Search

Background: Several factors contribute to ischemia/reperfusion injury (IRI), including activation of the NLRP3 inflammasome and its byproducts, such as interleukin-1β (IL-1β) and caspase-1. However, NLRP3 may paradoxically exhibit cardioprotective properties. This study aimed to assess the protective effects of the novel NLRP3 inhibitor, INF195, both in vitro and ex vivo., Methods: To investigate the relationship between NLRP3 and myocardial IRI, we synthetized a series of novel NLRP3 inhibitors, and investigated their putative binding mode via docking studies. Through in vitro studies we identified INF195 as optimal for NLRP3 inhibition. We measured infarct-size in isolated mouse hearts subjected to 30-min global ischemia/one-hour reperfusion in the presence of three different doses of INF195 (5, 10, or 20-μM). We analyzed caspase-1 and IL-1β concentration in cardiac tissue homogenates by ELISA. Statistical significance was determined using one-way ANOVA followed by Tukey's test., Results and Conclusion: INF195 reduces NLRP3-induced pyroptosis in human macrophages. Heart pre-treatment with 5 and 10-μM INF195 significantly reduces both infarct size and IL-1β levels. Data suggest that intracardiac NLRP3 activation contributes to IRI and that low doses of INF195 exert cardioprotective effects by reducing infarct size. However, at 20-μM, INF195 efficacy declines, leading to a lack of cardioprotection. Research is required to determine if high doses of INF195 have off-target effects or dual roles, potentially eliminating both harmful and cardioprotective functions of NLRP3. Our findings highlight the potential of a new chemical scaffold, amenable to further optimization, to provide NLRP3 inhibition and cardioprotection in the ischemia/reperfusion setting., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Despite recent progress, ischemic heart disease poses a persistent global challenge, driving significant morbidity and mortality. The pursuit of therapeutic solutions has led to the emergence of strategies such as ischemic preconditioning, postconditioning, and remote conditioning to shield the heart from myocardial ischemia/reperfusion injury (MIRI). These ischemic conditioning approaches, applied before, after, or at a distance from the affected organ, inspire future therapeutic strategies, including pharmacological conditioning. Gasotransmitters, comprising nitric oxide, hydrogen sulfide, sulfur dioxide, and carbon monoxide, play pivotal roles in physiological and pathological processes, exhibiting shared features such as smooth muscle relaxation, antiapoptotic effects, and anti-inflammatory properties. Despite potential risks at high concentrations, physiological levels of gasotransmitters induce vasorelaxation and promote cardioprotective effects. Noble gases, notably argon, helium, and xenon, exhibit organ-protective properties by reducing cell death, minimizing infarct size, and enhancing functional recovery in post-ischemic organs. The protective role of noble gases appears to hinge on their modulation of molecular pathways governing cell survival, leading to both pro- and antiapoptotic effects. Among noble gases, helium and xenon emerge as particularly promising in the field of cardioprotection. This overview synthesizes our current understanding of the roles played by gasotransmitters and noble gases in the context of MIRI and cardioprotection. In addition, we underscore potential future developments involving the utilization of noble gases and gasotransmitter donor molecules in advancing cardioprotective strategies., (© 2024. The Author(s).)

Despite impressive improvements in the care of patients with ST-segment elevation myocardial infarction, mortality remains high. Reperfusion is necessary for myocardial salvage, but the abrupt return of flow sets off a cascade of injurious processes that can lead to further necrosis. This has been termed myocardial ischemia-reperfusion injury and is the subject of this review. The pathologic and molecular bases for myocardial ischemia-reperfusion injury are increasingly understood and include injury from reactive oxygen species, inflammation, calcium overload, endothelial dysfunction, and impaired microvascular flow. A variety of pharmacologic strategies have been developed that have worked well in preclinical models and some have shown promise in the clinical setting. In addition, there are newer mechanical approaches including mechanical unloading of the heart prior to reperfusion that are in current clinical trials., Competing Interests: Funding Support and Author Disclosures Dr Welt has served as a consultant to Xenter Inc and Faraday Pharmaceuticals. Dr Drakos has served as a consultant to Abbott Laboratories; and has received research support from Novartis. Dr Kapur has received consulting/speaker fees from Abbott, Abiomed, Boston Scientific, Edwards, Getinge, LivaNova, Teleflex, and Zoll; and has received institutional research grants from Abbott, Abiomed, Boston Scientific, Getinge, LivaNova, and Teleflex. All other authors have reported that they have no relationships relevant to contents of this paper to disclose., (Copyright © 2024 American College of Cardiology Foundation. All rights reserved.)

Inspired by the recent advancements in understanding the binding mode of sulfonylurea-based NLRP3 inhibitors to the NLRP3 sensor protein, we developed new NLRP3 inhibitors by replacing the central sulfonylurea moiety with different heterocycles. Computational studies evidenced that some of the designed compounds were able to maintain important interaction within the NACHT domain of the target protein similarly to the most active sulfonylurea-based NLRP3 inhibitors. Among the studied compounds, the 1,3,4-oxadiazol-2-one derivative 5 (INF200) showed the most promising results being able to prevent NLRP3-dependent pyroptosis triggered by LPS/ATP and LPS/MSU by 66.3 ± 6.6% and 61.6 ± 11.5% and to reduce IL-1β release (35.5 ± 8.8% μM) at 10 μM in human macrophages. The selected compound INF200 (20 mg/kg/day) was then tested in an in vivo rat model of high-fat diet (HFD)-induced metaflammation to evaluate its beneficial cardiometabolic effects. INF200 significantly counteracted HFD-dependent "anthropometric" changes, improved glucose and lipid profiles, and attenuated systemic inflammation and biomarkers of cardiac dysfunction (particularly BNP). Hemodynamic evaluation on Langendorff model indicate that INF200 limited myocardial damage-dependent ischemia/reperfusion injury (IRI) by improving post-ischemic systolic recovery and attenuating cardiac contracture, infarct size, and LDH release, thus reversing the exacerbation of obesity-associated damage. Mechanistically, in post-ischemic hearts, IFN200 reduced IRI-dependent NLRP3 activation, inflammation, and oxidative stress. These results highlight the potential of the novel NLRP3 inhibitor, INF200, and its ability to reverse the unfavorable cardio-metabolic dysfunction associated with obesity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Nitric oxide (NO) and carbon monoxide (CO) represent a pair of biologically active gases with an increasingly well-defined range of effects on circulating platelets. These gases interact with platelets and cells in the vessels and heart and exert fundamentally similar biological effects, albeit through different mechanisms and with some peculiarity. Within the cardiovascular system, for example, the gases are predominantly vasodilators and exert antiaggregatory effects, and are protective against damage in myocardial ischemia-reperfusion injury. Indeed, NO is an important vasodilator acting on vascular smooth muscle and is able to inhibit platelet activation. NO reacts with superoxide anion (O 2 ( - •)) to form peroxynitrite (ONOO( - )), a nitrosating agent capable of inducing oxidative/nitrative signaling and stress both at cardiovascular, platelet, and plasma levels. CO reduces platelet reactivity, therefore it is an anticoagulant, but it also has some cardioprotective and procoagulant properties. This review article summarizes current knowledge on the platelets and roles of gas mediators (NO, and CO) in cardioprotection. In particular, we aim to examine the link and interactions between platelets, NO, and CO and cardioprotective pathways.

Experimentally, many strong cardioprotective treatments have been identified in different animal models of acute ischaemia/reperfusion injury (IRI) and coronary artery disease (CAD). However, the translation of these cardioprotective therapies for the benefit of the patients into the clinical scenario has been very disappointing. The reasons for this lack are certainly multiple. Indeed, many confounding factors we must deal in clinical reality, such as aging, sex and inflammatory processes are neglected in many experiments. Due to the pivotal role of aging, sex and inflammation in determining cardiac ischaemic disease, in this review, we take into account age as a modifier of tolerance to IRI in the two sexes, dissecting aging and myocardial reperfusion injury mechanisms and the sex differences in tolerance to IRI. Then we focus on the role of the gut microbiota and the NLRP3 inflammasome in myocardial IRI and on the possibility to consider NLRP3 inflammasome as a potential target in the treatment of CAD in relationship with age and sex. Finally, we consider the cardioprotective mechanisms and cardioprotective treatments during aging in the two sexes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Since coronary reperfusion was introduced into clinical practice in the late 1970s, the further translation of several successful animal experiments on cardioprotection into clinical practice has been disappointing to date. Animal experiments are often performed on young, healthy animals lacking the risk factors, co-morbidities and co-medications characteristic of acute myocardial infarction patients. Many hopes were kindled in 1986 when ischemic preconditioning was discovered. However, it is not yet known how long ischemia can last and what is the best modality for additional cardioprotection through conditioning to obtain benefits. There is a lack of experimental studies on the long-term effects of additional cardioprotection, in addition to the reduction in infarct size; in particular, there is a lack of studies on vessel protection, repair, inflammation, remodeling, and mortality. The reproducibility and robustness of experimental studies are often limited by species differences, the role of co-morbidities, vascular damage, inflammatory processes, and co-medications, which are not adequately considered. In particular, inflammatory processes, including NLRP3 inflammasome, play an important role in the long-term effects. Future studies should focus on interventions/agents with robust preclinical data and should recruit patients who truly have the potential to benefit from further cardioprotection. Here we focus on the main mechanisms and targets of cardioprotection during remote conditioning and their alteration by one of the most common co-morbidities, namely diabetes, in which microvascular lesions and inflammatory processes play extremely important roles., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ischemia-reperfusion injury (IRI) is one of the biggest challenges for cardiovascular researchers given the huge death toll caused by myocardial ischemic disease. Cardioprotective conditioning strategies, namely pre- and post-conditioning maneuvers, represent the most important strategies for stimulating pro-survival pathways essential to preserve cardiac health. Conditioning maneuvers have proved to be fundamental for the knowledge of the molecular basis of both IRI and cardioprotection. Among this evidence, the importance of signal transducer and activator of transcription 3 (STAT3) emerged. STAT3 is not only a transcription factor but also exhibits non-genomic pro-survival functions preserving mitochondrial function from IRI. Indeed, STAT3 is emerging as an influencer of mitochondrial function to explain the cardioprotection phenomena. Studying cardioprotection, STAT3 proved to be crucial as an element of the survivor activating factor enhancement (SAFE) pathway, which converges on mitochondria and influences their function by cross-talking with other cardioprotective pathways. Clearly there are still some functional properties of STAT3 to be discovered. Therefore, in this review, we highlight the evidence that places STAT3 as a promoter of the metabolic network. In particular, we focus on the possible interactions of STAT3 with processes aimed at maintaining mitochondrial functions, including the regulation of the electron transport chain, the production of reactive oxygen species, the homeostasis of Ca 2+ and the inhibition of opening of mitochondrial permeability transition pore. Then we consider the role of STAT3 and the parallels between STA3/STAT5 in cardioprotection by conditioning, giving emphasis to the human heart and confounders., (© 2021. The Author(s).)

During myocardial ischemia, timely reperfusion is critical to limit infarct area and the overall loss of cardiac contractile function. However, reperfusion further exacerbates the damage of the ischemic heart. This type of injury is known as ischemia-reperfusion injury (IRI). Ischemic conditioning is a procedure which consists of brief cycles of ischemia and reperfusion in order to protect the myocardium against IRI. Remote ischemic conditioning (RIC), namely transient brief episodes of ischemia at a remote site before a subsequent damaging ischemia/reperfusion procedure of the target organ (e.g., the heart), protects against IRI. However, how the stimulus of RIC is transduced from the remote organ to the ischemic heart is still unknown. Recently, extracellular vesicles (EVs) have been proposed to have a role in the RIC procedure. The endothelium releases EVs and is also one of the tissues mostly exposed to EVs during their journey to the target organ. Moreover, EVs may have important roles in angiogenesis and, therefore, in the remodeling of post-ischemic organs. Here we analyze how EVs may contribute to the overall cardioprotective effect and the implication of the endothelium and its EVs in RIC mediated acute cardioprotection as well as in angiogenesis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The relevance of extracellular vesicles (EV) as mediators of cardiac damage or recovery upon Ischemia Reperfusion Injury (IRI) and Remote Ischemic PreConditioning (RIPC) is controversial. This study aimed to investigate whether serum-derived EV, recovered from patients with Acute Coronary Syndrome (ACS) and subjected to the RIPC or sham procedures, may be a suitable therapeutic approach to prevent IRI during Percutaneous-Coronary-Intervention (PCI). A double-blind, randomized, sham-controlled study (NCT02195726) has been extended, and EV were recovered from 30 patients who were randomly assigned (1:1) to undergo the RIPC- (EV-RIPC) or sham-procedures (EV-naive) before PCI. Patient-derived EV were analyzed by TEM, FACS and western blot. We found that troponin (TnT) was enriched in EV, compared to healthy subjects, regardless of diagnosis. EV-naive induced protection against IRI, both in-vitro and in the rat heart, unlike EV-RIPC. We noticed that EV-naive led to STAT-3 phosphorylation, while EV-RIPC to Erk-1/2 activation in the rat heart. Pre-treatment of the rat heart with specific STAT-3 and Erk-1/2 inhibitors led us to demonstrate that STAT-3 is crucial for EV-naive-mediated protection. In the same model, Erk-1/2 inhibition rescued STAT-3 activation and protection upon EV-RIPC treatment. 84 Human Cardiovascular Disease mRNAs were screened and DUSP6 mRNA was found enriched in patient-derived EV-naive. Indeed, DUSP6 silencing in EV-naive prevented STAT-3 phosphorylation and cardio-protection in the rat heart. This analysis of ACS-patients' EV proved: (i) EV-naive cardio-protective activity and mechanism of action; (ii) the lack of EV-RIPC-mediated cardio-protection; (iii) the properness of the in-vitro assay to predict EV effectiveness in-vivo., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Diabetic patients are at increased risk of developing coronary artery disease and experience worse clinical outcomes following acute myocardial infarction. Novel therapeutic strategies are required to protect the myocardium against the effects of acute ischaemia-reperfusion injury (IRI). These include one or more brief cycles of non-lethal ischaemia and reperfusion prior to the ischaemic event (ischaemic preconditioning [IPC]) or at the onset of reperfusion (ischaemic postconditioning [IPost]) either to the heart or to extracardiac organs (remote ischaemic conditioning [RIC]). Studies suggest that the diabetic heart is resistant to cardioprotective strategies, although clinical evidence is lacking. We overview the available animal models of diabetes, investigating acute myocardial IRI and cardioprotection, experiments investigating the effects of hyperglycaemia on susceptibility to acute myocardial IRI, the response of the diabetic heart to cardioprotective strategies e.g. IPC, IPost and RIC. Finally we highlight the effects of anti-hyperglycaemic agents on susceptibility to acute myocardial IRI and cardioprotection. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.23/issuetoc., (© 2020 The British Pharmacological Society.)

Acute myocardial infarction causes lethal injury to cardiomyocytes during both ischaemia and reperfusion (IR). It is important to define the precise mechanisms by which they die in order to develop strategies to protect the heart from IR injury. Necrosis is known to play a major role in myocardial IR injury. There is also evidence for significant myocardial death by other pathways such as apoptosis, although this has been challenged. Mitochondria play a central role in both of these pathways of cell death, as either a causal mechanism is the case of mitochondrial permeability transition leading to necrosis, or as part of the signalling pathway in mitochondrial cytochrome c release and apoptosis. Autophagy may impact this process by removing dysfunctional proteins or even entire mitochondria through a process called mitophagy. More recently, roles for other programmed mechanisms of cell death such as necroptosis and pyroptosis have been described, and inhibitors of these pathways have been shown to be cardioprotective. In this review, we discuss both mitochondrial and mitochondrial-independent pathways of the major modes of cell death, their role in IR injury and their potential to be targeted as part of a cardioprotective strategy. This article is part of a special Issue entitled 'Mitochondria as targets of acute cardioprotection' and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The protection from ischaemia-reperfusion-associated myocardial infarction worsening remains a big challenge. We produced a bioartificial 3D cardiac patch with cardioinductive properties on stem cells. Its multilayer structure was functionalised with clinically relevant doses of adenosine. We report here the first study on the potential of these cardiac patches in the controlled delivery of adenosine into the in vivo ischaemic-reperfused pig heart. A Fourier transform infrared chemical imaging approach allowed us to perform a characterisation, complementary to the histological and biochemical analyses on myocardial samples after in vivo patch implantation, increasing the number of investigations and results on the restricted number of pigs (n = 4) employed in this feasibility step. In vitro tests suggested that adenosine was completely released by a functionalised patch, a data that was confirmed in vivo after 24 hr from patch implantation. Moreover, the adenosine-loaded patch enabled a targeted delivery of the drug to the ischaemic-reperfused area of the heart, as highlighted by the activation of the pro-survival signalling reperfusion injury salvage kinases pathway. At 3 months, though limited to one animal, the used methods provided a picture of a tissue in dynamic conditions, associated to the biosynthesis of new collagen and to a non-fibrotic outcome of the healing process underway. The synergistic effect between the functionalised 3D cardiac patch and adenosine cardioprotection might represent a promising innovation in the treatment of reperfusion injury. As this is a feasibility study, the clinical implications of our findings will require further in vivo investigation on larger numbers of ischaemic-reperfused pig hearts., (© 2019 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.)

Acute obstruction of a coronary artery causes myocardial ischaemia and if prolonged, may result in an ST-segment elevation myocardial infarction (STEMI). First-line treatment involves rapid reperfusion. However, a highly dynamic and co-ordinated inflammatory response is rapidly mounted to repair and remove the injured cells which, paradoxically, can further exacerbate myocardial injury. Furthermore, although cardiac remodelling may initially preserve some function to the heart, it can lead over time to adverse remodelling and eventually heart failure. Since the size of the infarct corresponds to the subsequent risk of developing heart failure, it is important to find ways to limit initial infarct development. In this review, we focus on the role of the innate immune system in the acute response to ischaemia-reperfusion (IR) and specifically its contribution to cell death and myocardial infarction. Numerous danger-associated molecular patterns are released from dying cells in the myocardium, which can stimulate pattern recognition receptors including toll like receptors and NOD-like receptors (NLRs) in resident cardiac and immune cells. Activation of the NLRP3 inflammasome, caspase 1, and pyroptosis may ensue, particularly when the myocardium has been previously aggravated by the presence of comorbidities. Evidence will be discussed that suggests agents targeting innate immunity may be a promising means of protecting the hearts of STEMI patients against acute IR injury. However, the dosing and timing of such agents should be carefully determined because innate immunity pathways may also be involved in cardioprotection. This article is part of a Cardiovascular Research Spotlight Issue entitled 'Cardioprotection Beyond the Cardiomyocyte', and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

During an ST-elevation myocardial infarction (STEMI), the myocardium undergoes a prolonged period of ischaemia. Reperfusion therapy is essential to minimize cardiac injury but can paradoxically cause further damage. Experimental procedures to limit ischaemia and reperfusion (IR) injury have tended to focus on the cardiomyocytes since they are crucial for cardiac function. However, there is increasing evidence that non-cardiomyocyte resident cells in the heart (as discussed in a separate review in this Spotlight series) as well as circulating cells and factors play important roles in this pathology. For example, erythrocytes, in addition to their main oxygen-ferrying role, can protect the heart from IR injury via the export of nitric oxide bioactivity. Platelets are well-known to be involved in haemostasis and thrombosis, but beyond these roles, they secrete numerous factors including sphingosine-1 phosphate (S1P), platelet activating factor, and cytokines that can all strongly influence the development of IR injury. This is particularly relevant given that most STEMI patients receive at least one type of platelet inhibitor. Moreover, there are large numbers of circulating vesicles in the blood, including microvesicles and exosomes, which can exert both beneficial and detrimental effects on IR injury. Some of these effects are mediated by the transfer of microRNA (miRNA) to the heart. Synthetic miRNA molecules may offer an alternative approach to limiting the response to IR injury. We discuss these and other circulating factors, focussing on potential therapeutic targets relevant to IR injury. Given the prevalence of comorbidities such as diabetes in the target patient population, their influence will also be discussed. This article is part of a Cardiovascular Research Spotlight Issue entitled 'Cardioprotection Beyond the Cardiomyocyte', and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Aim: Apelin, the ligand of the G-protein-coupled receptor (GPCR) APJ, exerts a post-conditioning-like protection against ischaemia/reperfusion injury through activation of PI3K-Akt-NO signalling. The pathway connecting APJ to PI3K is still unknown. As other GPCR ligands act through transactivation of epidermal growth factor receptor (EGFR) via a matrix metalloproteinase (MMP) or Src kinase, we investigated whether EGFR transactivation is involved in the following three features of apelin-induced cardioprotection: limitation of infarct size, suppression of contracture and improvement of post-ischaemic contractile recovery., Method: Isolated rat hearts underwent 30 min of global ischaemia and 2 h of reperfusion. Apelin (0.5 μm) was infused during the first 20 min of reperfusion. EGFR, MMP or Src was inhibited to study the pathway connecting APJ to PI3K. Key components of RISK pathway, namely PI3K, guanylyl cyclase or mitochondrial K + -ATP channels, were also inhibited. Apelin-induced EGFR and phosphatase and tensing homolog (PTEN) phosphorylation were assessed. Left ventricular pressure and infarct size were measured., Results: Apelin-induced reductions in infarct size and myocardial contracture were prevented by the inhibition of EGFR, Src, MMP or RISK pathway. The involvement of EGFR was confirmed by its phosphorylation. However, neither direct EGFR nor MMP inhibition affected apelin-induced improvement of early post-ischaemic contractile recovery, which was suppressed by Src and RISK inhibitors only. Apelin also increased PTEN phosphorylation, which was removed by Src inhibition., Conclusion: While EGFR and MMP limit infarct size and contracture, Src or RISK pathway inhibition suppresses the three features of cardioprotection. Src does not only transactivate EGFR, but also inhibits PTEN by phosphorylation thus playing a crucial role in apelin-induced cardioprotection., (© 2017 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Patients with ischaemic heart disease or chronic heart failure show altered levels of obestatin, suggesting a role for this peptide in human heart function. We have previously demonstrated that GH secretagogues and the ghrelin gene-derived peptides, including obestatin, exert cardiovascular effects by modulating cardiac inotropism and vascular tone, and reducing cell death and contractile dysfunction in hearts subjected to ischaemia/reperfusion (I/R), through the Akt/nitric oxide (NO) pathway. However, the mechanisms underlying the cardiac actions of obestatin remain largely unknown. Thus, we suggested that obestatin-induced activation of PI3K/Akt/NO and PKG signalling is implicated in protection of the myocardium when challenged by adrenergic, endothelinergic or I/R stress. We show that obestatin exerts an inhibitory tone on the performance of rat papillary muscle in both basal conditions and under β-adrenergic overstimulation, through endothelial-dependent NO/cGMP/PKG signalling. This pathway was also involved in the vasodilator effect of the peptide, used both alone and under stress induced by endothelin-1. Moreover, when infused during early reperfusion, obestatin reduced infarct size in isolated I/R rat hearts, through an NO/PKG pathway, comprising ROS/PKC signalling, and converging on mitochondrial ATP-sensitive potassium [mitoK(ATP)] channels. Overall, our results suggest that obestatin regulates cardiovascular function in stress conditions and induces cardioprotection by mechanisms dependent on activation of an NO/soluble guanylate cyclase (sGC)/PKG pathway. In fact, obestatin counteracts exaggerated β-adrenergic and endothelin-1 activity, relevant factors in heart failure, suggesting multiple positive effects of the peptide, including the lowering of cardiac afterload, thus representing a potential candidate in pharmacological post-conditioning., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Mechanisms underlying the pathogenesis of ischemia/reperfusion injury are particularly complex, multifactorial and highly interconnected. A complex and entangled interaction is also emerging between platelet function, antiplatelet drugs, coronary diseases and ischemia/reperfusion injury, especially in diabetic conditions. Here we briefly summarize features of antiplatelet therapy in type 2 diabetes (T2DM). We also treat the influence of T2DM on ischemia/reperfusion injury and how anti-platelet therapies affect post-ischemic myocardial damage through pleiotropic properties not related to their anti-aggregating effects. miRNA-based signature associated with T2DM and its cardiovascular disease complications are also briefly considered. Influence of anti-platelet therapies and different effects of healthy and diabetic platelets on ischemia/reperfusion injury need to be further clarified in order to enhance patient benefits from antiplatelet therapy and revascularization. Here we provide insight on the difficulty to reduce the cardiovascular risk in diabetic patients and report novel information on the cardioprotective role of widely used anti-aggregant drugs.

Background: Neuregulin1 (Nrg1) and its receptors ErbB are crucial for heart development and for adult heart structural maintenance and function and Nrg1 has been proposed for heart failure treatment. Infarct size is the major determinant of heart failure and the mechanism of action and the role of each ErbB receptor remain obscure, especially in the post-ischemic myocardium. We hypothesized that Nrg1 and ErbB are affected at transcriptional level early after ischemia/reperfusion (I/R) injury, and that the protective postconditioning procedure (PostC, brief cycles of ischemia/reperfusion carried out after a sustained ischemia) can influence this pathway., Methods and Results: The Langendorff's heart was used as an ex-vivo model to mimic an I/R injury in the whole rat heart; after 30min of ischemia and 2h of reperfusion, with or without PostC, Nrg1 and ErbB expression were analysed by quantitative real-time PCR and Western blot. While no changes occur for ErbB2, ErbB4 and Nrg1, an increase of ErbB3 expression occurs after I/R injury, with and without PostC. However, I/R reduces ErbB3 protein, whereas PostC preserves it. An in vitro analysis with H9c2 cells exposed to redox-stress indicated that the transient over-expression of ErbB3 alone is able to increase cell survival (MTT assay), limiting mitochondrial dysfunction (JC-1 probe) and apoptotic signals (Bax/Bcl-2 ratio)., Conclusions: This study suggests ErbB3 as a protective factor against death pathways activated by redox stress and supports an involvement of this receptor in the pro-survival responses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The heart of patients with hypertension and cardiac hypertrophy is more vulnerable to ischemia-reperfusion injury (IRI). Here we discuss the main mechanisms of IRI and possible targets for cardioprotection. In particular, we consider the viewpoint that hypertension and cardiac hypertrophy may act synergistically in increasing the predisposition to cardiovascular accidents and in worsening IRI. There is no doubt that hypertrophic hearts may be redirected to be less vulnerable to IRI. Some experimental evidences suggest that antihypertensive drugs may have beneficial effects, some of which are not directly related to hypertension-lowering effect. However, more thorough experimental and clinical studies are necessary to understand the mechanisms and to maximize the beneficial effects of reperfusion after a heart attack in the presence of comorbidities, such as hypertension and cardiac hypertrophy.

Excessive fatty acids and sugars intake is known to affect the development of cardiovascular diseases, including myocardial infarction. However, the underlying mechanisms are ill defined. Here we investigated the balance between prosurvival and detrimental pathways within the heart of C57Bl/6 male mice fed a standard diet (SD) or a high-fat high-fructose diet (HFHF) for 12 weeks and exposed to cardiac ex vivo ischemia/reperfusion (IR) injury. Dietary manipulation evokes a maladaptive response in heart mice, as demonstrated by the shift of myosin heavy chain isoform content from α to β, the increased expression of the Nlrp3 inflammasome and markers of oxidative metabolism, and the downregulation of the hypoxia inducible factor- (HIF-)2α and members of the Reperfusion Injury Salvage Kinases (RISK) pathway. When exposed to IR, HFHF mice hearts showed greater infarct size and lactic dehydrogenase release in comparison with SD mice. These effects were associated with an exacerbated overexpression of Nlrp3 inflammasome, resulting in marked caspase-1 activation and a compromised activation of the cardioprotective RISK/HIF-2α pathways. The common mechanisms of damage here reported lead to a better understanding of the cross-talk among prosurvival and detrimental pathways leading to the development of cardiovascular disorders associated with metabolic diseases.

Although the nucleotide-binding oligomerization domain- (NOD-) like receptor pyrin domain containing 3 (NLRP3) inflammasome has been recently detected in the heart, its role in cardiac ischemia/reperfusion (IR) is still controversial. Here, we investigate whether a pharmacological modulation of NLRP3 inflammasome exerted protective effects in an ex vivo model of IR injury. Isolated hearts from male Wistar rats (5-6 months old) underwent ischemia (30 min) followed by reperfusion (20 or 60 min) with and without pretreatment with the recently synthetized NLRP3 inflammasome inhibitor INF4E (50  μ M, 20 min before ischemia). INF4E exerted protection against myocardial IR, shown by a significant reduction in infarct size and lactate dehydrogenase release and improvement in postischemic left ventricular pressure. The formation of the NLRP3 inflammasome complex was induced by myocardial IR and attenuated by INF4E in a time-dependent way. Interestingly, the hearts of the INF4E-pretreated animals displayed a marked improvement of the protective RISK pathway and this effect was associated increase in expression of markers of mitochondrial oxidative phosphorylation. Our results demonstrate for the first time that INF4E protected against the IR-induced myocardial injury and dysfunction, by a mechanism that involves inhibition of the NLRP3 inflammasome, resulting in the activation of the prosurvival RISK pathway and improvement in mitochondrial function., Competing Interests: The authors declare that they have no competing interests.

The morbidity and mortality from coronary artery disease (CAD) remain significant worldwide. The treatment for acute myocardial infarction has improved over the past decades, including early reperfusion of culprit coronary arteries. Although it is mandatory to reperfuse the ischaemic territory as soon as possible, paradoxically this leads to additional myocardial injury, namely ischaemia/reperfusion (I/R) injury, in which redox stress plays a pivotal role and for which no effective therapy is currently available. In this review, we report evidence that the redox environment plays a pivotal role not only in I/R injury but also in cardioprotection. In fact, cardioprotective strategies, such as pre- and post-conditioning, result in a robust reduction in infarct size in animals and the role of redox signalling is of paramount importance in these conditioning strategies. Nitrosative signalling and cysteine redox modifications, such as S-nitrosation/S-nitrosylation, are also emerging as very important mechanisms in conditioning cardioprotection. The reasons for the switch from protective oxidative/nitrosative signalling to deleterious oxidative/nitrosative/nitrative stress are not fully understood. The complex regulation of this switch is, at least in part, responsible for the diminished or lack of cardioprotection induced by conditioning protocols observed in ageing animals and with co-morbidities as well as in humans. Therefore, it is important to understand at a mechanistic level the reasons for these differences before proposing a safe and useful transition of ischaemic or pharmacological conditioning. Indeed, more mechanistic novel therapeutic strategies are required to protect the heart from I/R injury and to improve clinical outcomes in patients with CAD., (© 2014 The British Pharmacological Society.)

Cardiovascular diseases (CVD) are the leading cause of death, chronic illness and disability in Western countries. The most common cause of CVD derives from the harmful effects of acute myocardial ischemia and subsequent reperfusion injury. Cardioprotection against acute ischemia/ reperfusion injury is made possible by the "conditioning protocols." Conditioning is obtained by applying a few periods of brief ischemia and reperfusion in the event of prolonged (index) ischemia that may cause myocardial infarction. Whilst the conditioning stimulus is applied before the index ischemia in ischemic pre-conditioning, it is applied after the event in post-conditioning. Pre and post- conditioning stimuli can be applied in a different/remote organ (remote pre- and post-conditioning); in this case conditioning stimulus can also be applied during the index event, in the so called remote per-conditioning. All these endogenous cardioprotective strategies recruit endogenous cytoprotective agents and factors that elicit specific cardioprotective pathways. Here, we discuss many of these cardioprotective factors compared to literature and highlight their main characteristics and mechanisms of action. Enphasis is given to endogenous cardioprotective agents acting or not on surface receptors, including chromogranin A derivatives, ghrelin-associated peptides, growth factors and cytokines, and to microvesicles and exosomes. Moreover the cardioprotective effects of gasotransmitters nitric oxide, hydrogen sulphide and carbon monoxide are reviewed. The possible clinical translation of these knowledge for future successful therapies is briefly and critically discussed.

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.

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 PKCε, 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 PKCε inhibitor (εV1-2), 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 εV1-2, 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 (PKCε 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.

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.

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.

Low concentrations of a hydrophilic nitric oxide donor (NOD) are reported to reduce myocardial reperfusion injury only when combined with a lipophilic antioxidant (AOX) to form a hybrid molecule (HYB). Here we tested whether liposoluble NOD requires to be combined with AOX to be protective. Isolated rat hearts underwent 30 minutes of ischemia and 120 minutes of reperfusion. To induce postconditioning, 1 μM solutions of the following liposoluble compounds were given during the first 20 minutes of reperfusion: NOD with weak (w-NOD) or strong NO-releasing potency (s-NOD); weak HYB built up with w-NOD and a per se ineffective AOX lead; strong HYB built up with s-NOD and the same AOX; mixtures of w-NOD plus AOX or s-NOD plus AOX. A significant reduction of infarct size with improved recovery of cardiac function was obtained only with weak HYB. We suggest that w-NOD requires the synergy with a per se ineffective AOX to protect. The synergy is possible only if the 2 moieties enter the cell simultaneously as a hybrid, but not as a mixture. It seems that strong HYB was ineffective because an excessive intracellular NO release produces a large amount of reactive species, as shown from the increased nitrotyrosine production.

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.

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&#95;ND) or 10 weeks (long&#95;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&#95;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&#95;ND hearts than the other groups. Although cardiac/body weight ratio markedly increased after long&#95;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&#95;ND increased phosphatases, non-phosphorylated and phosphorylated Akt, long&#95;ND reduced phosphatase-expression and Akt phosphorylation. Both short&#95;ND and long&#95;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&#95;ND improves post-ischemic myocardial performance in postconditioned hearts. However, long&#95;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.

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.

Nitric oxide (NO) and reactive oxygen species (ROS) are double-edged swords in reperfused hearts. The effects of a NO-donor and an antioxidant compound against ischemia/reperfusion were studied. The compounds were tested separately, as a mixture and as a new hybrid molecule containing both leads. Isolated rat hearts underwent 30 min global ischemia and 2 hrs reperfusion. Compounds were infused either at 1 or 10 microM concentrations during the first 20 min of reperfusion. Hybrid was also tested in the presence of mitochondrial K(+) ATP-sensitive (mKATP) channel blockade by 5-HD (100 microM). Reduction of infarct size and recovery of left ventricular developed pressure during reperfusion were evaluated. When given at 1 microM concentration, hybrid significantly improved all indices of protection; its beneficial effects were abolished by mKATP channel blockade. At the same concentration, mixture and NO-donor alone improved recovery of left ventricular developed pressure but did not reduce infarct size; antioxidant was ineffective. When given at 10 microM concentration, antioxidant and mixture improved all parameters of protection; NO-donor and hybrid were ineffective. Our data suggest that different signaling cascades could be elicited by low and high concentrations of antioxidant compound and/or NO-donor. It is likely that a different NO-induced release of reactive oxygen species via mKATP channel activation may play a pivotal role in affecting infarct size and post-ischemic contractile recovery.

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.

Whether cardioprotection by postconditioning (PostC) is gender dependent is not clear. We studied the effect of PostC in terms of both infarct size (IS) and post-ischemic systolic dysfunction (PSD) reduction. Isolated male and female rat hearts were subjected to 10- or 30-min of global ischemia and 120-min of reperfusion, with or without PostC (i.e., 5 cycles of 10-s reperfusion/ischemia immediately after the ischemia). Surprisingly, after 10-min ischemia, IS and PSD were greater in female than male hearts (IS: 21 +/- 2% Vs. 11 +/- 2%; P < 0.01), while PostC attenuated IS and PSD in female hearts only. After 30-min ischemia IS was smaller in female than male hearts (52 +/- 2% Vs. 61 +/- 3%; P < 0.05), whereas PSD was similar in these two groups. PostC reduced IS in both genders, though the effect was smaller (P < 0.05) in females. Yet, PostC reduced PSD in female, but not in male hearts. Contracture development paralleled IS in all groups. To check the effects of buffer perfusion over heart function, additional hearts underwent 150-min buffer perfusion only. Contractile function of these hearts was not significantly affected over time. In conclusion IS, contracture and PSD are differently affected by gender, depending on ischemia duration. Yet, reduction of IS induced by PostC depends on the extension of IS induced by index-ischemia. While in female hearts reduction of PSD paralleled IS reduction, in male it does not occur. Results suggest that improvement of systolic function is mainly due to the anti-necrotic rather than to the anti-stunning effect exerted by PostC.

A series of brief (a few minutes) ischemia/reperfusion cycles (ischemic preconditioning, IP) limits myocardial injury produced by a subsequent prolonged period of coronary artery occlusion and reperfusion. Postconditioning (PostC), which is a series of brief (a few seconds) reperfusion/ischemia cycles at reperfusion onset, attenuates also ischemia/reperfusion injury. In recent years the main idea has been that reactive oxygen species (ROS) play an essential, though double-edged, role in cardioprotection: they may participate in reperfusion injury or may play a role as signaling elements of protection in the pre-ischemic phase. It has been demonstrated that preconditioning triggering is redox-sensitive, using either ROS scavengers or ROS generators. We have shown that nitroxyl triggers preconditioning via pro-oxidative, and/or nitrosative stress-related mechanism(s). Several metabolites, including acetylcholine, bradykinin, opioids and phenylephrine, trigger preconditioning-like protection via a mitochondrial K(ATP)-ROS-dependent mechanism. Intriguingly, and contradictory to the above mentioned theory of ROS as an obligatory part of reperfusion-induced damage, some studies suggest the possibility that some ROS at low concentrations could protect ischemic hearts against reperfusion injury. Yet, we demonstrated that ischemic PostC is also a cardioprotective phenomenon that requires the intervention of redox signaling to be protective. Emerging evidence suggests that in a preconditioning scenario a redox signal is required during the first few minutes of myocardial reperfusion following the index ischemic period. Intriguingly, the ROS signaling in the early reperfusion appear crucial to both preconditioning- and postconditioning-induced protection. Therefore, our and others' results suggest that the role of ROS in reperfusion may be reconsidered as they are not only deleterious.

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-3beta 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.

Background: 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., Materials and Methods: 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., Results: In Control hearts after ischemia/reperfusion, infarct size was 64% +/- 4% of risk-area. PostC reduced infarct size to 28% +/- 3% (P < 0.01). 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; P < 0.01 versus PostC). Despite disparities on infarct size, post-ischemic systolic function was similar among groups., Conclusions: 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.

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 (omega3-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 1ml/day of fish oil for one month; 3) FE+omega3-FFA rats who were given 1ml/day of fish oil and forced to run on a motorized wheel for 30min 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 30min global ischemic event and 2h reperfusion, hearts of sedentary-omega3 animals recovered about 37% of left ventricular developed pressure, whereas FE, omega3+FE and CTRL-SED animals recovered only about 15%, 5% and 8% respectively. Similarly, heart infarct size was significantly lower in sedentary-omega3 animals compared to animals in the three other groups., Conclusions: Results indicate that one month of treatment with an omega3-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 omega3-FFA diet.

Beta(2)-adrenoreceptor overexpression is beneficial against ischemia/reperfusion (I/R) injury. Whether beta-adrenoreceptors are involved in postconditioning (PostC) is unknown. We investigated whether nandrolone-decanoate (ND)-pretreatment can modulate (1) beta-adrenoreceptor expression and (2) post-ischemic cardiac function in response to I/R and PostC. Finally, we tested whether cardioprotection can be prevented by the inhibition of beta(2)-adrenoreceptors. Isolated rat hearts from ND pretreated (15 mg/kg/day i.m., for 14 days) and untreated-animals underwent 30-min ischemia and 120-min reperfusion. In subgroups, at the end of ischemia a PostC protocol (five cycles of 10-s reperfusion and 10-s ischemia) was applied and/or a beta(2)-adrenoreceptor blocker, ICI-118.551 (10 microM), was infused. Left ventricular pressure (LVP) was measured with an electromanometer, and infarct-size was evaluated using nitro-blue-tetrazolium staining. ND-pretreatment increased beta(2)-adrenoreceptor expression, but did not alter cardiac-weight, LVP and maximum rate of increase of LVP (dP/dt(max)). After I/R, infarct-size was smaller in ND-pretreatment than in untreated-animals. Infarct-size was also reduced by PostC, both in untreated and ND-pretreated animals. Contracture was less marked in ND-pretreated animals. PostC reduced contracture in both ND-pretreated and untreated hearts. Moreover, PostC improved post-ischemic recovery of developed LVP and dP/dt(max) much more in earts of ND-pretreated than untreated-animals. ICI-118.551 abolished ND protection and PostC-protection both in ND-pretreated and untreated hearts. Data show that two-weeks ND-pretreatment induces 1) an overexpression of beta(2)-ARs without cardiac hypertrophy and 2) improves the post-ischemic diastolic and systolic cardiac function. Intriguingly, ND-pretreatment potentiates the improvement of systolic function induced by postconditioning via beta(2)-adrenoreceptor activation.

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% P < 0.001 Vs. Control). BK intermittent protocol to mimic PostC, attenuated infarct size (40% +/- 2% P < 0.01 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.

Ischaemic preconditioning limits the damage induced by subsequent ischaemia/reperfusion (I/R). However, preconditioning is of little practical use as the onset of an infarction is usually unpredictable. Recently, it has been shown that the heart can be protected against the extension of I/R injury if brief (10-30 sec.) coronary occlusions are performed just at the beginning of the reperfusion. This procedure has been called postconditioning (PostC). It can also be elicited at a distant organ, termed remote PostC, by intermittent pacing (dyssynchrony-induced PostC) and by pharmacological interventions, that is pharmacological PostC. In particular, brief applications of intermittent bradykinin or diazoxide at the beginning of reperfusion reproduce PostC protection. PostC reduces the reperfusion-induced injury, blunts oxidant-mediated damages and attenuates the local inflammatory response to reperfusion. PostC induces a reduction of infarct size, apoptosis, endothelial dysfunction and activation, neutrophil adherence and arrhythmias. Whether it reduces stunning is not clear yet. Similar to preconditioning, PostC triggers signalling pathways and activates effectors implicated in other cardioprotective manoeuvres. Adenosine and bradykinin are involved in PostC triggering. PostC triggers survival kinases (RISK), including Akappat and extracellular signal-regulated kinase (ERK). Nitric oxide, via nitric oxide synthase and non-enzymatic production, cyclic guanosine monophosphate (cGMP) and protein kinases G (PKG) participate in PostC. PostC-induced protection also involves an early redox-sensitive mechanism, and mitochondrial adenosine-5' -triphosphate (ATP)-sensitive K(+) and PKC activation. Protective pathways activated by PostC appear to converge on mitochondrial permeability transition pores, which are inhibited by acidosis and glycogen synthase kinase-3beta (GSK-3beta). In conclusion, the first minutes of reperfusion represent a window of opportunity for triggering the aforementioned mediators which will in concert lead to protection against reperfusion injury. Pharmacological PostC and possibly remote PostC may have a promising future in clinical scenario.

The acidic protein chromogranin A (CgA) is the precursor of several regulatory peptides generated by specific proteolytic processes. Human recombinant CgA NH(2)-terminal fragment STA-CgA(1-78) (hrSTA-CgA(1-78)), containing vasostatin-1 (CgA(1-76)) domain, exerts a negative inotropic effect and counteracts the beta-adrenergic positive inotropic effect on the rat heart. We hypothesized an involvement of nitric oxide (NO)-dependent pathway in both cardiodepression and cardioprotection by hrSTA-CgA(1-78). We also hypothesized an involvement of adenosine A(1) receptor and protein kinase C (PKC) in cardioprotection by hrSTA-CgA(1-78). Therefore, we evaluated whether 1) the cardioinhibition mediated by hrSTA-CgA(1-78) involves the G(i/o) proteins/NO-dependent signal transduction cascade, 2) hrSTA-CgA(1-78) induces ischemic preconditioning-like protective effects on the myocardium, and 3) inhibition of NO synthase (NOS), adenosine A(1) receptor, or PKC affects hrSTA-CgA(1-78) protection. Using the isolated rat heart, we found that the reduction of left ventricular pressure (LVP), rate-pressure product, and maximal values of the first derivative of LVP elicited by hrSTA-CgA(1-78) at 33 nM is abolished by blocking G(i/o) proteins with pertussis toxin, scavenging NO with hemoglobin, and blocking NOS activity with N(G)-monomethyl-l-arginine or N(5)-(iminoethyl)-l-ornithine, soluble guanylate cyclase with 1H-[1,2,4]oxadiazole-[4,4-a]quinoxalin-1-one, and protein kinase (PKG) with KT5823. Data suggest the involvement of the G(i/o) proteins/NO-cGMP-PKG pathway in the hrSTA-CgA(1-78)-dependent cardioinhibition. When given before 30 min of ischemia, hrSTA-CgA(1-78) significantly reduced the size of the infarct from 64 +/- 4 to 32 +/- 3% of the left ventricular mass. This protective effect was abolished by either NOS inhibition or PKC blockade and was attenuated, but not suppressed, by the blockade of A(1) receptors. These results suggest that hrSTA-CgA(1-78) activity triggers two different pathways: one of these pathways is mediated by A(1) receptors, and the other is mediated by NO release. As with repeated brief preconditioning ischemia, hrSTA-CgA(1-78) may be considered a stimulus strong enough to trigger both pathways, which may converge on PKC.

Post-ischemic reperfusion worsens myocardial injury. Ischemic preconditioning limits the damage by ischemia and reperfusion. Both adenosine and nitric oxide (NO) pathways are involved in protection. Preconditioning, however, is of little, if any, practical use as the onset of an infarction is usually unpredictable. Recently, it has been shown that the heart can be protected against the extension of reperfusion injuries if 3 or 4 brief (10-30 s) coronary occlusions are performed just at the beginning of the reperfusion. This procedure has been called post-conditioning. Post-conditioning reduces the oxidant-induced injury; moreover, it attenuates the local inflammatory response to reperfusion. Post-conditioning also activates triggers, signaling pathways and effectors implicated in other cardioprotective maneuvers, such as ischemic and pharmacological preconditioning. Post-conditioning seems to trigger the up-regulation of survival kinases principally known to attenuate the pathogenesis of apoptosis and possibly necrosis. As regards the possibility of pharmacological post-conditioning, several agents have been tested. We are testing NO donor(s), which can reduce infarct size in the rat in the absence of post-conditioning. Since during reperfusion there is a large production of reactive oxygen species, also the effect of administration of an antioxidant compound during reperfusion was studied. In the rat, such a procedure reduced the infarct size to a greater extent than post-conditioning. Moreover, an additive effect of NO donors and antioxidant compounds is possible.