[Effects of stimulation and blockade of the synthesis of endogenous hydrogen sulfide at myocardial ischemia-reperfusion].

In experiments on isolated rat hearts, perfused according to Langendorff method, the effects of stimulation of the synthesis and blockade of endogenous hydrogen sulfide in myocardial ischemia-reperfusion (20 min/40 min) was studied. L-cysteine (121 mg/kg), precursor of endogenous hydrogen sulfide was injected intraperitoneally 30 minutes before the experiment without and within 10 minutes after administration of DL-propargylglycine (11.3 mg/kg) ("Sigma", USA)--inhibitor of cystathionine-gamma-lyase. The heart function was assessed by measuring the LVDP, dP/dt, coronary flow, heart rate. The opening of mitochondria permeability transition (MPT) pore was estimated by releasing of a stable factor with UV absorbance (lambda(max) 250 nm) into the coronary outflow probes during the initial phase of reperfusion. Administration L-cysteine was accompanied by a decrease of reperfusion disorders in cardiac function compared to control rats. The results showed that L-cysteine pretreated hearts against the blockade of cystathionine-gamma-lyase with DL-propargylglycine exerted a powerful cardioprotective effect in ischemia-reperfusion injury. Significant post-ischemic recover of heart function and improving the efficiency of oxygen metabolism was accompanied with tiny quantity of mitochondrial factor releasing comparing to I/R group. Positive influence of the combined DL-propargylglycine and L-cysteine action was the prevention of MPT pore opening.

[Effect of hydrogen sulfide on isolated rat heart reaction under volume load and ischemia-reperfusion].

The present study was aimed to investigate the effect of H2S donor (NaHS) on heart function in conditions of functional loads and ischemia-reperfusion (I/R) injury by using Langendorf isolated heart perfusion. NaHS ("Sigma", 7,4 mg per kg) was dissolved in physiological solution and injected intraperitoneally 30 min before experiment. Rat isolated hearts were Langendorf-perfused and subjected to 20-minute non-flow ischemia followed by 40-minute reperfusion. The heart function was assessed by measuring the LVDP, dP/dt, coronary flow, heart rate. The opening of mitochondria permeability transition (MPT) pore was estimated by releasing of a stable factor with UV absorbance (lambdamax 250 nm) into the coronary outflow probes during the initial phase of reperfusion. The results showed that NaHS pretreated hearts developed greater LVDP without decreasing ofdP/dt min in response to an increase of left ventricle volume indicating greater functional reserves and effectiveness of Frank-Starling low realization. NaHS increased cardiac mitochondrial membrane potential but did not changed UCP3 gene expression. Significant post-ischemic recover of heart function in NaHS group was accompanied with tiny quantity ofmitochondrial factor releasing comparing to I/R group (p<0.001). Thus, NaHS do provides cardioprotective effect by inhibition of MPT pore opening.

[Mitochondria permeability transition as a target for ischemic preconditioning].

The ischemic preconditioning (IPC) limits myocardial injury provoked by a subsequent prolonged ischemia-reperfusion (I/ R). The underlying mechanisms of enhanced resistance of heart are actively studied, but for sure it was established that mitochondria play a major role in IPC-stimulated adaptation to ischemia. In this article we present and discuss evidences that cardioprotective effect of IPC is mediated by inhibition of mitochondria permeability transition pore (MPTP) opening. It was shown that IPC effectively prevents the excessive production of ROS by mitochondria during I/R and promotes a more complete restoration of function of isolated rat hearts. It was revealed that MPTP formation due to I/R was inhibited in IPC heart. Mitochondrial factor, the marker of MPTP opening found in outflow probes, was released in much lesser amounts in IPC hearts that in non-IPC. Furthermore, mitochondria isolated from IPC hearts showed a decreased sensitivity to calcium ions, a MPTP inductor, and, thus, massive MPTP-depended swelling of mitochondria was abrogated in IPC hearts. In our experiments we observed slight increase in inducible NOS activity right after short ischemic stimuli. We suppose that increased NO production by iNOS is involved in inhibition of MPTP and this may be one of the possible mechanisms of decreased sensitivity of mitochondria to calcium ions. It is concluded that among the processes involved in formation of cardioprotective effect of IPC, a reduction of membrane permeability due to the inhibition of MPTP opening plays a crucial role.

[Genipin--uncoupling protein inhibitor--reduces the protective effect of ischemic preconditioning].

The functional significance of uncoupling proteins UCP2 and UCP3 in the cell and the organism remains unknown. There have been reports about their involvement in cellular protection mechanisms underlying the phenomenon of ischemic preconditioning. The purpose of this study was to elucidate the role of uncoupling proteins UCP2 and UCP3 in the formation of cardioprotective effect of ischemic preconditioning. In experiments on isolated rat hearts we show here an increase in the level of UCP2 and UCP3 gene expression in the heart tissue under the influence of ischemic preconditioning with three episodes of 5 min stopping the flow perfusion. Similar effects were induced by a prolonged ischemia-reperfusion of myocardium. The blockade of the UCP2 activity by genipin (Wako Inc., USA, 10(-5) mol/L, isolated heart perfusion for 15 minutes) abolished the protective effect of adaptation to ischemia. It was concluded that uncoupling proteins take part in the cardioprotective effect ofischemic preconditioning.

[Role of nitric oxide in the development of the myocardial contractile reactions in trained animals].

Intensive constitutive production of nitric oxide (NO) during physical training improves vasodilatation and heart function. However, it remains unclear how NO takes part in myocardial adaptation to workload, which is accompanied by an increased heart inflow and intracellular calcium content. Using isolated rat heart by Langendorf preparation, we studied myocardial response to gradually increased left ventricular volume (Frank-Starling low) and increasing concentration of Ca2+ in the perfusion solution (from 1.7 mM to 12.5 mM) in trained and untrained rats. It was shown that 4 weeks swimming course improved heart function: heart rate was decreased; contractile activity (dP/dt max) and coronary flow were increased by 20% and 33%, respectively. Equal volume stretching of balloon in left ventricle provoked greater contraction in trained comparing to untrained hearts, demonstrating extended functional reserves after swimming course. Mitochondrial membrane potential was significantly increased in hearts of trained rats. Furthermore, training prevented fast increase of the end diastolic pressure during calcium upload. Mitochondrial factor release due to opening of mitochondrial permeability transition pore (MPTP) in trained hearts was detected at higher concentrations of calcium that reveals extended calcium capacity of mitochondria and lesser sensitivity of MPTP to its inductor-calcium. Blockade of NO synthesis with L-NAME application of (10(-4) M for 15 min) abolished reaction of trained heart during Frank-Starling and calcium upload. Thus, heart adaptation to physical training and extension of functional reserves in heart are provided by endogenous NO production. Key words: nitric oxide, Frank-Starling low, physical training, calcium upload, mitochondrial permeability transition, membrane potential.

[UCP2 and UCP3 gene expression, heart function and oxygen cost of myocardial work changes during aging and ischemia-reperfusion].

To examine the effects of ischemia/reperfusion on UCPs genes expression, heart function and oxygen cost of myocardial work, hearts of adult (6 mo) and old (24 mo) rats were perfused by Langendorf preparation and subjected to 20 min ischemia followed by 40 min reperfusion. Mitochondrial permeability transition due to ischemic stimuli was evaluated by release of mitochondrial factor (lambda 250 nm) which was previously shown as a marker of MPTP opening. Expression of UCPs was detected by reverse transcriptional polymerase chain reaction. Mitochondrial membrane potential (deltaphi(m)) and oxygen consumption in isolated heart mitochondria of adult and old rats were measured. It was shown that impaired function of aging rat hearts was accompanied with an increased oxygen cost of myocardial work and lower mitochondrial membrane potential compared with adult rats. Reperfusion disturbances of cardiodynamic, contractile activity of myocardium and noneffective oxygen utilization in early period of reperfusion were less intensive in aged hearts than in adult ones. Therefore, the levels of mRNA of UCP2 in aging hearts were higher and mRNA levels of UCP3 were tended to increase. At the same time ischemia/reperfusion increased the expression of UCP2 and UCP3 in adult myocardium: mRNA levels of UCPs were significantly higher that those in control, whereas there was no such effect in aging hearts. It is concluded that uncoupling proteins are implicated in the age-depended heart dysfunction and development of the pathological mechanisms during ischemia-reperfusion.

[Effect of UCP2 activity inhibitor genipin on heart function of aging rats].

To estimate the role of uncoupling proteins in aging rat heart recovery from prolonged ischemia we used genipin application during Langendorfpreparation. It was shown that genipin in dose-depended manner depressed coronary flow, heart rate and cardiac diastolic function. Such effect was similar to that observed during myocardial Ca2+ overload by gradually elevated CaCl2, in perfusion solution. Moreover, postischemic disturbances of cardiodynamic parameters, oxygen cost of myocardial work were much increased in genipin pretreated hearts that in control ones. Thus, genipin inhibition of UCP2 activity has cardiodepressive effects that imply UCPs in cardiac calcium regulation.

[Effects of uncoupling proteins on nitric oxide synthesis and oxidative stress development in ishemia-reperfusion of old rat hearts].

Genipin is aglycone of geniposide, one of the active compounds of Gardenia gasminoides Ellis. The gardenia fruit extract has been used in traditional Chinese medicine to relieve the symptoms of type 2 diabetes that is accompanied with extensive oxidative stress and endothelial dysfunction of NO production. Besides, genipin was shown to inhibit UCP-depended proton leak through the inner mitochondrial membrane that leads to increased membrane potential and ATP production. We studied the effects of genipin at ischemia/reperfusion-induced oxidative stress and activity of NOS isozymes using Langendorfperfused old rat heart model. Ischemia/reperfusion is well-known oxidative agent, and showed significant increasing of superoxide radical, hydrogen peroxide and hydroxyl radical. Genipin application in doze 10-5 mol/L for 15 min before prolonged ischemia exerted powerful antiradical and antilipoperoxidative effects. Heart ischemia/reperfusion was supported with peroxynitrite generation and nitrozative stress. We demonstrated the inhibitory property of genipin on INOS expression that possibly occurs via protein kinase A inhibition and stabilization of I-kappaB-NF-kappaB complex. Genipin stimulated cNOS activity seemingly activating PI3K/Akt signaling pathway. Although, post-ischemic recovery ofcardiodynamic parameters of old rat hearts were depressed due to "switching off" the NO production by inducible NOS which is important in early period of reperfusion. Thus, we conclude that genipin is powerfull antioxidant and posses insulin-like activity due to its property of managing the NO production at intracellular signal transduction cascade level.

[Inhibition of mitochondrial permeability transition pore is one of the mechanisms of cardioprotective effect of coenzyme Q10].

Protective properties of coenzyme Q10 (CoQ10) on the: (i) Langendorff isolated guinea pig heart's function under ischemia and reperfusion (I/R) and on the isolated mitochondria (ii) the mitochondrial permeability transition pore (MPTP) opening under exposure to calcium as natural MPTP inductor and phenylarsine oxide as oxidant--were studied. Physiological characteristic of contractile function, myocardial oxygen consumption and mitochondrial factor release as index of MPTP opening were compared before and after ischemia of isolated heart in control animals and animals with preliminary administration of CoQ10 per os. It have been shown that I/R disturbances of heart function were decreased and oxygen metabolism was normalised in animals treated with CoQ10 in compare to non-treated control. It was accompanied with substantial stabilization of mitochondrial membrane. Decreased I/R disturbances of isolated heart from CoQ10-treated animals were correlated to amount of mitochondrial factor released to coronary flow. Moreover, preliminary incubation of mitochondria, isolated from rat heart, with CoQ10 (10(-5) mol/l) substantially prevented calcium and phenylarsine-induced, cyclosporine A-sensitive mitochondrial swelling. This protective effect was increased in experiments with deenergizing mitochondria. Results of physiological and biochemical study reveal that one of the mechanisms of CoQ10's cardioprotective effect could be direct inhibition of mitochondrial permeability transition pore opening during ischemia and reperfusion of the heart.

[Factor released during myocardial ischemia reperfusion may become a marker of opening of the mitochondrial permeability transition pore]. / Faktor, iakyi vyvil'niuiet'sia pid chas reperfuziï ishemizovanoho sertsia, mozhe buty markerom vidkryttia mitokhondrial'noï pory.

In experiments on isolated hearts of guinea-pigs, on a model of ischemia-reperfusion cardiac reperfusion has been shown to result in a constriction of coronary vessels, arrhythmia, an inhibition of the contractile activity of the myocardium, and an increase in an oxygen cost of the myocardial work. Apart from that, a stable agent was detected in a reperfusion solution by spectrophotometry on the wave length of 250 nM. Similar deterioration of the heart function and the availability of the stable agent in the solution were observed under influence of the known activators of mitochondrial permeability transition pore-phenilarsine oxide and antimycin A. In anaesthetized animals a release of the stable factor into the blood was induced by either ischemia-reperfusion or those activators. Application of the known inhibitors of the mitochondrial permeability transition pore cyclosporin A or trolox (water-soluble vitamin E) decreased remarkably both the cardiodynamic deterioration and a release of the stable factor under heart reperfusion. Mitochondrial origin of the factor was confirmed in experiments on isolated mitochondria. Thus, the detected factor has been determined to be released from mitochondria at the opening of mitochondrial permeability transition pore and is thought to be the marker of its opening in experiments in vitro and in vivo.

[Study of a factor released during myocardial ischemia reperfusion and its effects on myocardial, coronary and peripheral vessels]. / Faktor, iakyi vyvil'niuiet'sia pid chas reperfuziï ishemizovanoho sertsia, doslidzhennia vplyvu na miokard, koronarni ta peryferychni sudyny.

The effect of reoxygenated coronary effluent of an isolated guinea-pig heart on a sequentially perfused second heart, right auricle trabecula and arterial vessel rings was studied after 20 min ischaemia of the first heart. It has been shown that a factor released after myocardial ischemia/reperfusion stimulates arrhythmia, decreases myocardial contractility of the second heart and depresses tonical tension of the right auricle trabecula and arterial vessel rings. Storage of the coronary effluent up to 24 h did not modify its effects. These results suggest stable factor released from an isolated heart after ischaemia at reperfusion to exert humoral effect not only on a myocardial contractility and coronary circulation but on the peripheral vessels tonus.

[The role of nitric oxide in changes in the oxygen consumption and the oxygen cost of the work of the cardiac muscle]. / Vyvchennia roli oksydu azotu u zminakh spozhyvannia kysniu ta kysnevoï vartosti roboty sertsevoho m'iaza.

In experiments on guinea-pig isolated hearts, perfused under Langendorff preparation, it has been shown that heart volume load and inotropic stimulation were accompanied with an increase of nitric oxide synthesis (by 46 and 51% accordingly) and with a decrease in an oxygen cost of myocardial work. L-arginine or sodium nitroprusside administration resulted in a reduce of the oxygen consumption by 29% and a decrease of oxygen cost of myocardial work. Inhibition of NO-synthase activity lead to an opposite effect--an increase in both--O2 consumption and an oxygen cost of the heart work by 15 and 19%. The data obtained evidence that NO is a regulator of the oxygen consumption by tissues and it determines the oxygen cost of the myocardial work. Its lack can play an important role in the developing of pathological states of the cardiovascular system, which are followed with its insufficient synthesis.

[The effect of a grape extract on the contractile activity of the myocardium and on the coronary flow of the isolated guinea pig heart]. / Vplyv ekstraktu vynohradu na skorotlyvu aktyvnist' miokarda ta koronarnyi potik izol'ovanoho sertsia mors'koï svynky.

Effects of one of the fraction of grape extract on myocardial contractility and coronary flow of isolated guinea pig heart have been investigated. It was shown that the administration of grape extract led to myocardial contractility index is increase by 77%, coronary flow increase by 13% and oxygen consumption increase by 30%. Inhibition of nitric oxide synthesis by L-NMMA resulted in decrease of grape extract-induced coronary flow increase. These data support of isolated strips investigation that grape has a compounds which induced endothelium-dependent vasodilation of coronary bed. They indicate that these substances have the strong cardiostimulating effect.