Teresa Pasqua

Nesfatin-1 as a novel cardiac peptide: identification, functional characterization, and protection against ischemia/reperfusion injury

Nesfatin-1 is an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide. It controls feeding behavior, water intake, and glucose homeostasis. If intracerebrally administered, it induces hypertension, thus suggesting a role in central cardiovascular control. However, it is not known whether it is able to directly control heart performance. We aimed to verify the hypothesis that, as in the case of other hypothalamic satiety peptides, Nesfatin-1 acts as a peripheral cardiac modulator. By western blotting and QT-PCR, we identified the presence of both Nesfatin-1 protein and NUCB2 mRNA in rat cardiac extracts. On isolated and Langendorff-perfused rat heart preparations, we found that exogenous Nesfatin-1 depresses contractility and relaxation without affecting coronary motility. These effects did not involve Nitric oxide, but recruited the particulate guanylate cyclase (pGC) known as natriuretic peptide receptor A (NPR-A), protein kinase G (PKG) and extracellular signal-regulated kinases1/2 (ERK1/2). Co-immunoprecipitation and bioinformatic analyses supported an interaction between Nesfatin-1 and NPR-A. Lastly, we preliminarily observed, through post-conditioning experiments, that Nesfatin-1 protects against ischemia/reperfusion (I/R) injury by reducing infarct size, lactate dehydrogenase release, and postischemic contracture. This protection involves multiple prosurvival kinases such as PKCε, ERK1/2, signal transducer and activator of transcription 3, and mitochondrial KATP channels. It also ameliorates contractility recovery. Our data indicate that: (1) the heart expresses Nesfatin-1, (2) Nesfatin-1 directly affects myocardial performance, possibly involving pGC-linked NPR-A, the pGC/PKG pathway, and ERK1/2, (3) the peptide protects the heart against I/R injury. Results pave the way to include Nesfatin-1 in the neuroendocrine modulators of the cardiac function, also encouraging the clarification of its clinical potential in the presence of nutrition-dependent physio-pathologic cardiovascular diseases.

The novel chromogranin A-derived serpinin and pyroglutaminated serpinin peptides are positive cardiac β-adrenergic-like inotropes

Three forms of serpinin peptides, serpinin (Ala26Leu), pyroglutaminated (pGlu)‐serpinin (pGlu23Leu), and serpinin‐Arg‐Arg‐Gly (Ala29Gly), are derived from cleavage at pairs of basic residues in the highly conserved C terminus of chromogranin A (CgA). Serpinin induces PN‐1 expression in neuroendocrine cells to up‐regulate granule biogenesis via a cAMP‐protein kinase A‐Sp1 pathway, while pGlu‐serpinin inhibits cell death. The aim of this study was to test the hypothesis that serpinin peptides are produced in the heart and act as novel β‐adrenergic‐like cardiac modulators. We detected serpinin peptides in the rat heart by HPLC and ELISA methods. The peptides included predominantly Ala29Gly and pGlu‐serpinin and a small amount of serpinin. Using the Langendorff perfused rat heart to evaluate the hemodynamic changes, we found that serpinin and pGlu‐serpinin exert dose‐dependent positive inotropic and lusitropic effects at 11–165 nM, within the first 5 min after administration. The pGlu‐serpinin‐induced contractility is more potent than that of serpinin, starting from 1 nM. Using the isolated rat papillary muscle preparation to measure contractility in terms of tension development and muscle length, we further corroborated the pGlu‐serpinin‐induced positive inotropism. Ala29Gly was unable to affect myocardial performance. Both pGlu‐serpinin and serpinin act through a β1‐adrenergic receptor/adenylate cyclase/cAMP/PKA pathway, indicating that, contrary to the β‐blocking profile of the other CgA‐derived cardiosuppressive peptides, vasostatin‐1 and catestatin, these two C‐terminal peptides act as β‐adrenergic‐like agonists. In cardiac tissue extracts, pGluserpinin increased intracellular cAMP levels and phosphorylation of phospholamban (PLN)Ser16, ERK1/2, and GSK‐3β. Serpinin and pGlu‐serpinin peptides emerge as novel β‐adrenergic inotropic and lusitropic modulators, suggesting that CgA and the other derived cardioactive peptides can play a key role in how the myocardium orchestrates its complex response to sympathochromaffin stimulation.—Tota, B., Gentile, S., Pasqua, T., Bassino, E., Koshimizu, H., Cawley, N. X., Cerra, M. C., Loh, Y. P., Angelone, T. The novel chromogranin A‐derived serpinin and pyroglutaminated serpinin peptides are positive cardiac β‐adrenergic‐like inotropes. FASEB J. 26, 2888–2898 (2012). www.fasebj.org

GPER Mediates Cardiotropic Effects in Spontaneously Hypertensive Rat Hearts

Estrogens promote beneficial effects in the cardiovascular system mainly through the estrogen receptor (ER)α and ERβ, which act as ligand-gated transcription factors. Recently, the G protein-coupled estrogen receptor (GPER) has been implicated in the estrogenic signaling in diverse tissues, including the cardiovascular system. In this study, we demonstrate that left ventricles of male Spontaneously Hypertensive Rats (SHR) express higher levels of GPER compared to normotensive Wistar Kyoto (WKY) rats. In addition, we show that the selective GPER agonist G-1 induces negative inotropic and lusitropic effects to a higher extent in isolated and Langendorff perfused hearts of male SHR compared to WKY rats. These cardiotropic effects elicited by G-1 involved the GPER/eNOS transduction signaling, as determined by using the GPER antagonist G15 and the eNOS inhibitor L-NIO. Similarly, the G-1 induced activation of ERK1/2, AKT, GSK3β, c-Jun and eNOS was abrogated by G15, while L-NIO prevented only the eNOS phosphorylation. In hypoxic Langendorff perfused WKY rat heart preparations, we also found an increased expression of GPER along with that of the hypoxic mediator HIF-1α and the fibrotic marker CTGF. Interestingly, G15 and L-NIO prevented the ability of G-1 to down-regulate the expression of both HIF-1α and CTGF, which were found expressed to a higher extent in SHR compared to WKY rat hearts. Collectively, the present study provides novel data into the potential role played by GPER in hypertensive disease on the basis of its involvement in myocardial inotropism and lusitropism as well as the expression of the apoptotic HIF-1α and fibrotic CTGF factors. Hence, GPER may be considered as a useful target in the treatment of some cardiac dysfunctions associated with stressful conditions like the essential hypertension.

Full-Length Human Chromogranin-A Cardioactivity: Myocardial, Coronary, and Stimulus-Induced Processing Evidence in Normotensive and Hypertensive Male Rat Hearts

Plasma chromogranin-A (CgA) concentrations correlate with severe cardiovascular diseases, whereas CgA-derived vasostatin-I and catestatin elicit cardiosuppression via an antiadrenergic/nitric oxide-cGMP mediated mechanism. Whether these phenomena are related is unknown. We here investigated whether and to what extent full-length CgA directly influences heart performance and may be subjected to stimulus-elicited intracardiac processing. Using normotensive and hypertensive rats, we evaluated the following: 1) direct myocardial and coronary effects of full-length CgA; 2) the signal-transduction pathway involved in its action mechanism; and 3) CgA intracardiac processing after β-adrenergic [isoproterenol (Iso)]- and endothelin-1(ET-1)-dependent stimulation. The study was performed by using a Langendorff perfusion apparatus, Western blotting, affinity chromatography, and ELISA. We found that CgA (1-4 nM) dilated coronaries and induced negative inotropism and lusitropism, which disappeared at higher concentrations (10-16 nM). In spontaneously hypertensive rats (SHRs), negative inotropism and lusitropism were more potent than in young normotensive rats. We found that perfusion itself, Iso-, and endothelin-1 stimulation induced intracardiac CgA processing in low-molecular-weight fragments in young, Wistar Kyoto, and SHR rats. In young normotensive and adult hypertensive rats, CgA increased endothelial nitric oxide synthase phosphorylation and cGMP levels. Analysis of the perfusate from both Wistar rats and SHRs of untreated and treated (Iso) hearts revealed CgA absence. In conclusion, in normotensive and hypertensive rats, we evidenced the following: 1) full-length CgA directly affects myocardial and coronary function by AkT/nitric oxide synthase/nitric oxide/cGMP/protein kinase G pathway; and 2) the heart generates intracardiac CgA fragments in response to hemodynamic and excitatory challenges. For the first time at the cardiovascular level, our data provide a conceptual link between systemic and intracardiac actions of full-length CgA and its fragments, expanding the knowledge on the sympathochromaffin/CgA axis under normal and physiopathological conditions.

Phosphodiesterase type-2 and NO-dependent S-nitrosylation mediate the cardioinhibition of the antihypertensive catestatin

The chromogranin A (CHGA)-derived peptide catestatin (CST: hCHGA(352-372)) is a noncompetitive catecholamine-release inhibitor that exerts vasodilator, antihypertensive, and cardiosuppressive actions. We have shown that CST directly influences the basal performance of the vertebrate heart where CST dose dependently induced a nitric oxide-cGMP-dependent cardiosuppression and counteracted the effects of adrenergic stimulation through a noncompetitive antagonism. Here, we sought to determine the specific intracardiac signaling activated by CST in the rat heart. Physiological analyses performed on isolated, Langendorff-perfused cardiac preparations revealed that CST-induced negative inotropism and lusitropism involve β(2)/β(3)-adrenergic receptors (β(2)/β(3)-AR), showing a higher affinity for β(2)-AR. Interaction with β(2)-AR activated phosphatidylinositol 3-kinase/endothelial nitric oxide synthase (eNOS), increased cGMP levels, and induced activation of phosphodiesterases type 2 (PDE2), which was found to be involved in the antiadrenergic action of CST as evidenced by the decreased cAMP levels. CST-dependent negative cardiomodulation was abolished by functional denudation of the endothelium with Triton. CST also increased the eNOS expression in cardiac tissue and human umbilical vein endothelial cells. cells, confirming the involvement of the vascular endothelium. In ventricular extracts, CST increased S-nitrosylation of both phospholamban and β-arrestin, suggesting an additional mechanism for intracellular calcium modulation and β-adrenergic responsiveness. We conclude that PDE2 and S-nitrosylation play crucial roles in the CST regulation of cardiac function. Our results are of importance in relation to the putative application of CST as a cardioprotective agent against stress, including excessive sympathochromaffin overactivation.

Receptor identification and physiological characterisation of glucagon-like peptide-2 in the rat heart.

BACKGROUND AND AIMS The anorexigenic glucagon-like peptide (GLP)-2 is produced by intestinal L cells and released in response to food intake. It affects intestinal function involving G-protein-coupled receptors. To verify whether GLP-2 acts as a cardiac modulator in mammals, we analysed, in the rat heart, the expression of GLP-2 receptors and the myocardial and coronary responses to GLP-2. METHODS AND RESULTS GLP-2 receptors were detected on ventricular extracts by quantitative real-time polymerase chain reaction (Q-RT-PCR) and Western blotting. Cardiac GLP-2 effects were analysed on Langendorff perfused hearts. Intracellular GLP-2 signalling was investigated on Langendorff perfused hearts and by Western blotting and enzyme-linked immunosorbent assay (ELISA) on ventricular extracts. By immunoblotting and Q-RT-PCR, we revealed the expression of ventricular GLP-2 receptors. Perfusion analyses showed that GLP-2 induces positive inotropism at low concentration (10-12 mol l(-1)), and negative inotropism and lusitropism from 10 to 10 mol l(-1). It dose-dependently constricts coronaries. The negative effects of GLP-2 were independent from GLP-1 receptors, being unaffected by exendin-3 (9-39) amide. GLP-2-dependent negative action involves Gi/o proteins, associates with a reduction of intracellular cyclic adenosine monophosphate (cAMP), an increase in extracellular signal regulated kinases 1 and 2 (ERK1/2) and a decrease in phospholamban phosphorylation, but is independent from endothelial nitric oxide synthase (eNOS) and protein kinase G (PKG). Finally, GLP-2 competitively antagonised β-adrenergic stimulation. CONCLUSIONS For the first time, to our knowledge, we found that: (1) the rat heart expresses functional GLP-2 receptors; (2) GLP-2 acts on both myocardium and coronaries, negatively modulating both basal and β-adrenergic stimulated cardiac performance; and (3) GLP-2 effects are mediated by G-proteins and involve ERK1/2.

Catestatin increases the expression of anti-apoptotic and pro-angiogenetic factors in the post-ischemic hypertrophied heart of SHR.

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

Akt/eNOS signaling and PLN S-sulfhydration are involved in H2S-dependent cardiac effects in frog and rat

Hydrogen sulfide (H₂S) has recently emerged as an important mediator of mammalian cardiovascular homeostasis. In nonmammalian vertebrates, little is known about the cardiac effects of H₂S. This study aimed to evaluate, in the avascular heart of the frog, Rana esculenta, whether and to what extent H₂S affects the cardiac performance, and what is the mechanism of action responsible for the observed effects. Results were analyzed in relation to those obtained in the rat heart, used as the mammalian model. Isolated and perfused (working and Langendorff) hearts, Western blot analysis, and modified biotin switch (S-sulfhydration) assay were used. In the frog heart, NaHS (used as H₂S donor, 10⁻¹²/10⁻⁷ M) dose-dependently decreased inotropism. This effect was reduced by glibenclamide (KATP channels blocker), NG-monomethyl-L-arginine (NOS inhibitor), 1H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (guanylyl cyclase inhibitor), KT₅₈₂₃ (PKG inhibitor), and it was blocked by Akt1/2 (Akt inhibitor) and by detergent Triton X-100. In the rat, in addition to the classic negative inotropic effect, NaHS (10⁻¹²/10⁻⁷ M) exhibited negative lusitropism. In both frog and rat hearts, NaHS treatment induced Akt and eNOS phosphorylation and an increased cardiac protein S-sulfhydration that, in the rat heart, includes phospholamban. Our data suggest that H₂S represents a phylogenetically conserved cardioactive molecule. Results obtained on the rat heart extend the role of H₂S also to cardiac relaxation. H₂S effects involve KATP channels, the Akt/NOS-cGMP/PKG pathway, and S-sulfhydration of cardiac proteins.

The NO stimulator, Catestatin, improves the Frank-Starling response in normotensive and hypertensive rat hearts.

The myocardial response to mechanical stretch (Frank-Starling law) is an important physiological cardiac determinant. Modulated by many endogenous substances, it is impaired in the presence of cardiovascular pathologies and during senescence. Catestatin (CST:hCgA352-372), a 21-amino-acid derivate of Chromogranin A (CgA), displays hypotensive/vasodilatory properties and counteracts excessive systemic and/or intra-cardiac excitatory stimuli (e.g., catecholamines and endothelin-1). CST, produced also by the myocardium, affects the heart by modulating inotropy, lusitropy and the coronary tone through a Nitric Oxide (NO)-dependent mechanism. This study evaluated the putative influence elicited by CST on the Frank-Starling response of normotensive Wistar-Kyoto (WKY) and hypertensive (SHR) hearts by using isolated and Langendorff perfused cardiac preparations. Functional changes were evaluated on aged (18-month-old) WKY rats and SHR which mimic human chronic heart failure (HF). Comparison to WKY rats, SHR showed a reduced Frank-Starling response. In both rat strains, CST administration improved myocardial mechanical response to increased end-diastolic pressures. This effect was mediated by EE/IP3K/NOS/NO/cGMP/PKG, as revealed by specific inhibitors. CST-dependent positive Frank-Starling response is paralleled by an increment in protein S-Nitrosylation. Our data suggested CST as a NO-dependent physiological modulator of the stretch-induced intrinsic regulation of the heart. This may be of particular importance in the aged hypertrophic heart, whose function is impaired because of a reduced systolic performance accompanied by delayed relaxation and increased diastolic stiffness.

Granin-derived peptides

The granin family comprises altogether 7 different proteins originating from the diffuse neuroendocrine system and elements of the central and peripheral nervous systems. The family is dominated by three uniquely acidic members, namely chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). Since the late 1980s it has become evident that these proteins are proteolytically processed, intragranularly and/or extracellularly into a range of biologically active peptides; a number of them with regulatory properties of physiological and/or pathophysiological significance. The aim of this comprehensive overview is to provide an up-to-date insight into the distribution and properties of the well established granin-derived peptides and their putative roles in homeostatic regulations. Hence, focus is directed to peptides derived from the three main granins, e.g. to the chromogranin A derived vasostatins, betagranins, pancreastatin and catestatins, the chromogranin B-derived secretolytin and the secretogranin II-derived secretoneurin (SN). In addition, the distribution and properties of the chromogranin A-derived peptides prochromacin, chromofungin, WE14, parastatin, GE-25 and serpinins, the CgB-peptide PE-11 and the SgII-peptides EM66 and manserin will also be commented on. Finally, the opposing effects of the CgA-derived vasostatin-I and catestatin and the SgII-derived peptide SN on the integrity of the vasculature, myocardial contractility, angiogenesis in wound healing, inflammatory conditions and tumors will be discussed.