values. Infusion of the acidified perfusate with insulin (the RIA group) resulted in neither improvement of high energy phosphate recovery nor preservation of myocardial amino acid and SigmaCr pools, compared with these indices in control. The lack of augmented metabolic effects of combined treatment with acidosis and insulin in the RIA group was accompanied by nearly the same recovery of majority of functional indices as was observed in RA and RI groups. The absence of additive protective effects of insulin and acidosis on early reperfusion was probably induced by acidosis induced inhibition of insulin-stimulated glucose uptake and glycolysis.

[Effects of dinitrosyl iron complex with glutathione and its components on ischemic rat heart during reperfusion].

The effects of the drug, a complex of dinitrosyl iron with glutathione lyophilized on dextrane, and its components: glutathione, nitrosoglutathione, dextrane, as well as nitric oxide released from dinitrosyl iron with glutathione--on the energy metabolism and functional recovery of isolated perfused rat heart subjected to global ischemia and reperfusion have been studied. The infusion of 100 nM dinitrosyl iron with glutathione after ischemia substantially enhanced the recovery of coronary flow, the cardiac contractile and pump functions during reperfusion with simultaneous preservation of myocardial high-energy phosphates, and cell membrane integrity. It was shown by the EPR method that these effects were associated with the transfer of Fe+(NO+)2 groups from dinitrosyl iron with glutathione to thiol-containing proteins of cardiomyocytes and coronary vessels. The combined infusion of 100 nM dinitrosyl iron with glutathione and 25 MM 2-(phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), a nitric oxide scavenger, after ischemia profoundly reduced the metabolic and functional recovery of reperfused hearts. After the postischemic administration of a 100 nM aliquot of hydrolysate of dinitrosyl iron with glutathione (a completely decomposed complex), the recovery of coronary flow, the majority of cardiac function indices, as well as the myocardial metabolic state and cell membrane injury did not differ from those in control or were significantly lower. The results obtained demonstrate that that inclusion of Fe+(NO+)2 groups into myocardial tissue and a spontaneous release of nitric oxide trigger the cardioprotective mechanisms of action of dinitrosyl iron with glutathione on ischemic heart.

Moderation of postischemic damage to cardiomyocytic membranes with reperfusion solution.

Isolated perfused hearts of Wistar rats subjected to total ischemia and reperfusion were used to examine the possibility of moderating damage to cardiomyocyte membranes with reperfusion solution containing l-aspartic acid, d-glucose, and d-mannitol. During the first 5 minutes of reperfusion, this solution significantly improved recovery of the pumping and contractile functions of the heart compared to the control and reduced the release of lactate dehydrogenase and systems generating short-living ROS into the effluent. To the end of reperfusion, the content of ATP and phosphocreatine was higher and the loss of total creatine was lower in hearts perfused with the test solution compared to the control. It is hypothesized that better integrity of the myocyte sarcolemma in hearts perfused with the test solution results from better preservation of macroergic phosphates and inhibition of ROS generation in this solution.

[Mechanisms of ischemic heart injury attenuation by means of a modified reperfusion].

Mechanisms of attenuation of membrane injury and metabolic disturbances in postischemic cardiomyocytes have been studied on a model of ischemic and reperfusion stress of rat heart using a modified early reperfusion. Optimization of reperfusion infusate composition augmented cardiac pump and contractile function recovery. This was accompanied by a reduced release of lactate dehydrogenase activity and systems generating short-lived reactive oxygen species into myocardial effluent and was associated with more efficient oxidative metabolism recovery and decreased losses of intracellular total creatine and amino acids pools. The results indicate availability of postischemic functional and metabolic myocardial injury correction by means of a controlled reperfusion.

Efficiency of cardioplegic solutions containing L-arginine and L-aspartic acid.

In experiments on rats we studied the effects of cardioplegic solutions with L-aspartic acid or L-arginine on functional recovery and metabolism of isolated working heart after 40-min normothermal global ischemia and 30-min reperfusion. After reperfusion of the hearts preventively protected with cardioplegic solution containing L-aspartic acid or L-arginine, coronary flow decreased in comparison with the initial values. As a component of cardioplegic solution, L-arginine was less efficient in recovery of contractility and cardiac output of the hearts in comparison with L-aspartic acid. In hearts protected with L-aspartic acid, the postischemic levels of ATP and phosphocreatine were significantly higher, and the level of lactate was significantly lower than in hearts protected with L-arginine. In comparison with L-arginine, L-aspartic acid is a more efficient component of cardioplegic solution in protection of the heart from metabolic and functional damages caused by global ischemia and reperfusion.

[Controlled reperfusion improves the metabolic and functional recovery of the isolated heart in rats after total ischemia].

Metabolic and functional effects of a hypocalcium reperfusion solution (RS) with low oxygen content containing d-glucose, trisamine, d-mannitol and I-aspartic acid have been studied in isolated rat hearts. The hearts were initially perfused for 20 min with the Krebs solution under constant left atrial filling pressure of 15 mm Hg and aortic perfusion pressure of 60 mm Hg. Then they were subjected to 30-min total normothermic ischemia followed by 30-min of reperfusion. The Krebs solution (control, n=16) or RS (n=11) were infused in retrograde mode with a rate of 4 ml/min during first 5 min of reperfusion. After that the hearts of both groups were reperfused in antegrade mode with the Krebs solution for 25 min under initial conditions. Short-term infusion of RS markedly improved postischemic functional recovery of cardiac function. After 30-min reperfusion coronary flow, an index of contractile function intensity, expressed as the left ventricle developed pressure-heart rate product, and cardiac work, calculated as the minute volume-aortic perfusion pressure product, recovered up to 92+/-1%, 77+/-1% and 61+/-1% of baseline values, respectively. In the control group the same indices were significantly lower and were 74+/-3%, 48+/-5% and 33+/-2%, respectively (p<0.001). At the end of reperfusion hearts treated with RS compared with the control hearts showed higher myocardial levels of ATP, phosphocreatine (PCr) and total creatine (SCr). These metabolic findings indicate better recovery of energy state and lesser sarcolemmal damage of postischemic cardiomyocytes after RS infusion. Thus, optimization of administration mode and composition of reperfusion solutions is a promising tool to attenuate functional and metabolic disturbances of the postischemic heart.

[Aspartic Acid and mannitol enhance protective efficiency of asanquineous cardioplegic solution]. / Asparaginovaia kislota i mannit uluchshaiut zashchitnye svoistva solevogo kardioplegicheskogo rastvora.

The aim of this work was to assess effects of a novel asanquineous cardioplegic solution (CP-5), buffered with trisamine (pH 7.6+/-0.1 at 22 degrees C) and containing 21.5 mM aspartic acid and 20.0 mM mannitol, on postischemic functional and metabolic recovery of isolated rat heart. A modified Ringer solution with 25 mM KCl (pH 7.6+/-0.1 at 22 degrees C) and the St. Thomas' cardioplegic solution (pH 7.8+/-0.1 at 22 degrees C) were used as controls. Osmolarity of all cardioplegic solutions were 340+/-5. After 20-min initial perfusion according to Neely (steady state) the hearts were subjected to 40-min normothermal total ischemia followed by 30-min antegrade reperfusion. Cardioplegic solutions were infused prior to ischemia at rate of the initial coronary flow for 5 min at room temperature. During reperfusion the hearts of CP-5 group completely recovered coronary flow and significantly enhanced restoration of the majority functional indices compared to the hearts in both control groups. This effect was combined with less lactate accumulation and preservation of higher ATP and phosphocreatine (PCr) levels in the heart tissue by the end of ischemia and, probably was induced by inclusion of aspartic acid into composition of CP-5. By the end of reperfusion the hearts treated with CP-5 completely recovered PCr content and restored ATP level up to 65.2+/-4.6% of initial one. A better energy state of reperfused hearts in CP-5 group was accompanied by reduction of myocardial lactate tissue to the preischemic value. Restoration of ATP, PCr and lactate content was significantly poor in both control groups during reperfusion. The least formation of a spin adduct of the short life oxygen radicals was found in the myocardial effluent of the hearts of CP-5 group at the early reperfusion using EPR technique. These data suggest a reduced release of oxygen radical generating systems from postischemic myocardium into perfusate due to antioxidant effect of mannitol. The obtained results substantiate addition of aspartic acid and mannitol to the asanquineous cardioplegic solution, buffered with trisamine, to enhance efficacy of myocardial protection against ischemia and reperfusion injury.

[Effects of metabolic substrates and mannitol on efficiency of cardioplegic protection in isolated rat heart]. / Vliianie metabolicheskikh substratov i mannita na éffektivnost' kardioplegicheskoi zashchity izolirovannogo serdtsa krysy.

The aim of this work was to study rationality of addition of aspartic acid, phosphocreatine, mannitol and tris(bydroxymethyl) aminomethane (trisamine) to a sanguineous cardioplegic solution. Isolated perfused rat hearts were subjected to 40-min normothermic total ischemia and 30-min reperfusion. Cardioplegic solutions were infused for 5 min prior to ischemia. A modified Ringer solution with 25 mM KCI was used as control. Osmolarity and pH of cardioplegic solutions were 340+/-5 mOms and 7.6+/-0.1 at 22 degreesC, respectively. Efficiency of myocardial protection was evaluated by recovery of contractile and pump function during reperfusion. The optimal solution contained aspartic acid (21.5 mM), mannitol (20.0 mM) and trisamine (5 mM). By the end of reperfusion the heart protected by this solution showed almost complete recovery of coronary flow (98+/-3% of the initial value vs. 77+/-3% in the control), and 2.6-fold higher recovery of stroke volume compared to the control. As a result, recovery of external cardiac work index, calculated as cardiac output-mean perfusion pressure, was 64+/-1% of the initial value vs. 24+/-5% in the control. Increase in buffer capacity of this cardioplegic solution by trisamine (up to 20.0 mM) as well as addition of phosphocreatine (10.0 mM) did not result in further augmentation of cardiac function recovery. The results suggest promising perspectives for development of medicinal form of this solution.

[Effect of antioxidant histochrome preparation on the contractile function and metabolism of the isolated rat heart under conditions of "calcium paradox", ischemia, and reperfusion]. / Vliianie antioksidantnogo preparata gistokhrom na sokratitel'nuiu funktsiiu i metabolizm izolirovannogo serdtsa krysy v usloviiakh "kal'tsievogo paradoksa", ishemii i reperfuzii.

A comparative study of protective effect of a new antioxidant histochrome on isolated rat heart at calcium paradox and ischemia-reperfusion was carried out. Perfusion with Ca-containing solution after 10-min perfusion with Ca-free medium caused depletion of high energy phosphates, loss of myoglobin, uncoupling of mitochondria, increase of left ventricular diastolic pressure. Pretreatment with histochrome led to decrease of cardiomyocyte damage evaluated by myoglobin efflux in perfused solution, attenuation of decrease ATP and phosphocreatine, and coupling of mitochondria. This resulted in a decrease of left ventricular diastolic pressure at calcium paradox. Less effect of histochrome treatment was noted in the case of ischemia-reperfusion model. It was concluded that histochrome effectively prevents heart from abnormalities caused by calcium imbalance in heart.

[Microdialysis study in vivo of the release of adenine nucleotide degradation products into intercellular space of canine myocardium during regional ischemia and reperfusion]. / Mikrodializnoe issledovanie in vivo vysvobozhdeniia produktov raspada adeninnukleotidov v mezhkletochnoe prostranstvo miokarda sobaki pri regional'noi ishemii i reperfuzii.

Intercellular concentrations of adenine nucleotide degradation products (ANDP)--adenosine inosine and hypoxanthine--in ischemic and control regions of the canine myocardium were measured by microdialysis technique during 20- and 40-min coronary artery occlusion and reperfusion. In hearts that fibrillated on reperfusion during the ischemic 40-min period catabolism of adenine nucleotides was more intensive, which could be the min cause of the reperfusion ventricular fibrillation. Reperfusion ventricular fibrillation was accompanied by an increase in the intercellular ANDP level in the control region, that indicated the development of the total myocardial ischemia. During the initial period of reperfusion after 20-min, a sharp increase in the interstitial ANDP level was observed in the ischemic region as compared with the end of the ischemia which could be explained as a result of demasking of reperfusion damage in such a case. The 40-min reperfusion induced slow reduction of the intercellular ANDP level in the ischemic region, while the regional blood flow already 5 min after the reperfusion did not differ from the blood flow in the control region. It is supposed that a slow washout of ANDP could be caused by the "no-reflow" phenomenon.

[Effect of gradual reperfusion on the restoration of the pump function and energy stores of the heart after ischemia]. / Vliianie gradual'noi reperfuzii na vosstanovlenie nasosnoi funktsii i énergeticheskie zapasy serdtsa posle iskhemii.

The contractile and pump function of guinea pig hearts was evaluated 40 min after total normothermic ischemia and 30 min reperfusion. A half of the hearts was reperfused with rapid mode restoration of the preischemic coronary flow ("sudden" reperfusion, SR), while the other half was reperfused with gradual mode restoration (GR) of coronary flow by 2 ml/min each 4 min till the initial level. The cardiac output and external work after SR constituted 49 and 28 of initial values, while after GR-87 and 71%, respectively. A distinct rise in minimal diastolic left ventricular pressure in the former group indicates a deteriorated filling of the heart. The total pool of adenine nucleotides and ATP content remained almost unchanged after GR: while after SR they decreased to 61 and 54% of the initial level, and myocardial lactate content was three times higher. The beneficial effect of GR seems to be due to better preservation or restoration of ATP content and more profound relaxation of the heart.

[Microdialysis study of release of adenine nucleotide breakdown products into the intercellular space of the myocardium in ischemia and reperfusion]. / Mikrodializnoe issledovanie vysvobozhdeniia v mezhkletochnoe prostranstvo miokarda produktov raspada adeninnukleotidov pri ishemii i reperfuzii.

Interstitial concentrations of adenine nucleotide breakdown products (ANBP)--adenosine (ADS), inosine (INS), hypoxanthine (HYP), in perfused rat hearts were measured during total ischemia and reperfusion by microdialysis technique. Progressive accumulation of ANBP in the interstitial space was observed during ischemia, but unlike INS and HYP, maximal ADS concentration was reached not by the end of ischemia but after the beginning of reperfusion. During reperfusion the qualitative accordance between changes in ANBP levels in the interstitial space and venous effluent was observed, but interstitial concentrations were several times greater.

[Relations of glutamate and aspartate contents of the heart and its energy state after ischemia]. / Vzaimosviaz' soderzhaniia glutamata i aspartata v serdtse s ego énergeticheskim sostoianiem posle ishemiii.

Ischemia and subsequent reperfusion with 5.5 mM glucose or sodium acetate were studied for impact on energy metabolism of the guinea pig isolated heart and glutamate, aspartate, and alanine levels in it and myocardial outflow. Acetate reperfusion resulted in a more significant reduction in the pool of adenine nucleotides and total creatine (phosphocreatine + creatine) by 48 and 60% of the baselines, respectively than did glucose reperfusion (as much as 65 and 76% reduction, respectively). The total glutamate and aspartate pool was twice as less as the baseline after reperfusion with any of the substrates, with acetate, tissue glutamate concentration was decreased by 42% of the baseline, whereas with glucose, it was reduced by as much as 62%. The consumption of amino acids was largely associated with their implication in alanine synthesis, which was stimulated by glycolysis/glucogenolysis at the early stage of reperfusion. The residue glutamate and aspartate contents in the reperfused hearts positively correlated with the pool of adenine nucleotides, total creatine, and the recovery of myocardial contractility. The findings suggest that the myocardial levels of these amino acids are closely associated with its energy state following ischemia and thus may affect the recovery of cardiac contractility.

[Relation between the energy state of the myocardium and the release of products of anaerobic metabolism]. / [Sviaz' mezhdu énergeticheskim sostoianiem miokarda i vykhodom iz nego produktov anaérobnogo obmena.

The relationships between cellular energy parameters and succinate, alanine and creatine release from isolated guinea pig hearts were studied during a 50 min perfusion (0.2 ml/min) with 5.5 mM glucose or 5 mM sodium acetate. Compared to glucose-perfused hearts, a more rapid ATP depletion accompanied by an increased succinate and creatine release was observed during underperfusion with acetate. The succinate and alanine accumulation in the myocardial effluent was related to a decrease in tissue ATP; the creatine release showed a close inverse correlation with the tissue phosphocreatine/creatine ratio. Hyperbolic and linear relationships were found between these indices for glucose- and acetate-perfused hearts, respectively. The logarithm of tissue ATP had negative linear correlations with the perfusate succinate/creatine ratio for the both substrates. The experimental results suggest that succinate, creatine and alanine assays in the myocardial effluent may be used for the assessment of the energy state of ischemic heart.