Donatella Gattullo

Post–conditioning induced cardioprotection requires signaling through a redox–sensitive mechanism, mitochondrial ATP–sensitive K+ channel and protein kinase C activation

Post–conditioning (Post–C) induced cardioprotection involves activation of guanylyl–cyclase. In the ischemic preconditioning scenario, the downstream targets of cGMP include mitochondrial ATP–sensitive K+ (mKATP) channels and protein kinase C (PKC), which involve reactive oxygen species (ROS) production. This study tests the hypothesis that mKATP, PKC and ROS are also involved in the Post–C protection. Isolated rat hearts underwent 30 min global ischemia (I) and 120 min reperfusion (R) with or without Post–C (i.e., 5 cycles of 10 s R/I immediately after the 30 min ischemia). In 6 groups (3 with and 3 without Post–C) either mKATP channel blocker, 5– hydroxydecanoate (5–HD), or PKC inhibitor, chelerythrine (CHE) or ROS scavenger, N–acetyl–cysteine (NAC), were given during the entire reperfusion (120 min). In other 6 groups (3 with and 3 without Post–C), 5–HD, CHE or NAC were infused for 117 min only starting after 3 min of reperfusion not to interfere with the early effects of Post–C and/or reperfusion. In an additional group NAC was given during Post–C maneuvers (i.e., 3 min only). Myocardial damage was evaluated using nitro–blue tetrazolium staining and lactate dehydrogenase (LDH) release. Post–C attenuated myocardial infarct size (21 ± 3% vs. 64 ± 5% in control; p < 0.01). Such an effect was abolished by 5–HD or CHE given during either the 120 or 117 min of reperfusion as well as by NAC given during the 120 min or the initial 3 min of reperfusion. However, delayed NAC (i.e., 117 min infusion) did not alter the protective effect of Post– C (infarct size 32 ± 5%; p < 0.01 vs. control, NS vs. Post–C). CHE, 5–HD or NAC given in the absence of Post–C did not alter the effects of I/R. Similar results were obtained in terms of LDH release. Our data show that Post–C induced protection involves an early redox–sensitive mechanism as well as a persistent activation of mKATP and PKC, suggesting that the mKATP/ROS/PKC pathway is involved in post–conditioning.

Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning.

Nitric oxide (NO) donors mimic the early phase of ischemic preconditioning (IPC). The effects of nitroxyl (HNO/NO(-)), the one-electron reduction product of NO, on ischemia/reperfusion (I/R) injury are unknown. Here we investigated whether HNO/NO(-), produced by decomposition of Angelis salt (AS; Na(2)N(2)O(3)), has a cardioprotective effect in isolated perfused rat hearts. Effects were examined after intracoronary perfusion (19 min) of either AS (1 microM), the NO donor diethylamine/NO (DEA/NO, 0.5 microM), vehicle (100 nM NaOH) or buffer, followed by global ischemia (30 min) and reperfusion (30 min or 120 min in a subset of hearts). IPC was induced by three cycles of 3 min ischemia followed by 10 min reperfusion prior to I/R. The extent of I/R injury under each intervention was assessed by changes in myocardial contractility as well as lactate dehydrogenase (LDH) release and infarct size. Postischemic contractility, as indexed by developed pressure and dP/dt(max), was similarly improved with IPC and pre-exposure to AS, as opposed to control or DEA/NO-treated hearts. Infarct size and LDH release were also significantly reduced in IPC and AS groups, whereas DEA/NO was less effective in limiting necrosis. Co-infusion in the triggering phase of AS and the nitroxyl scavenger, N-acetyl-L-cysteine (4 mM) completely reversed the beneficial effects of AS, both at 30 and 120 min reperfusion. Our data show that HNO/NO(-) affords myocardial protection to a degree similar to IPC and greater than NO, suggesting that reactive nitrogen oxide species are not only necessary but also sufficient to trigger myocardial protection against reperfusion through species-dependent, pro-oxidative, and/or nitrosative stress-related mechanisms.

Post-conditioning reduces infarct size in the isolated rat heart: role of coronary flow and pressure and the nitric oxide/cGMP pathway.

AbstractWe aimed to assess the role of the nitric oxide (NO)–cGMP pathway in cardioprotection by brief intermittent ischemias at the onset of reperfusion (i.e., post–conditioning (Post–con)). We also evaluated the role of coronary flow and pressure in Post–con. Rat isolated hearts perfused at constant– flow or –pressure underwent 30 min global ischemia and 120 min reperfusion. Post–con obtained with brief ischemias of different duration (modified, MPost–con) was compared with Post–con obtained with ischemias of identical duration (classical, C–Post–con) and with ischemic preconditioning (IP). Infarct size was evaluated using nitro–blue tetrazolium staining and lactate dehydrogenase (LDH) release. In the groups, NO synthase (NOS) or guanylyl–cyclase (GC) was inhibited with LNAME and ODQ, respectively. In the subgroups, the enzyme immunoassay technique was used to quantify cGMP release. In the constant–flow model, M–Post–con and C–Post–con were equally effective, but more effective than IP in reducing infarct size. The cardioprotection by M–Post–con was only blunted by the NOS–inhibitor, but was abolished by the GC–antagonist. Post–ischemic cGMP release was enhanced by MPost–con. In the constant–pressure model IP, M–Post–con and C–Post–con were equally effective in reducing infarct size. Post–con protocols were more effective in the constant–flow than in the constant–pressure model. In all groups, LDH release during reperfusion was proportional to infarct size. In conclusion, Post–con depends upon GC activation, which can be achieved by NOS–dependent and NOS–independent pathways. The benefits of M– and CPost–con are similar. However, protection by Post–con is greater in the constant–flow than in the constant–pressure model.

Ischemic preconditioning: from the first to the second window of protection.

In many species one or more brief coronary occlusions limit the injuries which a subsequent ischemia-reperfusion can produce in the myocardium. A similar protection has been observed in the majority of organ systems. A first period or window of protection can lasts up to 3 hours and is followed by a second window of protection (SWOP) which begins about 24 hours after the brief coronary occlusions and lasts about 72 hours. Increase of the release of endogenous agents such as adenosine and nitric oxide (NO) may be responsible for both windows through the activation of a protein-kinase C (PKC) which in turn activates ATP sensitive potassium (K+(ATP)) channels. Nitric oxide is also reported to act directly on K+(ATP) channels. Recently, it has been suggested that the channels involved in the protection are mitochondrial rather than sarcolemmal. In SWOP the origin of NO is attributed to the activity of an inducible NO-synthase. Free oxygen radicals released during preconditioning are likely to take part in the delayed protection through the production of peroxynitrite which activates PKC and through the increase of the activity of antioxidant enzymes such as Mn superoxide-dismutase. The production of heat shock proteins is considered a marker rather than a mechanism of SWOP.

Ischaemic preconditioning changes the pattern of coronary reactive hyperaemia in the goat: role of adenosine and nitric oxide

OBJECTIVES After ischaemic preconditioning (IP), obtained by short episodes of ischaemia, cardiac protection occurs due to a reduction in myocardial metabolism through the activation of A1 adenosine receptors. The antiarrhythmic effect of IP is attributed to an increase in the release of nitric oxide (NO) by the endothelium. On the basis of the above consideration the present investigation studies the changes induced by preconditioning in coronary reactive hyperaemia (RH) and how blockade of A1 receptors and inhibition of NO synthesis can modify these changes. METHODS In anaesthetised goats, an electromagnetic flow-probe was placed around the left circumflex coronary artery. Preconditioning was obtained with two episodes of 2.5 min of coronary occlusion, separated by 5 min of reperfusion. RH was obtained with a 15 s occlusion. In a control group (n = 7) RH was studied before and after IP. In a second group (n = 7), 0.2 mg kg-1 of 8-cyclopentyl-dipropylxanthine, an A1 receptor blocker, and in a third group (n = 7) 10 mg kg-1 of NG-nitro-L-arginine (LNNA), an NO inhibitor, were given before IP. Reactive hyperaemia was again obtained before and after IP. RESULTS In the control group, after IP, the time to peak hyperaemic flow and total hyperaemic flow decreased by about 50% and 25%, respectively. The A1 receptor blockade alone did not change RH. During A1 blockade, IP reduced the time to peak of RH similar as in control (45%), but did not alter total hyperaemic flow. LNNA alone reduced resting flow and total hyperaemic flow. After NO inhibition, IP only reduced total hyperaemic flow by about 15%, but the time to peak flow was not affected. CONCLUSIONS IP alters RH by decreasing total hyperaemic flow and reducing the time to peak hyperaemic flow. While the former effect is attributed to a reduction in myocardial metabolism through the activation of the A1 receptors, the latter is likely to be due to an increased endothelial release of NO, suggesting that in addition to a protective effect on the myocardium, IP also exerts a direct effect on the responsiveness of the coronary vasculature (vascular preconditioning).

The Gaboon viper, Bitis gabonica: Hemorrhagic, metabolic, cardiovascular and clinical effects of the venom

The effects of Bitis gabonica venom have been studied in several animal species, including the monkey, dog, rabbit, rat and guinea pig. Further information has been provided by observations on the effects of snake bite in man. Bitis gabonica venom exerts a number of cytotoxic and cardiovascular effects: cytotoxic effects include widespread hemorrhage, caused by the presence of two hemorrhagic proteins. These hemorrhagins bring about separation of vascular endothelial cells and extravasation of blood into the tissue spaces. Metabolic alterations include decreased oxygen utilization by tissues and increased plasma glucose and lactate concentrations. Metabolic non-compensated acidosis has also been seen in the rat as a consequence of the cytotoxicity of the venom. Cardiovascular effects include disturbances in atrio-ventricular conduction and reduction in amplitude and duration of the action potential brought about by a decreased calcium membrane conductance. A progressive decrease in myocardial contractility can also be attributed to the decreased calcium conductance, which together with the severe acidosis may cause death in experimental animals. A severe, though reversible, vasodilatation was observed after envenomation due to unidentified compounds in the venom. In man, envenomation causes a variable clinical picture depending on the time course and severity of envenomation. Frequently seen effects include hypotension, hemorrhage at the site of the bite and elsewhere and disseminated intravascular coagulation. Envenomation can be satisfactorily treated with antivenom.

NEW INSIGHTS INTO NITRIC OXIDE AND CORONARY CIRCULATION

Since its discovery over 20 years ago as an intercellular messenger, nitric oxide (NO), has been extensively studied with regard to its involvement in the control of the circulation and, more recently, in the prevention of atherosclerosis. The importance of NO in coronary blood flow control has also been recognized. NO-independent vasodilation causes increased shear stress within the blood vessel which, in turn, stimulates endothelial NO synthase activation, NO release and prolongation of vasodilation. Reactive hyperemia, myogenic vasodilation and vasodilator effects of acetylcholine and bradykinin are all mediated by NO. Ischemic preconditioning, which protects the myocardium from cellular damage and arrhythmias, is itself linked with NO and both the first and second windows of protection may be due to NO release. Exercise increases NO synthesis via increases in shear stress and pulse pressure and so it is likely that NO is an important blood flow regulatory mechanism in exercise. This phenomenon may account for the beneficial effects of exercise seen in atherosclerotic individuals. Whilst NO plays a protective role in preventing atherosclerosis via superoxide anion scavenging, risk factors such as hypercholesterolemia reduce NO release leading the way for endothelial dysfunction and atherosclerotic lesions. Exercise reverses this process by stimulating NO synthesis and release. Other factors impacting on the activity of NO include estrogens, endothelins, adrenomedullin and adenosine, the last appearing to be a compensatory pathway for coronary control in the presence of NO inhibition. These studies reinforce the pivotal role played by the substance in the control of coronary circulation.

THE ROLE OF NITRIC OXIDE IN THE INITIATION AND IN THE DURATION OF SOME VASODILATOR RESPONSES IN THE CORONARY CIRCULATION

In the coronary bed vasodilatation can be mediated by several mechanisms including endothelium-produced nitric oxide. To examine the contribution of nitric oxide, three different techniques to cause vasodilatation in the coronary vessels were used in the anaesthetized dog: intracoronary injection of 1 μg; acetylcholine, sudden reduction of the aortic blood pressure inducing a myogenic response and transient occlusion followed by release of the left circumflex coronary artery causing reactive hyperaemia. Each manoeuvre was performed before and after intracoronary adminstration of 100 mg N-nitro-l-arginine, an inhibitor of the synthesis of nitric oxide. In contrast to previous investigations, the inhibition of nitric oxide synthesis was prevented from causing an increase in blood pressure by the use of a blood-pressure-compensating device. The results observed during each of the three techniques, suggest that the initial cause of the vasodilatation is not the result of the increase of the production of nitric oxide. However, subsequent to the initiation of vasodilatation, an increase in the shear stress can result in an increase in the release of nitric oxide from the vascular endothelium, thus prolonging the vasodilatation obtained using each technique.

Myocardial, neural and vascular aspects of ischemic preconditioning

Ischemic preconditioning can be obtained with brief coronary occlusions. It has been studied in different animal species including dogs, pigs, rabbits and rats. The suggested duration of the occlusions ranges from four periods of 5 min, separated from each other by 5 min of reperfusion, to one period of 2.5 min. In addition to the reduction of the size of a subsequent infarction, preconditioning is responsible for the attenuation of the ischemia-reperfusion injury. The protection has a short duration and does not exceed two hours. Myocardial, neural and endothelial factors are involved in preconditioning. The myocardial component includes an increased release of adenosine with activation of A1 adenosine receptors, the activation of a protein-kinase C and possibly of antioxidant enzymes. The neural component includes a reduction in the release of noradrenaline from the postganglionic sympathetic fibers and a reduced myocardial sensitivity to noradrenaline. The increased myocardial release of adenosine, together with the reduced adrenergic activity, is consistent with the reduction in myocardial metabolism which has been observed after preconditioning. The coronary vascular endothelium is concerned in an increased release of nitric oxide which seems to be responsible for a prevention of reperfusion arrhythmias. In addition to the protective effect exerted on the myocardium, ischemic preconditioning seems to be responsible for a change in the coronary responsiveness to short periods of occlusion followed by release. This change in responsiveness is mainly represented by a greater velocity of the increase in flow occurring in the coronary reactive hyperemia.

Control of coronary blood flow by endothelial release of nitric oxide.

1. Nitric oxide (NO) is released from vascular endothelium following conversion of l‐arginine to l‐citrulline by calcium‐calmodulin‐dependent ‘constitutive’ NO‐synthase.