Gianni Losano

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.

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.

Apelin-13 limits infarct size and improves cardiac postischemic mechanical recovery only if given after ischemia

We studied whether apelin-13 is cardioprotective against ischemia/reperfusion injury if given as either a pre- or postconditioning mimetic and whether the improved postischemic mechanical recovery induced by apelin-13 depends only on the reduced infarct size or also on a recovery of function of the viable myocardium. We also studied whether nitric oxide (NO) is involved in apelin-induced protection and whether the reported ischemia-induced overexpression of the apelin receptor (APJ) plays a role in cardioprotection. Langendorff-perfused rat hearts underwent 30 min of global ischemia and 120 min of reperfusion. Left ventricular pressure was recorded. Infarct size and lactate dehydrogenase release were determined to evaluate the severity of myocardial injury. Apelin-13 was infused at 0.5 μM concentration for 20 min either before ischemia or in early reperfusion, without and with NO synthase inhibition by N(G)-nitro-l-arginine (l-NNA). In additional experiments, before ischemia also 1 μM apelin-13 was tested. APJ protein level was measured before and after ischemia. Whereas before ischemia apelin-13 (0.5 and 1.0 μM) was ineffective, after ischemia it reduced infarct size from 54 ± 2% to 26 ± 4% of risk area (P < 0.001) and limited the postischemic myocardial contracture (P < 0.001). l-NNA alone increased postischemic myocardial contracture. This increase was attenuated by apelin-13, which, however, was unable to reduce infarct size. Ischemia increased APJ protein level after 15-min perfusion, i.e., after most of reperfusion injury has occurred. Apelin-13 protects the heart only if given after ischemia. In this protection NO plays an important role. Apelin-13 efficiency as postconditioning mimetic cannot be explained by the increased APJ level.

Effect of endothelins on the cardiovascular system.

Endothelins (ETs) exert a persistent constrictor effect on the vessels via an increase in intracellular Ca2+ concentration due to the activation of Na+/H+ and Na+/Ca2+ exchangers of the vascular smooth muscle fibres. They also produce a transient dilator effect via the activation of endothelial nitric oxide synthase mediated by protein kinase B/Akt. ETA and ETB2 receptors are involved in vasoconstriction, whereas transient vasodilatation depends on the activation of ETB1 receptors. Depending on animal species and experimental conditions, ETs can also play a role in cardiac muscle contraction and induce either an increase or a decrease in contractility. It is likely that only ETA, and not ETB, receptors are involved in the ET-induced increase in myocardial contractility. As in the case of vasoconstriction, this inotropic effect depends on an increase in intracellular Ca2+ concentration induced by Na+/H+ and Na+/Ca2+ exchangers. Activation of the Na+/H+ exchanger is stimulated by protein kinase C, which is activated by diacylglycerol released in response to ET activity. It has also been proposed that the positive inotropic effect can occur without the contribution of the Na+/Ca2+ exchanger, if the cell alkalinisation produced by the Na+/H+ exchanger improves myofibrillar Ca2+ sensitivity. A reduction in contractility has been attributed to the involvement of the Gi protein/protein kinase G pathway or to the activation of protein kinase C without an increase in intracellular Ca2+ concentration or in myofibrillar Ca2+ sensitivity. The chronic effect of ETs on the myocardium results in hypertrophy and prevention of apoptosis, two processes that are together responsible for the contradictory effect of ETs in heart failure.

Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes.

In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST‐BODIPY‐Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X‐Rhod‐1. Immunofluorescence experiments showed the presence of connexin‐43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage‐dependent calcium signaling in cocultured MSCs. MSCs showed a time‐dependent increase of labeling of ST‐BODIPY‐Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non‐fluorescent DHP, Nifedipine, completely abolished ST‐BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization‐induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage‐dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte‐like phenotype. J. Cell. Biochem. 100: 86–99, 2007.

Effect of generalised sympathetic activation by cold pressor test on cerebral haemodynamics in healthy humans.

There is no general agreement regarding several aspects of the role of the sympathetic system on cerebral haemodynamics such as extent of effectiveness, operational range and site of action. This study was planned to identify the effect of a generalised sympathetic activation on the cerebral haemodynamics in healthy humans before it is masked by secondary corrections, metabolic or myogenic in nature. A total of 35 healthy volunteers aged 20-35 underwent a 5 min lasting cold pressor test (CPT) performed on their left hand. The cerebral blood flow (CBF) velocity in the middle cerebral arteries and arterial blood pressure were recorded with transcranial Doppler sonography and with a non-invasive finger-cuff method, respectively. The ratio of arterial blood pressure to mean blood velocity (ABP/Vm) and Pulsatility Index (PI) were calculated throughout each trial. CPT induced an increase in mean ABP (range 2-54 mmHg depending on the subject) and only a slight, though significant, increase in blood velocity in the middle cerebral artery (+2.4 and +4.4% on ipsi- and contralateral side, respectively). During CPT, the ratio ABP/Vm increased and PI decreased in all subjects on both sides. These changes began simultaneously with the increase in blood pressure. The increase in ABP/Vm ratio is attributed to an increase in the cerebrovascular resistance, while the concomitant reduction in PI is interpreted as due to the reduction in the compliance of the middle cerebral artery. The results suggest that generalised increases in the sympathetic discharge, causing increases in ABP, can prevent concomitant increases in CBF by acting on both small resistance and large compliant vessels. This effect is also present when a slight increase in blood pressure occurs, which suggests a moderate increase in the sympathetic discharge, i.e. when ABP remains far below the upper limit of CBF autoregulation.

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.

Early homing of adult mesenchymal stem cells in normal and infarcted isolated beating hearts

Little is known on the early homing features of transplanted mesenchymal stem cells (MSCs). We used the isolated rat heart model to study the homing of MSCs injected in the ventricular wall of a beating heart. In this model all types of cells and matrix elements with their interactions are represented, while external interferences by endothelial/neutrophil interaction and neurohormonal factors are excluded. We studied the morphology and marker expression of MSCs implanted in normal hearts and in the border‐zone of infarcted myocardium. Early morphological adaptation of MSC homing differs between normal and infarcted hearts over the first 6 hrs after transplantation. In normal hearts, MSCs migrate very early through the interstitial milieu and begin to show morphological changes. Yet, in infarcted hearts MSCs remain in the site of injection forming clusters of round‐shaped cells in the border‐zone of the infarcted area. Both in normal and infarcted hearts, immuno‐histochemistry and confocal imaging showed that, besides the proliferative marker proliferating cell nuclear agent (PCNA), some transplanted cells early express myoblastic maker GATA‐4, and some of them show a VWF immunopositivity. Moreover, a few hours after injection connexin‐43 is well evident between cardiomy‐ocytes and injected cells. This study indicates for the first time that the isolated beating heart is a good model to study early features of MSC homing without external interferences. The results show (i) that MSCs start to change marker expression few hours after injection into a beating heart and (ii) that infarcted myocardium influences transplanted MSC morphology and mobility within the heart.