Saveria Femminò

Pharmacological Inhibition of NLRP3 Inflammasome Attenuates Myocardial Ischemia/Reperfusion Injury by Activation of RISK and Mitochondrial Pathways

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 (30u2009min) followed by reperfusion (20 or 60u2009min) with and without pretreatment with the recently synthetized NLRP3 inflammasome inhibitor INF4E (50u2009μM, 20u2009min 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.

Catestatin exerts direct protective effects on rat cardiomyocytes undergoing ischemia/reperfusion by stimulating PI3K-Akt-GSK3β pathway and preserving mitochondrial membrane potential.

Catestatin (Cst) is a 21-amino acid peptide deriving from Chromogranin A. Cst exerts an overall protective effect against an excessive sympathetic stimulation of cardiovascular system, being able to antagonize catecholamine secretion and to reduce their positive inotropic effect, by stimulating the release of nitric oxide (NO) from endothelial cells. Moreover, Cst reduces ischemia/reperfusion (I/R) injury, improving post-ischemic cardiac function and cardiomyocyte survival. To define the cardioprotective signaling pathways activated by Cst (5 nM) we used isolated adult rat cardiomyocytes undergoing simulated I/R. We evaluated cell viability rate with propidium iodide labeling and mitochondrial membrane potential (MMP) with the fluorescent probe JC-1. The involvement of Akt, GSK3β, eNOS and phospholamban (PLN) cascade was studied by immunofluorescence. The role of PI3K-Akt/NO/cGMP pathway was also investigated by using the pharmacological blockers wortmannin (Wm), L-NMMA and ODQ. Our experiments revealed that Cst increased cell viability rate by 65% and reduced cell contracture in I/R cardiomyocytes. Wm, L-NMMA and ODQ limited the protective effect of Cst. The protective outcome of Cst was related to its ability to maintain MMP and to increase AktSer473, GSK3βSer9, PLNThr17 and eNOSSer1179 phosphorylation, while treatment with Wm abolished these effects. Thus, the present results show that Cst is able to exert a direct action on cardiomyocytes and give new insights into the molecular mechanisms involved in its protective effect, highlighting the PI3K/NO/cGMP pathway as the trigger and the MMP preservation as the end point of its action.

From molecular mechanisms to clinical management of antineoplastic drug-induced cardiovascular toxicity: A translational overview

Significance: Antineoplastic therapies have significantly improved the prognosis of oncology patients. However, these treatments can bring to a higher incidence of side-effects, including the worrying cardiovascular toxicity (CTX). Recent Advances: Substantial evidence indicates multiple mechanisms of CTX, with redox mechanisms playing a key role. Recent data singled out mitochondria as key targets for antineoplastic drug-induced CTX; understanding the underlying mechanisms is, therefore, crucial for effective cardioprotection, without compromising the efficacy of anti-cancer treatments. Critical Issues: CTX can occur within a few days or many years after treatment. Type I CTX is associated with irreversible cardiac cell injury, and it is typically caused by anthracyclines and traditional chemotherapeutics. Type II CTX is generally caused by novel biologics and more targeted drugs, and it is associated with reversible myocardial dysfunction. Therefore, patients undergoing anti-cancer treatments should be closely monitored, and patients at risk of CTX should be identified before beginning treatment to reduce CTX-related morbidity. Future Directions: Genetic profiling of clinical risk factors and an integrated approach using molecular, imaging, and clinical data may allow the recognition of patients who are at a high risk of developing chemotherapy-related CTX, and it may suggest methodologies to limit damage in a wider range of patients. The involvement of redox mechanisms in cancer biology and anticancer treatments is a very active field of research. Further investigations will be necessary to uncover the hallmarks of cancer from a redox perspective and to develop more efficacious antineoplastic therapies that also spare the cardiovascular system.

Obestatin regulates cardiovascular function and promotes cardioprotection through the nitric oxide pathway

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.

α-Cyclodextrin and α-Cyclodextrin Polymers as Oxygen Nanocarriers to Limit Hypoxia/Reoxygenation Injury: Implications from an In Vitro Model

The incidence of heart failure (HF) is increasing worldwide and myocardial infarction (MI), which follows ischemia and reperfusion (I/R), is often at the basis of HF development. Nanocarriers are interesting particles for their potential application in cardiovascular disease. Impaired drug delivery in ischemic disease is challenging. Cyclodextrin nanosponges (NS) can be considered innovative tools for improving oxygen delivery in a controlled manner. This study has developed new α-cyclodextrin-based formulations as oxygen nanocarriers such as native α-cyclodextrin (α-CD), branched α-cyclodextrin polymer (α-CD POLY), and α-cyclodextrin nanosponges (α-CD NS). The three different α-CD-based formulations were tested at 0.2, 2, and 20 µg/mL to ascertain their capability to reduce cell mortality during hypoxia and reoxygenation (H/R) in vitro protocols. H9c2, a cardiomyoblast cell line, was exposed to normoxia (20% oxygen) or hypoxia (5% CO2 and 95% N2). The different formulations, applied before hypoxia, induced a significant reduction in cell mortality (in a range of 15% to 30%) when compared to samples devoid of oxygen. Moreover, their application at the beginning of reoxygenation induced a considerable reduction in cell death (12% to 20%). α-CD NS showed a marked efficacy in controlled oxygenation, which suggests an interesting potential for future medical application of polymer systems for MI treatment.

Cardioprotective effects of calcitonin gene-related peptide in isolated rat heart and in H9c2 cells via redox signaling

The calcitonin-gene-related-peptide (CGRP) release is coupled to the signaling of Angelis salt in determining vasodilator effects. However, it is unknown whether CGRP is involved in Angelis salt cardioprotective effects and which are the mechanisms of protection. We aimed to determine whether CGRP is involved in myocardial protection induced by Angelis salt. We also analyzed the intracellular signaling pathway activated by CGRP. Isolated rat hearts were pre-treated with Angelis salt or Angelis salt plus CGRP8-37, a specific CGRP-receptor antagonist, and subjected to ischemia (30-min) and reperfusion (120-min). Moreover, we studied CGRP-induced protection during oxidative stress (H2O2) and hypoxia/reoxygenation protocols in H9c2 cardiomyocytes. Cell vitality and mitochondrial membrane potential (ΔYm, MMP) were measured using MTT and JC-1 dyes. Angelis salt reduced infarct size and ameliorated post-ischemic cardiac function via a CGRP-receptor-dependent mechanism. Pre-treatment with CGRP increased H9c2 survival upon challenging with either H2O2 (redox stress) or hypoxia/reoxygenation (H/R stress). Under these stress conditions, reduction in MMP and cell death were partly prevented by CGRP. These CGRP beneficial effects were blocked by CGRP8-37. During H/R stress, pre-treatment with either CGRP-receptor, protein kinase C (PKC) or mitochondrial KATP channel antagonists, and pre-treatment with an antioxidant (2-mercaptopropionylglycine) blocked the protection mediated by CGRP. In conclusion, CGRP is involved in the cardioprotective effects of Angelis salt. In H9c2 cardiomyocytes, CGRP elicits PKC-dependent and mitochondrial-KATP-redox-dependent mechanisms. Hence, CGRP is an important factor in the redox-sensible cardioprotective effects of Angelis salt.

Cardioprotective properties of human platelets are lost in uncontrolled diabetes mellitus: A study in isolated rat hearts

Platelets affect myocardial damage from ischemia/reperfusion. Redox-dependent sphingosine-1-phosphate production and release are altered in diabetic platelets. Sphingosine-1-phosphate is a double-edged sword for ischemia/reperfusion injury. Therefore, we aimed to verify whether: (1) human healthy- or diabetic-platelets are cardioprotective, (2) sphingosine-1-phosphate receptors and downstream kinases play a role in platelet-induced cardioprotection, and (3) a correlation between platelet redox status and myocardial ischemia/reperfusion injury exists. Isolated rat hearts were subjected to 30-min ischemia and 1-h reperfusion. Infarct size was studied in hearts pretreated with healthy- or diabetic-platelets. Healthy-platelets were co-infused with sphingosine-1-phosphate receptor blocker, ERK-1/2 inhibitor, PI3K antagonist or PKC inhibitor to ascertain the cardioprotective mechanisms. In platelets we assessed (i) aggregation response to ADP, collagen, and arachidonic-acid, (ii) cyclooxygenase-1 levels, and (iii) AKT and ERK-phosphorylation. Platelet sphingosine-1-phosphate production and platelet levels of reactive oxygen species (ROS) were quantified and correlated to infarct size. Infarct size was reduced by about 22% in healthy-platelets pretreated hearts only. This cardioprotective effect was abrogated by either sphingosine-1-phosphate receptors or ERK/PI3K/PKC pathway blockade. Cyclooxygenase-1 levels and aggregation indices were higher in diabetic-platelets than healthy-platelets. Diabetic-platelets released less sphingosine-1-phosphate than healthy-platelets when mechanical or chemically stimulated in vitro. Yet, ROS levels were higher in diabetic-platelets and correlated with infarct size. Cardioprotective effects of healthy-platelet depend on the platelet’s capacity to activate cardiac sphingosine-1-phosphate receptors and ERK/PI3K/PKC pathways. However, diabetic-platelets release less S1P and lose cardioprotective effects. Platelet ROS levels correlate with infarct size. Whether these redox alterations are responsible for sphingosine-1-phosphate dysfunction in diabetic-platelets remains to be ascertained.

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

Hans Erik Bøtker1 · Derek Hausenloy2,3,4,5,6 · Ioanna Andreadou7 · Salvatore Antonucci8 · Kerstin Boengler9 · Sean M. Davidson2 · Soni Deshwal8 · Yvan Devaux10 · Fabio Di Lisa8 · Moises Di Sante8 · Panagiotis Efentakis7 · Saveria Femminò11 · David García‐Dorado12 · Zoltán Giricz13,14 · Borja Ibanez15 · Efstathios Iliodromitis16 · Nina Kaludercic8 · Petra Kleinbongard17 · Markus Neuhäuser18,19 · Michel Ovize20,21 · Pasquale Pagliaro11 · Michael Rahbek‐Schmidt1 · Marisol Ruiz‐Meana12 · Klaus‐Dieter Schlüter9 · Rainer Schulz9 · Andreas Skyschally17 · Catherine Wilder2 · Derek M. Yellon2 · Peter Ferdinandy13,14 · Gerd Heusch17

Redox Aspects of Chaperones in Cardiac Function

Molecular chaperones are stress proteins that allow the correct folding or unfolding as well as the assembly or disassembly of macromolecular cellular components. Changes in expression and post-translational modifications of chaperones have been linked to a number of age- and stress-related diseases including cancer, neurodegeneration, and cardiovascular diseases. Redox sensible post-translational modifications, such as S-nitrosylation, glutathionylation and phosphorylation of chaperone proteins have been reported. Redox-dependent regulation of chaperones is likely to be a phenomenon involved in metabolic processes and may represent an adaptive response to several stress conditions, especially within mitochondria, where it impacts cellular bioenergetics. These post-translational modifications might underlie the mechanisms leading to cardioprotection by conditioning maneuvers as well as to ischemia/reperfusion injury. In this review, we discuss this topic and focus on two important aspects of redox-regulated chaperones, namely redox regulation of mitochondrial chaperone function and cardiac protection against ischemia/reperfusion injury.

Mitochondria in cardiac postconditioning

Mitochondria play a pivotal role in cardioprotection. Here we report some fundamental studies which considered the role of mitochondrial components (connexin 43, mitochondrial KATP channels and mitochondrial permeability transition pore) in postconditioning cardioprotection. We briefly discuss the role of mitochondria, reactive oxygen species and gaseous molecules in postconditioning. Also the effects of anesthetics—used as cardioprotective substances—is briefly considered in the context of postconditioning. The role of mitochondrial postconditioning signaling in determining the limitation of cell death is underpinned. Issues in clinical translation are briefly considered. The aim of the present mini-review is to discuss in a historical perspective the role of main mitochondria mechanisms in cardiac postconditioning.