[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.

[Involvement of NO-dependent mechanisms of apelin action in myocardial protection against ischemia/reperfusion damage].

Apelin 12 (A-12) was synthesized by the automatic solid phase method with the use of Fmoc technology. The synthesized peptide was purified by preparative HPLC and identified by 1H-NMR spectroscopy and mass spectrometry. Acute myocardial infarction was induced by 40-min LAD occlusion followed by 60-min reperfusion in narcotized Wistar rats. A-12 was administrated at the onset of the reperfusion at doses of 0.07, 0.35 and 0.70 micromole/kg; N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, was applied at a dose of 10 mg/kg 10 min prior to reperfusion alone or before A-12 administration (0.35 micromole/kg); saline was used in control. The indicated A-12 doses induced a transient reduction of the arterial systolic blood pressure (ASBP) to 85, 58, and 56% of the initial level, respectively, which was accompanied by its recovery by the end of reperfusion. All A-12 doses significantly limited myocardial infarct size by 26, 40 and 33%, respectively, compared to the value in control. After administration of A-12 at dose of 0.35 micromol/kg, this effect was combined with reduction of MB-creatine kinase (MB-CK) and lactate dehydrogenase (LDH) activities in plasma at the end of reperfusion by 56 and 47%, respectively, compared to the values in control. Inhibition of NO formation by L-NAME increased SABP but did not affect myocardial infarct size compared with that in control. Coadministration of L-NAME and A-12 resulted in lesser reduction of ASBP during reperfusion than injection of A-12 alone. This intervention led to an increase in infarct size by 26% with concomitant 1.8- and 1.5-times elevation of MB-CK and LDH activities, respectively, compared to the values in the A-12 group. The results indicate that NO is involved as a mediator of the effects of A-12 on the overall protection consisting in a limitation of infarct size and reduction of postischemic cardiomyocyte membrane damage. Cardioprotective mechanisms of apelin action are discussed.

[Dinitrosyl complexes of iron with glutathione in the rat myocardial tissue during regional ischemia and postischemic reperfusion].

The injection of dinitrosyliron iron complexes with glutathione at the onset of 40-min rat regional myocardial ischemia was shown to exert a clear cardioprotective action by decreasing the infarct size and suppressing the cardiac rhythm disturbance. After the introduction of the preparation, its effective accumulation with protein thiol-containing ligands in the myocardial tissue was registered be the EPR method. It was also found that, as a result of postischemic reperfusion, the rate of the decrease in the content of these complexes in the ischemic area increases, which demonstrates the effective scavenging of short-lived reactive oxygen species by molecules of dinitrosyl iron complexes.

[Dietary SkQ1 supplement reduces myocardial ischemia- reperfusion injury in rats in vivo].

To examine whether nutritional supplementation with SkQ1 can reduce myocardial ischemia-reperfusion injury in vivo, Wistar rats were fed a regular diet supplemented with different doses of SkQ1 for two or three weeks. Control groups of rats were fed the same diet supplemented with NaBr. Anaesthetized rats were subjected to 40-min regional myocardial ischemia and 1-h reperfusion. Myocardial infarct size was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining method. SkQ1-fed rats (125 nmol/kg/day for two weeks and 250 nmol/kg/day for two and three weeks) revealed significantly smaller myocardial infarction and less lactate dehydrogenase (LDH) and creatine kinase-MB fraction (CK-MB) activity elevations in plasma at the end of reperfusion compared with the controls. This effect was combined with improvement of energy state of the area at risk at the end of reperfusion, namely, augmentation of adenine nucleotide content, two-fold increase in phosphocreatine, reduction of lactate accumulation and decrease of lactate/pyruvate ratio in myocardial tissue. Therefore, nutritional supplementation with SkQ1 renders the hearts resistant to ischemia-reperfusion injury affecting oxidative metabolism of postischemic cardiomyocytes.

[Myocardial infarction remodeling in rats with dinitrosyl iron complex with glutathione].

Effects of dinitrosyl iron complex with reduced glutathione (DNIC-GS) on hemodynamics, metabolic state of the heart and myocardial infarction size were studied in vivo in rats subjected to 40-min occlusion of the anterior descending coronary artery (ADCA) and subsequent 60-min reperfusion. Intravenous bolus injection of DNIC-GS (3.10 or 0.78 micromol/kg body wt) was performed before ADCA occlusion or at the first minute of the reperfusion; the same volume of saline was infused in the control group. Nitroglycerine and nicorandil were used as reference preparations. DNIC-GS administration significantly reduced the mean arterial pressure, thus indicating vasodilative effect of NO releasing. Administration of both doses of DNIC-GS (before ADCA occlusion or at the first minute of the reperfusion) significantly limited the infarction size as compared with that in the control. Preischemic administration of 3.10 micromol DNIC-GS/kg body wt. substantially reduced activities of lactate dehydrogenase and MB-fraction of creatine kinase in blood plasma at the end of the reperfusion compared with these indices in the control. This effect was accompanied with essential improvement of energy state of the area at risk (AR) and with a lack of DNIC-GS influence on metabolism of non-ischemic area of the heart. The obtained results demonstrate that infarction remodelling after intravenous DNIC-GS administration is related to augmented preservation of aerobic metabolism and membrane integrity in post-ischemic cardiomyocytes of the AR.

[The relationship between the production of nitric oxide and the injury of cardiomyocytes caused by in vivo rat regional myocardial ischemia and reperfusion].

Changes in nitric oxide concentration in rat myocardium in vivo during temporary occlusion of the anterior descending coronary artery, followed by reperfusion were studied by microdialysis assay in risk and intact areas by using an NO spin trap (complex of ferrous ions with N-methyl-D, L-glucamine dihiocarbamate, Fe3+-MGD2). The amplitude of the EPR signal of the NO spin adduct NO-Fe2+-MGD2 in the risk area increased during the 40-min occlusion and remained higher than the initial level during 60-min postischemic reperfusion, indicating a substantial nitric oxide production. The size of the infarction in the risk area by the end of reperfusion was 47 +/- 3 %, the contents of ATP, phosphocreatine, and total creatine decreased to 44 +/- 4, 51 +/- 5, and 60 +/- 3 %, correspondingly, as compared with initial values, and the level of lactate was six times higher than the initial one. In the intact area of the left ventricle, the level of nitric oxide and high-energy metabolites did not change throughout the experiment. It was shown that the intensive nitric oxide production, in acute regional ischemia and reperfusion are related to the disturbance of energy metabolism, the damage to cytoplasmic membranes, and the death of cardiomyocytes.

[The study of vasodilative and antiischemic function of nicorandil in regional ischemia and reperfusion of rat heart in vivo].

Aim of the work was to study effect of nicorandil [N-(2-nitrooxiethyl) nicotinamide, SG75] on blood pressure (BP), heart rate (HR) and rhythm disturbances during regional ischemia and reperfusion of the heart in rats in vivo and its ability to limit acute myocardial infarction (MI). Nicorandil was obtained by nitrating nicotinamide ethanol using produced by industry ethylnicotinate. MI in Wistar rats was modeled by 40-min occlusion of anterior descending coronary artery (ADCA) and subsequent 60-min reperfusion. Nicorandil (3,2 mmol/kg) was administered intravenously before occlusion. Nitroglycerine was used as preparation of comparison; it was administered in the same dose. MI area and zone at risk (ZR) were measured by computer planimetry after staining of left ventricular sections with 2, 3, 5-triphenyltetrazolium chloride. Lowering of mean BP under influence of nicorandil during ADCA occlusion and subsequent reperfusion were deeper and longer than under influence of nitroglycerine. Contrary to nitroglycerine administration of nicorandil did not cause decrease of HR. Administration of both drugs postponed origination of rhythm disturbances during ischemia but did not affect their duration. MI dimension assessed by MI/ZR ratio after administration of nicorandil and nitroglycerine was significantly lowered down to 22 +/- 4 and 32 +/- 3%, respectively, compared with 47 +/- 3% in control. The results obtained evidence that in this model of ischemic and reperfusion damage of the heart vasodilating properties of nicorandil combined with decrease of postischemic loss of cardiomyocytes in ZR are comparable with effects of nitroglycerine.

[Cardioprotective efficacy of dinitrosyl iron complex with L-cysteine in rats in vivo].

The effect ofdinitrosyl iron complex (DNIC) with L-cysteine on the hemodynamic indices and the size of myocardial infarction, which was induced by 40-min regional ischemia and subsequent 60-min reperfusion, have been studied in rats in vivo. Intravenous bolus injection of DNIC (3.1 micromol/kg body weight in 0.5 ml saline) was performed before regional ischemia; the control group was administered the same volume of saline. DNIC administration significantly decreased the mean blood pressure throughout the experiment. DNIC reduced the duration of cardiac arrhythmias to 170 +/- 10 s as against 445 +/- 30 s in control. The myocardial infraction size significantly decreased in the DNIC group compared to control (38.0 +/- 1.4 and 48.0 +/- 3.9% of the area at risk, respectively; p < 0.05). A combination of the vasodilatory effect of DNIC with the reduction of the damaging effect of cardiac ischemia and reperfusion encourage the development of hypotensive and antiischemic drugs on this class of NO donors.

[Attenuation of irreversible rat heart injury by reperfusion with metabolic protectors].

The aim of this study was to evaluate the effects of intravenous infusion of potassium-magnesium aspartate (K-Mg-Asp), a glucose-insulin-potassium cocktail (GIK), a combination of glucose, insulin and potassium aspartate (GIKAsp), and insulin (I) alone during reperfusion after myocardial regional ischemia on metabolism of the risk area (AR) and cardiomyocyte membrane damage in rats in vivo. Acute myocardial infarction (MI) was induced by 40-min occlusion of the anterior descending coronary artery followed by 60-min reperfusion. At the onset of reperfusion, K-Mg-Asp, GIK, GIKAsp, I or the physiological solution (control) was infused into the jugular vein at a rate of 1 ml/kg/h. After reperfusion, MI size was estimated by myocardial staining with 2,3,5-triphenyltetrazolium chloride. In separate series transmural biopsies from the AR were taken for metabolite analysis. MI size in all experimental groups was less than that in the control and reduced in the following rank: K-Mg-Asp > GIKAsp > I > GIK. By the end of reperfusion with metabolic protectors, ATP and phosphocreatine levels in the AR were 2-2,5 times higher that in the control (56.3 +/- 3.4 and 81.8 +/- 7.9% of the initial values, respectively). The losses of aspartate and glutamate pool and lactate and glucose accumulation in the AR were significantly lower in the experimental groups than in control. At the end of the reperfusion, the total creatine content in the AR decreased to 32.3 +/- 2.3% of the initial value in control, but restored to 78.0 +/- 5.7; 76.7 +/- 5.5 and 62.4 +/- 5.6% under effects of GIK, I and K-Mg-Asp, respectively. The recovery of the majority indices of aerobic metabolism and cell membrane integrity was maximal in the GIK and I groups and insignificantly lower after reperfusion with K-Mg-Asp. Therefore the metabolic efficacy of the protectors on reperfusion corresponded to MI size limitation. The results suggest that myocardial reperfusion with GIK, I and K-Mg-Asp is a promising adjunctive therapy in patients with acute MI.

[Protection of rat heart myocardium with a selective Na(+)/H(+) exchange inhibitor and ischemic preconditioning].

Aim of this study was to compare effects of BIIB-722, a novel Na(+)/H(+) exchanger-1 inhibitor, and ischemic preconditioning (IP) on infarct size and metabolism of area at risk in rats. Regional ischemia was induced by 40-min occlusion of a diagonal branch of left anterior descending coronary artery (LAD); it was followed by 60-min reperfusion. Intravenous bolus injection of BIIB-722 (3 mg/kg) dissolved in 280 mM xylitol was performed before regional ischemia or during the first minute of reperfusion. In the control group 280 mM xylitol was infused before ischemia or at the beginning of reperfusion at the same mode. IP was initiated by two cycles of 5-min LAD occlusion followed by 5-min reperfusion prior to sustained regional ischemia. Microdialysis technique was used to monitor pH and inorganic phosphate (P(i)) in the interstitial fluid of the area at risk. Metabolic state of the area at risk was assessed by ATP, phosphocreatine (PCr) and lactate levels; cellular membrane damage was evaluated by total creatine (SigmaCr=PCr+Cr) tissue content. Myocardial infarct size was determined by computer planimetry after staining of left ventricular slices with 2,3,5-triphenyltetrazolium chloride. BIIB-722 administration before or after ischemia, as well as IP, had no effect on cardiac hemodynamics and acid-base indices of arterial blood throughout the experiments. The infarct size/area at risk ratio was 43.5+/-5.2% in control and was reduced to 11.4+/-3.1% with IP, and to 17.0+/-3.6% and 25.8+/-2.6% with BIIB-722 infused on early reperfusion and before ischemia, respectively. BIIB-722 administration during the first minute of reperfusion as well as IP significantly augmented ATP and PCr contents, reduced lactate level and decreased ECr loss at the area at risk by the end of reperfusion as compared with values in control. Additionally significantly higher rates of pH recovery and reduction of P(i) concentration in the interstitial fluid were observed during reperfusion compared with these indices in control. BIIB-722 administration before ischemia had much effects on contents of energy and carbohydrate metabolites at area at risk. The results obtained indicate that ability of BIIB-722 to limit infarct size and improve metabolism in the area at risk is comparable to cardioprotective effects of IP. Therefore this study substantiates a possibility of application of a novel Na(+)/H(+) exchange inhibitor for clinical investigations.

[Effects of Na(+)/H(+)-exchanger inhibition on metabolism of area at risk and myocardial infarct size in dogs]. / Vliianie ingibirovaniia Na(+)/H(+)-obmena na metabolizm zony riska i razmery infarkta miokarda u sobak.

The aim of this work was to study cardioprotective effects of BIIB 722, a novel Na(+)/H(+) exchanger-1 inhibitor, during regional ischemia and reperfusion in canine myocardium. The experiments were carried out on anaesthetized dogs intubated and artificially ventilated with room air enriched with oxygen. Regional ischemia was induced by 30-min occlusion of a diagonal branch of left anterior descending coronary artery (LAD), which was followed by 60-min reperfusion. BIIB 722, dissolved in 280 mM xylitol, was infused intracoronary for 10 min prior to LAD occlusion and at the beginning of reperfusion at the rate of 1 ml/min (30 microg/g myocardial tissue). In the control group, 280 mM xylitol was used for intracoronary administration with the same regimen. Microdialysis probes were implanted in the region of LAD occlusion to monitor interstitial pH, inorganic phosphate (Pi) and hydroxyl radical adduct. Energy state of the area at risk was evaluated by ATP and phosphocreatine (PCr) contents, cell membrane damage was assessed by total creatine (SigmaCr=PCr+Cr) tissue content. Myocardial infarct size was determined by staining with Evans Blue dye and further incubation of left ventricular slices in 2,3,5-triphenyltetrazolium chloride. The percentage ratio of infarct size to area at risk was calculated by computer planimetry. BIIB 722 administration had no effect on cardiac hemodynamics and acid-base indices of arterial blood throughout the experiments but induced 1.8-fold reduction of myocardial infarct size comparing with control. Treatment with BIIB 722 decreased acidification of the interstitial fluid following ischemia and facilitated recovery of pH to initial value on reperfusion. This effect was combined with significantly less Pi formation in the area at risk during LAD occlusion and reduction of this index to the initial value during reperfusion. At the end of reperfusion, the treated group showed augmented recovery of ATP and PCr tissue levels and higher content of SigmaCr comparing with the control. Additionally, BIIB 722 treatment markedly decreased generation of free oxygen radicals following LAD occlusion and completely avoided their formation on early reperfusion. The results indicate that BIIB 722 ability to limit myocardial infarct size in dogs is tightly connected with its influence on energy metabolism and oxygen radical generation.

[Free radical centers in the dog myocardial tissue in regional ischemia]. / Svobodnoradikal'nye tsentry v tkani miokarda sobaki v usloviiakh regional'noi ishemii.

The effect of regional ischemia on canine myocardial in situ free radical species was studied by the EPR method. Rapid fixation of heart muscle samples by freezeclamping was performed at the following physiological states: native myocardial blood circulation, regional ischemia with the presence of collateral circulation, total ischemia, and postischemic reperfusion. EPR spectra of the samples at -40 degrees C exhibited two free radical signals from the semireduced forms of ubiquinone and flavine coenzymes. Upon transition from normal blood supply to regional ischemia, an increase in the contribution of the flavine signal was registered, but reperfusion resulted in the recovery of the characteristics of EPR signals. It was found that the increase in the intensity of collateral circulation in the ischemic area led to an increase in the portion of ubisemiquinone in the integral EPR signal, whereas in total ischemia this signal was not registered. It was shown that the changes in spectral characteristics of integral free radical signals are accompanied by changes in their relaxation parameters.

[Registration of residual oxygen in the area of regional myocardial ischemia by ESR]. / Registratsiia ostatochnogo kislodoroda v oblasti regional'noi ishemii miokarda metodom EPR.

An experimental approach for recording molecular oxygen in the area of regional myocardial ischemia by using the method of microdialysis and spin label CTPO is presented.

[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.

[A method of modeling of occlusion-reperfusion induced myocardial infarction and quantitative evaluation of the effectiveness of limitation of its size]. / Metod modelirovaniia okkliuzionno-reperfuzionnogo infarkta miokarda i kolichestvennoi otsenki éffektivnosti ogranicheniia ego razmerov.

An experimental mathematical model was proposed to assess the efficiency of experimental myocardial infarction (MI) size limitation. A canine model of occlusion-reperfusion myocardial lesion was used in an acute experiment with an open chest. 90-minute occlusion and 4-hour reperfusion were performed by carotid coronary bypass surgery. The necrotic zone and the risk area were visualized by double perfusion with tetrazolium staining. Retrograde coronary blood flow was used as a measure of collateral blood flow. The multiple linear regression equation with values of the risk zone and retrograde blood flow/risk area ratio used as independent variables enabled the size of myocardial infarction to be highly accurately predicted. Comparison of the true size of MI with the "expected" one provided methods for quantitative assessment of pharmacological limitation of MI sizes. Calculating an individual value for each animal made it possible to examine its relation to coronary circulation parameters and facilitated comparison of benefits from various agents.

[Effect of a decrease in perfusion pressure on the alpha-adrenergic reactions of the coronary vessels]. / Vliianie snizheniia perfuzionnogo davleniia na al'fa-adrenergicheskie reaktsii koronarnykh sosudov.

Acute dog experiments involving the perfusion of donor coronary arteries demonstrated that alpha-adrenoreceptor stimulation in the presence of a beta-adrenergic block resulted in a 30% drop of coronary flow. This response is maintained at low perfusion coronary blood pressure values (up to 25 +/- 13 mmHg, p less than 0.02), whereas coronary dilatation reserve is already fully depleted at 60 +/- 8 mmHg, suggesting that the degree of coronary arterial stenosis associated with coronary insufficiency may be a decisive factor shaping coronary response to alpha-adrenoreceptor stimulation.

[Correction of contractile function and metabolism in canine ischemic myocardium due to exogenous glutamic acid]. / Korrektsiia sokratitel'noi funktsii i obmena ishemizirovannogo miokarda sobaki pod vliianiem ékzogennoi glutaminovoi kisloty.

The effect of intravenous glutamic acid infusion (3 mg/kg/min) was studied during myocardial ischemia and reperfusion in anesthetized dogs. Left ventricular ischemia was induced by underperfusion of the anterior descending and circumflex coronary arteries. Glutamic acid reduced the ischemic contractile depression 2 min after a 60%-reduction of the coronary blood flow. The left ventricular systolic pressure was decreased by 9% versus 22%, dP/dt by 16% versus 29%, left ventricular systolic pressure heart rate product by 16% versus 31%. Reperfusion with glutamic acid improved the recovery of cardiac performance without any increase in myocardial oxygen consumption. Glutamic acid infusion resulted in a 2-fold augmentation of glutamate uptake by the ischemic myocardium. It led to cessation of ammonia release by the heart due to activation of glutamine synthesis, enhancement of alanine formation coupled with pyruvate utilization and did not change lactate production. The mechanisms of the protective action of glutamic acid are discussed.

[Diffusion capacity of the myocardial capillary bed in graded decrease in the coronary blood flow]. / Diffuznaia sposobnost' kapilliarnogo rusla miokarda pri dozirovannom snizhenii koronarnogo krovotoka.

In closed chest dogs with controlled perfusion of the left coronary artery coronary blood flow was reduced by 10, 20, 30, 40, 50 and 70%. Diffusion capacity of myocardial capillary bed (permeability surface area product) decreased in all but the first (10%) series of experiments. 23-64% fall of the product occurred irrespective of coronary vessel reserve state but with respect to the degree of blood flow reduction. The amount of coronary influx possibly determined the number of perfused capillaries in the heart. Thus, 20% and more reduction of coronary blood flow cannot be compensated at the level of myocardial capillary bed.