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Previous studies have shown that the pial microcirculation remodeling improves neurological outcome after middle cerebral artery occlusion (MCAO), accompanied by higher expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS), modulating in vivo angiogenesis.This study was aimed to assess the effects of bone marrow mesenchymal stem cells (BM-MSCs) infused after MCAO on rat pial microcirculation.Animals were subjected to 2 hour MCAO followed by BM-MSCs infusion into internal carotid artery. Pial microcirculation was observed at different reperfusion times by fluorescence microscopy. Geometric characteristics of arteriolar networks, permeability increase, leukocyte adhesion, perfused capillary density, VEGF and endothelial nitric oxide synthase (e-NOS) expression were evaluated. Green fluorescent protein (GFP)-BM-MSCs were used to evaluate their distribution and cell phenotype development during reperfusion. BM-MSCs stimulated a geometric rearrangement of pial networks with formation of new anastomotic vessels sprouting from preexistent arterioles in the penumbra at 7-14- 28 days of reperfusion. At the same time VEGF and eNOS expression increased. GFP-BM-MSCs appear to be involved in endothelial and smooth muscle cell programming in the infarcted area. In conclusion, transient MCAO induced pial vascular remodeling characterized by arteriolar anastomotic arcades (originated from pre-existent arterioles in penumbra area) able to overlap the ischemic core supplying blood to the neuronal tissue. BM-MSCs appear to accelerate angiogenic processes facilitating new vessel formation; this mechanism was promoted by an increase in VEGF and eNOS expression.

In some pathophysiological conditions myocardial metabolism can switch from mainly long chain fatty acid (LCFA) oxidation to mainly glucose oxidation. Whether the predominant fatty acid or glucose oxidation affects cardiac performance has not been defined. In a buffer perfused isovolumetrically contracting rat heart, oxidation of endogenous pool LCFA was avoided by inhibiting carnitine-palmitoyl-transferase I (CPT-I) with oxfenicine (2 mM). In order to restore fatty acid oxidation, hexanoate (1 mM), which bypasses CPT-I inhibition, was added to the perfusate. Three groups of hearts were subjected to either an increase in left ventricular volume (VV, +25%) or an increase in coronary flow (CF, +50%), or inotropic stimulation with isoproterenol (10(-8) and 10(-6) m). The increase in VV (the Frank-Starling mechanism) increased rate-pressure product (RPP) by 21 +/- 2% under control conditions, but only by 6 +/- 2% during oxfenicine-induced CPT-I inhibition. The contractile response to changes in VV recovered after the addition of hexanoate. Similar results were obtained in hearts, in which an increase in CF was elicited (the Gregg phenomenon). Isoproterenol caused a similar increase in contractility regardless of the presence of oxfenicine or hexanoate. In all groups, a commensurate increase in oxygen consumption accompanied the increase in contractility. The fatty acid oxidation is necessary for an adequate contractile response of the isolated heart to increased pre-load or flow, whereas the inotropic response to adrenergic beta-receptor stimulation is insensitive to changes in substrate availability.

The present study was planned to investigate the effect of left ventricular pressure and inotropic state on coronary arterial inflow in systole in the anaesthetized dog. A wide range of left ventricular systolic pressures, including the physiological range, were studied. Experiments were done under conditions of maximal vasodilatation and low perfusion pressure in order to avoid vascular autoregulative interference and to keep the microvascular pressure within the normal range. In five anaesthetized dogs, perfused with extracorporeal circulation system, ventricular volume was changed from 20 to 50 ml in steps of 10 ml by filling an intraventricular latex balloon, and the related changes in left ventricular pressure and coronary flow were measured. The volume was then extended to 70 ml to obtain an overstretch which induced a transient decrease in cardiac contractility. During the period of low cardiac contractility the volume was brought back to 20 ml in steps of 10 ml. Systolic ventricular pressure changed with volume but was lower during the period of low contractility. For systolic pressures below 100 mmHg there was no significant relationship between pressure and coronary systolic flow, but the relationship shifted to higher flows during low contractility. For systolic pressures above 100 mmHg systolic coronary flow decreased significantly when systolic pressure increased. In this case the slopes of the relationships were not significantly different before and after the reduction in contractility. These findings suggest that for systolic pressures less than 100 mmHg (i.e. below the physiological range) the shielding effect of the contracting ventricle prevents the ventricular pressure from being transmitted in the myocardial wall. When systolic pressure exceeds 100 mmHg the shielding effect is overcome and the amplitude of the systolic flow reduction varies with ventricular pressure.

See article by Misao et al. [16] (pages 455–465) in this issue. For many years, the regeneration of the myocardium in which cells have been lost as a result of myocardial infarction (MI) and other forms of cardiac pathology has been a utopian goal of cardiologists. Recent experimental studies and early-phase clinical trials supported the possibility of enhancing cardiac repair as an achievable therapeutic target. Yet, disappointing, negative results have been reported both in experimental and clinical studies [1–3]. There can be several reasons for negative results. Certainly, the fact that we do not know the exact physiology of stem cells is one of the reasons. We do not know yet the ideal cell type: bone marrow stem cells (BMSCs), cardiomyoplasty cardiomyocytes, skeletal myoblasts, and resident stem cells have been tested, and among BMSCs several cell lines (MSCs, EPCs, etc.) have been proposed [1–3]. We do not know the underlying mechanism of cellular regeneration (transdifferentiation, fusion, and/or paracrine effects). Finally, we do not know whether myocardial repair can be better achieved via cellular transplantation (intramyocardial, intracoronary, and/or systemic delivery) or mobilization of primitive BMSCs by cytokines. Each of these approaches may be adequate for specific patients. Undoubtedly, mobilization of BMSCs might offer a noninvasive therapeutic approach that is appealing for both physicians and patients. The bone marrow is a large reservoir of adult stem cells distal from the heart that can be enrolled for cell-mediated cardiac repair via regeneration of healthy myocardial tissue and recovery of cardiac function. These could be accomplished by neo-angiogenesis, cardiogenesis, and/or paracrine effects. In its … * Tel.: +39 011 6705430; fax: +39 011 9038639. Email address: pasquale.pagliaro{at}unito.it

Background In the presence of comorbidities the effectiveness of many cardioprotective strategies is blunted. The goal of this study was to assess in a hypertensive rat model if the early reperfusion with anti-hypertensive and pro-angiogenic Chromogranin A-derived peptide, Catestatin (CST:hCgA352–372; CST-Post), protects the heart via Reperfusion-Injury-Salvage-Kinases (RISK)-pathway activation, limiting infarct-size and apoptosis, and promoting angiogenetic factors (e.g., hypoxia inducible factor, HIF-1α, and endothelial nitric oxide synthase, eNOS, expression). Methods and Results The effects of CST-Post on infarct-size, apoptosis and pro-angiogenetic factors were studied in isolated hearts of spontaneously hypertensive rats (SHR), which underwent the following protocols: (a) 30-min ischemia and 120-min reperfusion (I/R); (b) 30-min ischemia and 20-min reperfusion (I/R-short), both with and without CST-Post (75 nM for 20-min at the beginning of reperfusion). In unprotected Wistar-Kyoto hearts, used as normal counterpart, infarct-size resulted smaller than in SHR. CST-Post reduced significantly infarct-size and improved post-ischemic cardiac function in both strains. After 20-min reperfusion, CST-Post induced S-nitrosylation of calcium channels and phosphorylation of RISK-pathway in WKY and SHR hearts. Yet specific inhibitors of the RISK pathway blocked the CST-Post protective effects against infarct in the 120-min reperfusion groups. Moreover, apoptosis (evaluated by TUNEL, ARC and cleaved caspase) was reduced by CST-Post. Importantly, CST-Post increased expression of pro-angiogenetic factors (i.e., HIF-1α and eNOS expression) after two-hour reperfusion. Conclusions CST-Post limits reperfusion damages and reverses the hypertension-induced increase of I/R susceptibility. Moreover, CST-Post triggers antiapoptotic and pro-angiogenetic factors suggesting that CST-Post can be used as an anti-maladaptive remodeling treatment.

We studied the effects of zoledronic acid (ZA) and leuprore- lin acetate (LA) separately and combined on a human cell line derived from a bone metastasis of prostatic adenocarcinoma (PC3). In particu- lar, we focused on the effects that drugs given singularly or in associ- ation may play on tumor evolution and metastatization. Cell prolifera- tion, 2D- and 3D-migration were studied in basal conditions and under attractive stimuli exerted by bone marrow mesenchymal stem cells (BM-MSC). Either drug decreased PC3 proliferation, though ZA was much more cytotoxic than LA. However, LA cytotoxic concentrations are higher than those usually reached in vivo. Subtoxic concentration of either drug inhibited migration especially under BM-MSC medium stimuli via an Akt-dependent mechanism. The capability of either drug to inhibit cellular migration is in line with their well-known effect in limiting metastatization. Intriguingly, no additive effect on the antipro- liferative activity or in hindering migration is observed when the drugs are administered concomitantly, compared to the effects of each drug alone.

Despite the enormous progress in the treatment of coronary artery diseases, they remain the most common cause of heart failure in the Western countries. New translational therapeutic approaches explore cardiomyogenic differentiation of various types of stem cells in combination with tissue-engineered scaffolds. In this study we fabricated PHBHV/gelatin constructs mimicking myocardial structural properties. Chemical structure and molecular interaction between material components induced specific properties to the substrate in terms of hydrophilicity degree, porosity and mechanical characteristics. Viability and proliferation assays demonstrated that these constructs allow adhesion and growth of mesenchymal stem cells (MSCs) and cardiac resident non myocytic cells (NMCs). Immunofluorescence analysis demonstrated that stem cells cultured on these constructs adopt a distribution mimicking the three-dimensional cell alignment of myocardium. qPCR and immunofluorescence analyses showed the ability of this construct to direct initial MSC and NMC lineage specification towards cardiomyogenesis: both MSCs and NMCs showed the expression of the cardiac transcription factor GATA-4, fundamental for early cardiac commitment. Moreover NMCs also acquired the expression of the cardiac transcription factors Nkx2.5 and TBX5 and produced sarcomeric proteins. This work may represent a new approach to induce both resident and non-resident stem cells to cardiac commitment in a 3-D structure, without using additional stimuli. © 2013 Elsevier Ltd.

Scientific objectives Despite enormous progresses in the treatment of coronary artery disease, it remains the most common cause of heart failure and the leading cause of death in the Western countries. New translational therapeutic approaches based on personalized and regenerative medicine explore cardiomyogenic differentiation of various types of stem cells by electrical stimulation, biochemical inducers, or cell co-culturing [1-3]. In this study we fabricated bioartificial constructs mimicking anisotropic structure and mechanical properties of the myocardium [4].

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.

Melusin is a muscle-specific protein which interacts with β1 integrin cytoplasmic domain and acts as chaperone protein. Its overexpression induces improved resistance to cardiac overload delaying left ventricle dilation and reducing the occurrence of heart failure. Here, we investigated possible protective effect of melusin overexpression against acute ischemia/reperfusion (I/R) injury with or without Postconditioning cardioprotective maneuvers. Melusin transgenic (Mel-TG) mice hearts were subjected to 30-min global ischemia followed by 60-min reperfusion. Interestingly, infarct size was reduced in Mel-TG mice hearts compared to wild-type (WT) hearts (40.3 ± 3.5 % Mel-TG vs. 59.5 ± 3.8 % WT hearts; n = 11 animals/group; P < 0.05). The melusin protective effect was also demonstrated by measuring LDH release, which was 50 % lower in Mel-TG compared to WT. Mel-TG hearts had a higher baseline level of AKT, ERK1/2 and GSK3β phosphorylation, and displayed increased phospho-kinases level after I/R compared to WT mice. Post-ischemic Mel-TG hearts displayed also increased levels of the anti-apoptotic factor phospho-BAD. Importantly, pharmacological inhibition of PI3K/AKT (Wortmannin) and ERK1/2 (U0126) pathways abrogated the melusin protective effect. Notably, HSP90, a chaperone known to protect heart from I/R injury, showed high levels of expression in the heart of Mel-TG mice suggesting a possible collaboration of this molecule with AKT/ERK/GSK3β pathways in the melusin-induced protection. Postconditioning, known to activate AKT/ERK/GSK3β pathways, significantly reduced IS and LDH release in WT hearts, but had no additive protective effects in Mel-TG hearts. These findings implicate melusin as an enhancer of AKT and ERK pathways and as a novel player in cardioprotection from I/R injury.

Coronary artery disease is a major cause of morbidity and mortality in the Western countries. Acute myocardial infarction is a serious and often lethal consequence of coronary artery disease, resulting in contractile dysfunction and cell death. It is well known that unbalanced and high steady state levels of reactive oxygen and nitrogen species (ROS/RNS) are responsible for cytotoxicity, which in heart leads to contractile dysfunction and cell death. Pre- and post-conditioning of the myocardium are two treatment strategies that reduce contractile dysfunction and the amount of cell death considerably. Paradoxically, ROS and RNS have been identified as a part of cardioprotective signaling molecules, which are essential in pre- and post-conditioning processes. S-nitrosylation of proteins is a specific posttranslational modification that plays an important role in cardioprotection, especially within mitochondria. In fact, mitochondria are of paramount importance in either promoting or limiting ROS/RNS generation and reperfusion injury, and in triggering kinase activation by ROS/RNS signaling in cardioprotection. These organelles are also the targets of acidosis, which prevents mitochondrial transition pore opening, thus avoiding ROS-induced ROS release. Therefore, we will consider mitochondria as either targets of damage or protection from it. The origin of ROS/RNS and the cardioprotective signaling pathways involved in ROS/RNS-based pre- and post-conditioning will be explored in this article. A particular emphasis will be given to new aspects concerning the processes of S-nitrosylation in the cardioprotective scenario.

Postconditioning (PostC) can be obtained either with brief cycles of ischemia/reperfusion (I-PostC) or with a direct targeting of mitochondria with Diazoxide (pharmacological PostC, P-PostC). I-PostC may induce the activation of RISK and SAFE pathways and may favor nitric oxide production with S-Nitrosylation of proteins and redox signaling. It is not clear whether Diazoxide can lead to similar effects. We compared the effects of I-PostC and P-PostC on (a) kinases of RISK- and SAFE pathway, (b) S-Nitrosylation of mitochondrial proteins and (c) reduction of death signals (PKCδ, cleaved caspase-3 and Beclin-1) in cytosolic and mitochondrial fractions. Isolated rat hearts underwent (1) perfusion without ischemia (Sham), (2) ischemia/reperfusion (30-min ischemia plus 2-h reperfusion), (3) I-PostC (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia), (4) P-PostC (Diazoxide 30 μM in the first of 3-min of reperfusion) or (5) I-PostC + MPG or P-PostC + MPG (MPG, 2-mercaptopropionylglycine 300 μM). Using Western blot and biotin switch assay, we found that P-PostC induced a redox sensible phosphorylation/translocation of Akt, ERK1/2 and GSK3β into the mitochondria, but not of phospho-STAT3, which was translocated into the mitochondria by I-PostC only. Either I-PostC or P-PostC increased mitochondrial S-Nitrosylated proteins (e.g., VDAC) and reduced the levels of phospho-PKCδ, cleaved caspase-3 and Beclin-1. Therefore, direct targeting of mitochondria with Diazoxide (a) activates the RISK pathway via a redox signaling, (b) favors discrete mitochondrial protein S-Nitrosylation, including VDAC and (c) decreases signals of death. Intriguingly, phospho-STAT3 translocation is induced by I-PostC, but not by P-PostC, thus suggesting a redox-independent mechanism in the SAFE pathway.

GHRH stimulates GH synthesis and release from the pituitary and exerts direct effects in extrapituitary tissues. We have previously shown that pretreatment with GHRH reduces cardiomyocyte apoptosis and improves heart function in isolated rat hearts subjected to ischemia/reperfusion (I/R). Here, we determined whether GHRH given at reperfusion reduces myocardial reperfusion injury and investigated the molecular mechanisms involved in GHRH effects. Isolated rat hearts subjected to I/R were treated at the onset of reperfusion with: 1) GHRH; 2) GHRH+GHRH antagonist JV-1-36; 3) GHRH+mitochondrial ATP-dependent potassium channel inhibitor 5-hydroxydecanoate; 4) GHRH+mitochondrial permeability transition pore opener atractyloside; 5) GHRH+ phosphoinositide 3-kinase/Akt inhibitor Wortmannin (WM); and 6) GHRH+signal transducer and activator of transcription-3 inhibitor tyrphostin-AG490 (AG490). GHRH reduced infarct size at the end of reperfusion and reverted contractility dysfunction in I/R hearts. These effects were inhibited by either JV-1-36, 5-hydroxydecanoate, atractylosid, WM, or AG490. Western blot analysis on left ventricles showed GHRH-induced phosphorylation of either the reperfusion injury salvage kinases (RISK), phosphoinositide 3-kinase/Akt, ERK1/2, and glycogen synthase kinase-3β or signal transducer and activator of transcription-3, as part of the survivor activating factor enhancement (SAFE) pathway. GHRH-induced activation of RISK and SAFE pathways was blocked by JV-1-36, WM, and AG490. Furthermore, GHRH increased the phosphorylation of endothelial nitric oxide synthase and AMP-activated protein kinase and preserved postischemic nicotinamide adenine dinucleotide (NAD+) levels. These results suggest that GHRH protects the heart from I/R injury through receptor-mediated mechanisms, leading to activation of RISK and SAFE pathways, which converge on mitochondria and possibly on AMP-activated protein kinase.

Background Acidic perfusion (AP) performed at the onset of reperfusion (i.e., acid postconditioning) is cardioprotective. We investigated the effect of AP on postischemic cardiac function and on the activity of endogenous superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase. The role of exogenous CAT or SOD on AP cardioprotection was also investigated. Phosphorylation of redox-sensitive survival kinases (protein kinase C [PKC] e and extracellular signal–regulated kinase [ERK] 1/2) was also checked. Materials and methods Isolated rat hearts underwent ischemia and reperfusion (I/R) for 30 and 120 min, respectively. AP was obtained by lowering [ HCO 3 − ] in the perfusion buffer. Infarct size and left ventricular pressure were measured. Protocols include I/R only, I/R plus acidic perfusion in early reperfusion (I/R + AP), and I/R plus AP and CAT (I/R + AP + CAT) or SOD (I/R + AP + SOD). I/R + SOD and I/R + CAT additional hearts served as controls. AP and/or antioxidants were given in the initial 3 min of reperfusion. Enzyme activities were studied in postischemic phase (seventh minute of reperfusion) in I/R or I/R + AP and Sham (buffer-perfused) hearts. Results AP with (I/R + AP + CAT or I/R + AP + SOD) or without (I/R + AP) antioxidant enzymes resulted in a larger reduction of infarct size compared with I/R, I/R + SOD, or I/R + CAT. Compared with I/R, the postischemic systolic and diastolic recoveries of the cardiac function were markedly improved by the addition of AP and a lesser extent by AP + SOD or AP + CAT. AP increased the postischemic activity of CAT and lowered that of SOD and glutathione peroxidase compared with I/R only. Also, the phosphorylation and activity of ERK1/2 and PKCe were increased by AP. Conclusions Acid postconditioning affects the activity of endogenous antioxidant enzymes, activates ERK1/2–PKCe pathways, and protects against myocardial I/R injury. The combination of AP and exogenous SOD or CAT still provides cardioprotection. It is likely that intracellular (not extracellular) redox condition plays a pivotal role in acidic protection.

The history began in the 19th century with Ascanio Sobrero (1812-1888), the discoverer of glycerol trinitrate (nitroglycerine, NTG), and with Angelo Angeli (1864-1931), the discoverer of sodium trioxodinitrate (Angeli's salt). It is likely that Angeli and Sobrero never met, but their two histories will join each other more than a century later. In fact, it has been discovered that both NTG and Angeli's salt are able to induce a preconditioning effect. As NTG has a long history as an antianginal drug its newly discovered property as a preconditioning agent has also been tested in humans. Angeli's salt properties as a preconditioning and inotropic agent have only been tested in animals so far.

Catestatin (CST) limits myocardial ischaemia/reperfusion (I/R) injury with unknown mechanisms. Clearly phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) isoforms, including intra-mitochondrial PKCe, mitochondrial KATP (mitoKATP) channels and subsequent reactive oxygen species (ROS)-signalling play important roles in postconditioning cardioprotection, preventing mitochondrial permeability transition pore (mPTP) opening. Therefore, we studied the role of these extra- and intra-mitochondrial factors in CST-induced protection. Isolated rat hearts and H9c2 cells underwent I/R and oxidative stress, respectively. In isolated hearts CST (75nM, CST-Post) given in early-reperfusion significantly reduced infarct size, limited post-ischaemic contracture, and improved recovery of developed left ventricular pressure. PI3K inhibitor, LY-294002 (LY), large spectrum PKC inhibitor, Chelerythrine (CHE), specific PKCe inhibitor (eV1-2), mitoKATP channel blocker, 5-Hydroxydecanoate (5HD) or ROS scavenger, 2-mercaptopropionylglycine (MPG) abolished the infarct-sparing effect of CST. Notably the CST-induced contracture limitation was maintained during co-infusion of 5HD, MPG or eV1-2, but it was lost during co-infusion of LY or CHE. In H9c2 cells challenged with H2O2, mitochondrial depolarization (an index of mPTP opening studied with JC1-probe) was drastically limited by CST (75nM). Our results suggest that the protective signalling pathway activated by CST includes mitoKATP channels, ROS signalling and prevention of mPTP opening, with a central role for upstream PI3K/Akt and PKCs. In fact, all inhibitors completely abolished CST-infarct-sparing effect. Since CST-anti-contracture effect cannot be explained by intra-mitochondrial mechanisms (PKCe activation and mitoKATP channel opening) or ROS signalling, it is proposed that these downstream signals are part of a reverberant loop which re-activates upstream PKCs, which therefore play a pivotal role in CST-induced protection.

We recently reported that heart expresses functional receptors for the anorexigenic glucagon-like peptide (GLP)-2. Activation of these cardiac receptors affected basal heart performance through extracellular regulated kinase (ERK1/2) activation. Since ERK1/2 is considered one of the prosurvival kinases of postconditioning cardioprotective pathways, we hypothesized that GLP-2 directly protects the heart against ischemia/reperfusion (I/R) injury via prosurvival kinases. Wistar rat hearts were retrogradely perfused on a Langendorff perfusion apparatus. After 40-min stabilization, hearts underwent 30-min global ischemia and 120-min reperfusion (I/R group). In GLP-2 group, the hearts received 20-min GLP-2 (10(-7) M) infusion at the beginning of the 120-min reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated by nitroblue-tetrazolium staining. Compared with the I/R group, GLP-2-treated hearts showed a significant reduction of infarct size and of postischemic diastolic LVP (index of contracture), together with a sharp improvement of developed LVP recovery (index of contractility). The protective effects were abolished by co-infusion with phosphatidylinositol 3-kinase inhibitor, Wortmannin (WT), the ERK1/2 inhibitor, PD98059, or the mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate. GLP-2 effects were accompanied by increased phosphorylation of protein kinase B (PKB/Akt), ERK1/2 and glycogen synthase kinase (GSK3β). After 7-min reperfusion, WT blocked Akt and GSK3β phosphorylation. After 30-min reperfusion, WT inhibited phosphorylation of all kinases. In conclusion, the data suggest that GLP-2, given in early reperfusion, as postconditioning, protects against myocardial I/R injury, limiting infarct size, and improving post-ischemic mechanical recovery. It seems that the GLP-2-protection of rat heart involves multiple prosurvival kinases and mitochondrial K(ATP) channels.

Chromogranin A (CgA) is produced by cells of the sympathoadrenal system and by human ventricular myocardium. In the clinical setting CgA has been mainly used as a marker of neuroendocrine tumors, but in the last decade a plenty of data have been published on the role of CgA and its derived peptides, particularly catestatin and vasostatin, in the regulation of cardiovascular function and diseases, including heart failure and hypertension. CgA-derived peptides, namely catestatin and vasostatin, may exert negative inotropic and lusitropic effects on mammalian hearts. As such CgA and its derived peptides may be regarded as mediators of a complex feedback system able to modulate the exaggerated release of catecholamines. This system may be also interpreted as an attempt for compensatory cardioprotective response against myocardial injury in the pre and postischemic scenarios. In fact, while vasostatin can trigger cardioprotective effects akin ischemic preconditioning (protection is triggered before ischemia), catestatin is a potent cardioprotective agent in the early post-ischemic phase, acting like a postconditioning agent (protection is triggered at the onset of reperfusion). Admittedly, the exact mechanism of cardioprotection of this system is far from being fully understood. Interestingly, both vasostatin and catestatin have shown to be able to activate multiple cardioprotective pathways. In particular, these two CgA-derived peptides may induce nitric oxide dependent pathway, which may play a pivotal role in cardioprotection against ischemia/reperfusion injury. Here, we review the literature about the cardiac effects of catestatin and vasostatin, the mechanisms of myocardial injury and protection and the role of CgA derived peptides in cardioprotection.

The effects of transient changes in coronary transmural pressure on the coronary vasomotor tone were studied in 23 anesthetized dogs. Increases and decreases of the coronary transmural pressure were obtained by constrictions of various duration (2 to 20 s) of the descending thoracic aorta. The maneuvers were performed in animals with intact cardiac innervation, with the vagi sectioned and with vagal section together with beta-blockade. In the absence of beta-blockade the increase in the transmural pressure caused a transient increase in the coronary vasomotor tone attributable to a myogenic contractile response and the extravascular compression. This contractile response was not observed when the transmural pressure was increased in the presence of high vasomotor tone after beta-blockade. In all animals a transient hyperemia was seen with its peak 8 to 12 s after the release of the aortic constriction. Since its timing and amplitude were independent of the duration of the constriction, the metabolic effect of the increased ventricular afterload, although it may have contributed to the decrease of the coronary resistance, cannot be considered entirely responsible for the hyperemia, which was otherwise compatible with a myogenic vasodilatory response triggered by the sudden fall of the transmural pressure at the release of the constriction. It is concluded that, in the coronary circulation of the intact dog, transient changes in transmural pressure can induce vasomotor responses in which myogenic and metabolic mechanisms combine together in regulating the coronary flow. Changes in extravascular compression can also affect the flow when the experimental maneuver implies changes in the diastolic left ventricular pressure and volume. With the present experimental procedure the myogenic responses have been evidenced when the metabolic factors would have been expected to produce opposite changes in the vasomotor tone.

The present study was planned to investigate whether or not, after complete suppression of vasomotor tone, increases in intravascular blood pressure distend the coronary vasculature causing passive decreases in the resistance to the coronary arterial inflow during the diastole. In anaesthetized dogs, aortic and left ventricular pressures and flow in the left circumflex coronary artery were recorded. Coronary flow was derived using an electromagnetic flowmeter. Transient (10 s) increases in intravascular blood pressure in a range above 70 mm Hg were produced by mechanical constriction of the descending thoracic aorta. In the presence of a normal vasomotor tone the increase in blood pressure caused an autoregulatory increase in the mean diastolic coronary inflow resistance. After maximal vasodilatation by dipyridamole, no change in inflow resistance was induced by the increase in intravascular blood pressure. It may be argued that while a non-maximal vasodilatation is reported to increase coronary distensibility, at a blood pressure of 70 mm Hg the complete suppression of the vasomotor tone brings the vascular radius to a size which cannot be further distended by an increase in blood pressure.

It is well recognised that regular exercise is cardioprotective. On the other hand, sedentariety is an established risk factor for cardiovascular diseases. Furthermore, several clues suggest that exercise capacity is a strong predictor of risk of death from any cause in both healthy subjects and in those with cardiovascular diseases. However, the exact mechanisms through which regular physical activity confers cardiovascular protection are not yet well understood. Exercise probably acts at multiple levels. One possibility is that regular exercise modifies genes expression, thereby changing the production of bioactive molecules such as proteins and enzymes; another potential site of action is on the mechanisms of cardiovascular regulation during effort, which are improved after periods of training; finally, exercise can affect endothelial and platelet functions, thus reducing the risks of atherosclerosis and consequently the risk of infarction and stroke. This special issue focuses on potential beneficial mechanisms by which exercise operates to confer protection against cardiovascular diseases. The interesting paper by S. Heber and I. Volf reviews the effects of inactivity and training on platelet functions. In their literature review, they conclude that regular physical activity diminishes or prevents platelet activation in response to acute exercise and that habitual physical activity also positively modulates platelet functions. They also conclude that these effects support the well-recognised relation between exercise and the risk for cardiovascular events, as a physically active lifestyle dramatically reduces cardiovascular mortality. In another intriguing review, C.-Y. Hsu et al. focus on the important topic of the effects of exercise training on autonomic function in chronic heart failure (CHF). Exercise intolerance is one of the major and disturbing symptoms in these patients. Increased sympathetic tone and decreased parasympathetic activity have been often reported in CHF and these phenomena are associated with a poor survival. Data from this review indicate that participation in exercise training programs induces beneficial effects on autonomic function in CHF patients. They also point out that further research could examine additional aspects of the effects of exercise training in this population, such as the impact on the responses to exercise training in different levels of CHF severity, the possibility that a threshold intensity may be needed to affect cardiac autonomic function, and the type of exercise that should be recommended to achieve the highest positive effects on sympathovagal balance. In a third review by A. Crisafulli et al., the complex issue of cardiovascular reflexes during exercise is addressed. During exercise, the neural mechanisms controlling the cardiovascular apparatus regulate cardiac output and arteriolar tone in order to counteract the exercise-induced vasodilation due to functional sympatholysis in the working muscle. These cardiovascular adjustments guarantee adequate perfusion to vital organs (the brain and the heart) and to the working muscles as well as adequate washout of exercise-induced by-products. Moreover, these mechanisms prevent excessive increments in blood pressure. In this review, authors summarise neural reflexes operating during dynamic exercise, particularly their interaction. They point out that cardiovascular regulation during exercise is achieved through the contemporary integration and interaction of input arising from motor cortex (central command), skeletal muscle receptors (exercise pressor reflex), and arterial baroreceptors. They also conclude that further research is warranted to better understand how these reflexes interact during effort. The last interesting review by R. B. Batacan Jr. et al. is about the effects of light intensity activity on cardiovascular risk factors. These authors claim that there is little support for the role of light intensity activity to reduce cardiovascular disease risk factors and that further studies are needed to establish the value of light intensity physical activity in reducing cardiovascular risk factors. Finally, the research paper by N. G. Rocha et al. aims at evaluating the acute effects of exercise on endothelial functions in early metabolic syndrome. They find that these subjects, despite being free of symptoms, present with an early impairment of endothelial function. Moreover, they put forward the hypothesis that the analysis of some biomarkers changes could be potentially useful to develop preventive measures before the onset of overt metabolic syndrome. Collectively, these manuscripts confirm that sedentariety is harmful for the cardiovascular system. Moreover, exercise not only exerts positive effect on cardiovascular functions and reduces the risks of cardiovascular diseases (primary prevention), but could also revert cardiovascular disease and play a preventive role in the progression of pathological conditions (secondary/tertiary prevention).

Reperfusion therapy must be applied as soon as possible to attenuate the ischemic insult of acute myocardial infarction (AMI). However reperfusion is responsible for additional myocardial damage, which likely involves opening of the mitochondrial permeability transition pore (mPTP). In reperfusion injury, mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability. Major mechanisms of mitochondrial dysfunction include the long lasting opening of mPTPs and the oxidative stress resulting from formation of reactive oxygen species (ROS). Several signaling cardioprotective pathways are activated by stimuli such as preconditioning and postconditioning, obtained with brief intermittent ischemia or with pharmacological agents. These pathways converge on a common target, the mitochondria, to preserve their function after ischemia/reperfusion. The present review discusses the role of mitochondria in cardioprotection, especially the involvement of adenosine triphosphate-dependent potassium channels, ROS signaling, and the mPTP. Ischemic postconditioning has emerged as a new way to target the mitochondria, and to drastically reduce lethal reperfusion injury. Several clinical studies using ischemic postconditioning during angioplasty now support its protective effects, and an interesting alternative is pharmacological postconditioning. In fact ischemic postconditioning and the mPTP desensitizer, cyclosporine A, have been shown to induce comparable protection in AMI patients.

Postconditioning (PostC) modifies the early post-ischemic pH, redox environment, and activity of enzymes. We hypothesized that early acidosis in PostC may affect superoxide dismutase (SOD) and catalase (CAT) activities, may reduce 3-nitrotyrosine (3-NT) protein levels, and may increase S-nitrosylated (SNO) protein levels, thus deploying its protective effects. To verify this hypothesis, we studied the early (7(th) min) and late (120(th) min) phases of reperfusion (a) endogenous SOD and CAT activities and (b) 3-NT protein levels and SNO protein levels. Isolated rat hearts underwent 30-min ischemia/120-min reperfusion (I/R) or PostC (5 cycles of 10-s I/R at the beginning of 120-min reperfusion) either with or without exogenous CAT or SOD infused during the initial 3 min of reperfusion. The effects of early reperfusion with acid buffer (AB, pH 6.8) on endogenous antioxidant enzymes were also tested. Pressure, infarct size, and lactate dehydrogenase release were also measured. At the 7(th) min, PostC induced a significant decrease in SOD activity with no major change both in Mn and Cu/Zn SOD levels and in CAT activity and level. PostC also reduced 3-NT and increased SNO levels. Exogenous SOD, but not CAT, abolished PostC cardioprotection. In late reperfusion (120-min), I/R increased SOD activity but decreased CAT activity and Cu/Zn SOD levels; these effects were reversed by PostC; 3-NT was not affected, but SNO was increased by PostC. AB reproduced PostC effects on antioxidant enzymes. The conclusions are as follows: PostC downregulates endogenous SOD and preserves CAT activity, thus increasing SNO and reducing 3-NT levels. These effects are triggered by early post-ischemic acidosis. Yet acidosis-induced SOD downregulation may limit denitrosylation, thus contributing to PostC triggering. Hence, exogenous SOD, but not CAT, interferes with PostC triggering. Prolonged SOD downregulation and SNO increase may contribute to PostC and AB beneficial effects.

The nitric oxide (NO center dot) sibling, nitroxyl or nitrosyl hydride (HNO), is emerging as a molecule whose pharmacological properties include providing functional support to failing hearts. HNO also preconditions myocardial tissue, protecting it against ischemia-reperfusion injury while exerting vascular antiproliferative actions. In this review, HNO's peculiar cardiovascular assets are discussed in light of its unique chemistry that distinguish HNO from NO center dot as well as from reactive oxygen and nitrogen species such as the hydroxyl radical and peroxynitrite. Included here is a discussion of the possible routes of HNO formation in the myocardium and its chemical targets in the heart. HNO has been shown to have positive inotropic/lusitropic effects under normal and congestive heart failure conditions in animal models. The mechanistic intricacies of the beneficial cardiac effects of HNO are examined in cellular models. In contrast to beta-receptor/cyclic adenosine monophosphate/protein kinase A-dependent enhancers of myocardial performance, HNO uses its "thiophylic'' nature as a vehicle to interact with redox switches such as cysteines, which are located in key components of the cardiac electromechanical machinery ruling myocardial function. Here, we will briefly review new features of HNO's cardiovascular effects that when combined with its positive inotropic/lusitropic action may render HNO donors an attractive addition to the current therapeutic armamentarium for treating patients with acutely decompensated congestive heart failure. Antioxid. Redox Signal. 14, 1687-1698.

We hypothesized that nandrolone (ND)-abuse induces cardiac hypertrophy, increases myocardial susceptibility to ischemia/reperfusion (I/R) injury, and reduces responsiveness to postconditioning (PostC) cardioprotection. Wistar-rats were ND treated for 2 weeks (short_ND) or 10 weeks (long_ND). Vehicle-treated rats served as controls. Hearts were retrogradely perfused and left ventricular pressure (LVP) was measured before and after 30-min global ischemia. In subgroups of hearts, to induce cardioprotection a PostC protocol (five cycles of 10-s reperfusion and 10-s ischemia) was performed. β-adrenoreceptors, kinases (Akt and GSK-3β) and phosphatases (PP2A sub A and PP2A sub B) were examined by Western blot before and after ischemia. After 120-min reperfusion, infarct size was measured. Short_ND slightly increased cardiac/body weight ratio, but did not affect cardiac baseline nor post-ischemic contractile function or infarct size when compared to vehicle hearts. However, PostC limited cardiac dysfunction much more in short_ND hearts than the other groups. Although cardiac/body weight ratio markedly increased after long_ND, baseline LVP was not affected. Yet, post-ischemic contracture and infarct size were exacerbated and PostC was unable to reduce infarct size and ventricular dysfunction. While short_ND increased phosphatases, non-phosphorylated and phosphorylated Akt, long_ND reduced phosphatase-expression and Akt phosphorylation. Both short_ND and long_ND had no effect on the GSK-3β-phosphorylation but increased the expression of β(2)-adrenoreceptors. In reperfusion, PostC increased Akt phosphorylation regardless of protective effects, but reduced phosphatase-expression in protected hearts only. In conclusion, short_ND improves post-ischemic myocardial performance in postconditioned hearts. However, long_ND increases myocardial susceptibility to I/R injury and abolishes cardioprotection by PostC. This increased susceptibility might be related to steroid-induced hypertrophy and/or to altered enzyme expression/phosphorylation.

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 NG-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.

There appears to be no agreement as to whether or not an increase in diastolic left ventricular pressure and/or volume can cause a decrease in diastolic coronary blood flow. We investigated the problem in the anaesthetized dog using a flaccid freely distensible latex balloon inserted into the left ventricle with the animal on extracorporeal circulation and the coronary perfusion pressure constant at about 45 mm Hg. Maximal vasodilatation and suppression of autoregulation in coronary vasculature was obtained by the intracoronary infusion of dipyridamole (10-40 mg/h). Ventricular volume was changed in steps of 10 ml from 10 to 70 ml and back to 10 ml, whilst recording coronary blood flow and left ventricular pressure in the left circumflex coronary artery. Over a range of ventricular volumes from 20 to 50 ml and a concomitant rise in diastolic ventricular pressure to about 20 mm Hg there was no change in the diastolic coronary flow. Only when the ventricular volume was more than two times the control value (i.e. exceeded 50 ml) and left ventricular pressure was more than 20 mm Hg, was there a decrease in coronary flow. During the return of the volume to the control level there was a fall in diastolic flow and ventricular contractility with respect to the values obtained when the volume was increased; these two effects were transient lasting less than 10 min. It was not considered that any of the three models of the coronary circulation, waterfall, intramyocardial pump or varying elastance model could explain our results.(ABSTRACT TRUNCATED AT 250 WORDS)

In the heart, ischemia/reperfusion damage occurs mainly during the first minutes of reperfusion. Recently, it has been shown that the heart can be protected against the extension of ischemia/reperfusion injury if brief coronary occlusions are performed just at the beginning of the reperfusion. This procedure has been called postconditioning (PostC). It can also be elicited by pharmacological intervention, that is, pharmacological PostC. In particular, PostC limits infarct size, apoptosis, endothelial dysfunction, neutrophil adherence, and arrhythmias. Similar to preconditioning, PostC may trigger signaling pathways, including reperfusion injury salvage kinase and survivor activating factor enhancement pathways. PostC-induced protection also involves intracellular acidosis and early redox-sensitive mechanisms. However, controversies exist on the nature of receptors and main pathway(s) involved in PostC. Protective pathways activated by PostC appear to converge on mitochondria and, in particular, on mitochondrial permeability transition pores. Preliminary clinical data indicate that drugs targeting mitochondrial permeability transition pore or reperfusion injury salvage kinases may confer benefits to patients with acute myocardial infarction above that provided by myocardial reperfusion alone. Future studies must define the principal protective cascades, the interdependence of the signaling pathways, and the optimal pharmacological target and agent(s) for protection. These studies must also consider the possible confounding effects of comorbidities and their drug treatments.

Post-ischemic reperfusion may result in reactive oxygen species (ROS) generation, reduced availability of nitric oxide (NO•), Ca(2+)overload, prolonged opening of mitochondrial permeability transition pore, and other processes contributing to cell death, myocardial infarction, stunning, and arrhythmias. With the discovery of the preconditioning and postconditioning phenomena, reperfusion injury has been appreciated as a reality from which protection is feasible, especially with postconditioning, which is under the control of physicians. Potentially cooperative protective signaling cascades are recruited by both pre- and postconditioning. In these pathways, phosphorylative/dephosphorylative processes are widely represented. However, cardioprotective modalities of signal transduction also include redox signaling by ROS, S-nitrosylation by NO• and derivative, S-sulfhydration by hydrogen sulfide, and O-linked glycosylation with beta-N-acetylglucosamine. All these modalities can interact and regulate an entire pathway, thus influencing each other. For instance, enzymes can be phosphorylated and/or nitrosylated in specific and/or different site(s) with consequent increase or decrease of their specific activity. The cardioprotective signaling pathways are thought to converge on mitochondria, and various mitochondrial proteins have been identified as targets of these post-transitional modifications in both pre- and postconditioning.

Brief periods (a few seconds) of cyclic coronary occlusions applied early in reperfusion induce a cardioprotection against infarct size, called postconditioning (PostC) in which B(2)-bradykinin receptors play a pivotal role. Since angiotensin-converting enzyme (ACE) inhibitors reduce degradation of kinins, we studied the effects of PostC on infarct size and postischemic myocardial dysfunction in both normotensive (WKY) and spontaneously hypertensive rats (SHR) acutely or chronically treated with the ACE inhibitor Captopril. Isolated hearts from SHR and WKY rats were subjected to the following protocols: (a) ischemia for 30- and 120-min reperfusion (I/R); (b) I/R + PostC protocol (5-cycles 10-s I/R); (c) pretreatment with Captopril for 4-weeks before to subject the hearts to I/R with or without PostC maneuvers. Some SHR hearts were treated with Captopril during the 20- or 40-min early reperfusion with or without PostC maneuvers. Cardiac function was assessed in vivo with echocardiography. Left ventricular pressure and infarct size were measured ex vivo. Chronic Captopril significantly reduced left ventricular hypertrophy in SHR, and reduced infarct size in both WKY and SHR hearts. PostC maneuvers significantly reduced infarct size in WKY, but not in SHR hearts. Yet, PostC slightly improved postischemic systolic function in untreated SHR. Captopril given in reperfusion was unable to limit I/R injury in SHR hearts. Data show that PostC protection against infarct size is blunted in SHR and that PostC is unable to add its protective effect to those of chronic Captopril, which per se reduces cardiac hypertrophy and heart susceptibility to I/R insult.

AIM Platelet-activating factor (PAF) triggers cardiac pre-conditioning against ischemia/reperfusion injury. The actual protection of ischaemic pre-conditioning occurs in the reperfusion phase. Therefore, we studied in this phase the kinases involved in PAF-induced pre-conditioning. METHODS Langendorff-perfused rat hearts underwent 30 min of ischaemia and 2 h of reperfusion (group 1, control). Before ischaemia, group 2 hearts were perfused for 19 min with PAF (2 x 10(-11) M); groups 3-5 hearts were co-infused during the initial 20 min of reperfusion, with the protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M) or the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (5 x 10(-5) M) and atractyloside (2 x 10(-5) M), a mitochondrial permeability transition pore (mPTP) opener respectively. Phosphorylation of PKCepsilon, PKB/Akappat, GSK-3beta and ERK1/2 at the beginning of reperfusion was also checked. Left ventricular pressure and infarct size were determined. RESULTS PAF pre-treatment reduced infarct size (33 +/- 4% vs. 64 +/- 5% of the area at risk of control hearts) and improved pressure recovery. PAF pre-treatment enhanced the phosphorylation/activation of PKCepsilon, PKB/Akappat and the phosphorylation/inactivation of GSK-3beta at reperfusion. Effects on ERK1/2 phosphorylation were not consistent. Infarct-sparing effect and post-ischaemic functional improvement induced by PAF pre-treatment were abolished by post-ischaemic infusion of either chelerythrine, LY294002 or atractyloside. CONCLUSIONS The cardioprotective effect exerted by PAF pre-treatment involves activation of PKC and PI3K in post-ischaemic phases and might be mediated by the prevention of mPTP opening in reperfusion via GSK-3beta inactivation.

Background and aim The beneficial effects of exercise in reducing the incidence of cardiovascular diseases are well known. Several studies have demonstrated that forced exercise (FE) could activate a stress response similar to a restrain stress. Previous studies suggest that heart protection to ischemic events would be improved by an omega 3 free fatty acid (ω3-FFA)-enriched diet. Here, we investigate the impact of stressful FE and an omega 3-FFA-enriched diet on cardiac tolerance to ischemic events over one month. Methods and results Twenty-four Wistar rats were randomly assigned to one of the following protocols: 1) Sedentary (SED) animals who were regularly fed; 2) sedentary animals who were given 1 ml/day of fish oil for one month; 3) FE + ω3-FFA rats who were given 1 ml/day of fish oil and forced to run on a motorized wheel for 30 min every day, both for one month; and 4) FE animals were forced to exercise as group 3 and fed with a regular diet. At the end of the treatments an isolated heart preparation was performed. After a 30 min global ischemic event and 2 h reperfusion, hearts of sedentary-ω3 animals recovered about 37% of left ventricular developed pressure, whereas FE, ω3 + FE and CTRL-SED animals recovered only about 15%, 5% and 8% respectively. Similarly, heart infarct size was significantly lower in sedentary-ω3 animals compared to animals in the three other groups. Conclusions Results indicate that one month of treatment with an ω3-FFA-enriched diet improves cardioprotection upon ischemic events, whereas FE leads to a reduced heart tolerance to ischemic events, which cannot be reversed by an ω3-FFA diet.

Postconditioning (PostC) maneuvers allow post-ischemic accumulation of autacoids, which may trigger protection. Intermittent infusion of either bradykinin or diazoxide during early reperfusion triggered PostC protection via redox signaling. Here we tested whether intermittent adenosine (ADO) may trigger PostC-like cardioprotection.Isolated rat hearts underwent 30 min ischemia and 120 min reperfusion. PostC (5 cycles of 10-s reperfusion/ischemia) or short-term ADO treatment were performed immediately after the 30 min ischemia. Non-selective ADO receptor-antagonist (8-SPT) was infused during PostC maneuvers. Left ventricular pressure was monitored, and infarct size was evaluated by using nitro-blue-tetrazolium staining.In Control hearts after ischemia/reperfusion, infarct size was 64% +/- 4% of risk-area. PostC reduced infarct size to 28% +/- 3% (P0.01). PostC protection was abolished by 3 min of the infusion of 8-SPT during PostC maneuvers. Since 3 min of ADO infusion (1 mum or 30 mum) did not trigger PostC protection, protocol with intermittent ADO infusion (5 cycles of 10-s buffer-no-ADO/buffer-plus-ADO) was used to mimic PostC. Also intermittent ADO did not attenuate infarct size (75% +/- 2%; P = NS versus ADO and Control; P0.01 versus PostC). Despite disparities on infarct size, post-ischemic systolic function was similar among groups.Data suggest a strong ADO-dependent anti-infarct effect, but no an anti-stunning effect, by ischemic PostC. Neither intermittent ADO nor 3 min continuous ADO can trigger protection against infarct size extension. Yet, 3 min ADO antagonist can prevent PostC protection. It is thus likely that endogenous ADO binding with receptors during early reperfusion is necessary, but nonsufficient to induce protection against infarct size extension.

The mechanical and electrical effects of the venom of Bitis nasicornis were studied on the guinea-pig Langendorff and left atrial myocardium preparations. While Langendorff preparations were treated with individual doses of 0.1, 0.6 and 1.4 mg, isolated left atria were treated using concentrations of 2.0, 20 and 200 μg/ml of venom in the perfusion solution. In the Langendorff preparation, transient increases in left ventricular systolic pressure (LVSP) and heart rate (HR) were seen after 0.1 mg of venom. When 0.6 mg of venom was given, the increases were followed by decreases, while 1.4 mg doses simply induced decreases in LVSP and HR. After both 0.6 and 1.4 mg doses the decreases were accompanied by increases in left ventricular diastolic pressure. In addition to these mechanical effects, transient increases in HR with atrio-ventricular blocks, ventricular extrasystoles and tachycardia were observed after each dose. In the left atrium the 2 μg/ml venom concentration produced an increase, followed by a decrease, in the maximum tension developed, which was only seen to decrease with higher concentrations of 20 and 200 μg/ml of venom. A dose dependent significant reduction in the action potential duration was observed for the doses of 0.6 and 1.4 mg in the ventricle and for all three concentrations in the atrium.

1. Intravenous venom (4 mg/kg) caused a non-compensated metabolic acidosis. 2. Bicarbonate concentration, base excess, standard base excess and pH all fell dramatically. 3. A respiratory impairment occurred characterized by pulmonary oedema and a fall in arterial pO2. 4. Acidosis occurred soon after venom when pO2 was still normal, indicating that changes in tissue metabolism contributed to the acidosis independently of reduced oxygen availability.

Postconditioning (PostC) may limit mitochondrial damage and apoptotic signaling. We studied markers of apoptosis and mitochondrial protection in isolated rat hearts, which underwent a) perfusion without ischemia (Sham), b) 30-min ischemia (I) plus 2-hour reperfusion (R), or c) PostC protocol (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia). Markers were studied in cytosolic (CF) and/or mitochondrial (MF) fractions. In CF, while pro-apoptotic factors (cytochrome c and caspase-3) were reduced, the anti-apoptotic markers (Bcl-2 and Pim-1) were increased by PostC, compared to the I/R group. Accordingly, phospho-GSK-3β and Bcl-2 levels increased in mitochondria of PostC group. Moreover, I/R reduced the level of mitochondrial structural protein (HSP-60) in MF and increased in CF, thus suggesting mitochondrial damage and HSP-60 release in cytosol, which were prevented by PostC. Electron microscopy confirmed that I/R markedly damaged cristae and mitochondrial membranes; damage was markedly reduced by PostC. Finally, total connexin-43 (Cx43) levels were reduced in the CF of the I/R group, whereas phospho-Cx43 level resulted in higher levels in the MF of the I/R group than the Sham group. PostC limited the I/R-induced increase of mitochondrial phospho-Cx43. Data suggest that PostC i) increases the levels of anti-apoptotic markers, including the cardioprotective kinase Pim-1, ii) decreases the pro-apoptotic markers, e.g. cytochrome c, iii) preserves the mitochondrial structure, and iv) limits the migration of phospho-Cx43 to mitochondria.

Postconditioning (PostC), obtained with brief intermittent cycles of ischemia alternating with reperfusion applied after the ischemic event, has been shown to reduce infarct size. Recently, we have shown that PostC triggering includes B2 receptor activation and its downstream pathway. Moreover, we showed that BK intermittent infusion induces a cardioprotection similar to PostC. The aim of this study was to investigate the involvement of cyclooxygenase-(COX)-derivated prostaglandins, such as prostacyclin (PGI2) pathway in the cardioprotective action mediated by intermittent BK infusion. Isolated rat hearts underwent 30 min ischemia and 120 min reperfusion. Myocardial damage was evaluated using nitro-blue-tetrazolium staining. The production of metabolite of PGI2, 6-keto-PGF1alpha, was evaluated with EIA assay on the samples collected during reperfusion. The perfusion pressure and the left ventricular pressure were monitored. In Control hearts, the infarct size was 64% +/- 4% of risk area. PostC reduced significantly the infarct size (28% +/- 4% P0.001 Vs. Control). BK intermittent protocol to mimic PostC, attenuated infarct size (40% +/- 2% P0.01 Vs. Control). The BK-intermittent and PostC protections were abolished with COX-inhibition. Intermittent BK and PostC enhanced the release of prostacyclin metabolite, 6-keto-PGF1alpha, in the late phase of reperfusion (i.e., 6-keto-PGF1alpha peaked 30 min after protective maneuvers). Also the stable PGI2 analogue, Iloprost, given in the early reperfusion reduced infarct size and improved post-ischemic heart function. In conclusion, protection by PostC and intermittent BK requires COX activation and PGI2 release during late reperfusion. These data suggest that COX must not be inhibited to have PostC protection. This finding should be kept present by future clinical studies on PostC.

The muscle metaboreflex is enhanced in chronic heart failure (CHF) patients, and this fact has been associated with the early fatigue shown by these patients in response to exercise. In animal studies of CHF, it was found that the limited capacity to enhance ventricular performance is responsible for a functional shift from a cardiac output to a systemic vascular resistance (SVR) increase in the mechanism by which the cardiovascular system raises blood pressure in response to the metaboreflex. However, the existence of this functional shift is still unknown in humans. The present study was undertaken to test the hypothesis that a similar hemodynamic response was also present in humans with CHF. The hemodynamic response to metaboreflex activation obtained through postexercise ischemia was assessed in nine patients with CHF and nine healthy controls (CTL) by means of impedance cardiography. The main results were that 1) the blood pressure rise due to the metaboreflex was similar in the two groups; 2) the CTL group achieved the blood pressure response via cardiac output increase, and the CHF group, via SVR increase; and 3) stroke volume was enhanced in the CTL group and decreased in the CHF group. This study demonstrates that in CHF patients, metaboreflex recruitment causes a functional shift from flow increase to peripheral vasoconstriction in the mechanism through which blood pressure is increased. The incapacity to enhance cardiac performance and stroke volume is probably the primary cause of this cardiovascular alteration.