Modulation of Endothelial Function by TMAO, a Gut Microbiota-Derived Metabolite.

Endothelial function is essential in the maintenance of systemic homeostasis, whose modulation strictly depends on the proper activity of tissue-specific angiocrine factors on the physiopathological mechanisms acting at both single and multi-organ levels. Several angiocrine factors take part in the vascular function itself by modulating vascular tone, inflammatory response, and thrombotic state. Recent evidence has outlined a strong relationship between endothelial factors and gut microbiota-derived molecules. In particular, the direct involvement of trimethylamine N-oxide (TMAO) in the development of endothelial dysfunction and its derived pathological outcomes, such as atherosclerosis, has come to light. Indeed, the role of TMAO in the modulation of factors strictly related to the development of endothelial dysfunction, such as nitric oxide, adhesion molecules (ICAM-1, VCAM-1, and selectins), and IL-6, has been widely accepted. The aim of this review is to present the latest studies that describe a direct role of TMAO in the modulation of angiocrine factors primarily involved in the development of vascular pathologies.

Trimethylamine N-Oxide (TMAO) Impairs Purinergic Induced Intracellular Calcium Increase and Nitric Oxide Release in Endothelial Cells.

Trimethylamine N-oxide (TMAO) is a diet derived compound directly introduced through foodstuff, or endogenously synthesized from its precursors, primarily choline, L-carnitine, and ergothioneine. New evidence outlines high TMAO plasma concentrations in patients with overt cardiovascular disease, but its direct role in pathological development is still controversial. The purpose of the study was to evaluate the role of TMAO in affecting key intracellular factors involved in endothelial dysfunction development, such as reactive oxygen species, mitochondrial health, calcium balance, and nitric oxide release using bovine aortic endothelial cells (BAE-1). Cell viability and oxidative stress indicators were monitored after acute and prolonged TMAO treatment. The role of TMAO in interfering with the physiological purinergic vasodilatory mechanism after ATP stimulation was defined through measurements of the rise of intracellular calcium, nitric oxide release, and eNOS phosphorylation at Ser1179 (eNOSSer1179). TMAO was not cytotoxic for BAE-1 and it did not induce the rise of reactive oxygen species and impairment of mitochondrial membrane potential, either in the basal condition or in the presence of a stressor. In contrast, TMAO modified the purinergic response affecting intracellular ATP-induced calcium increase, nitric oxide release, and eNOSSer1179. Results obtained suggest a possible implication of TMAO in impairing the endothelial-dependent vasodilatory mechanism.

Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110γ.

Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining ß-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in ß-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes ß-adrenergic receptor density and improves contractility in failing hearts.

Trimethylamine N-Oxide Does Not Impact Viability, ROS Production, and Mitochondrial Membrane Potential of Adult Rat Cardiomyocytes.

Trimethylamine N-oxide (TMAO) is an organic compound derived from dietary choline and L-carnitine. It behaves as an osmolyte, a protein stabilizer, and an electron acceptor, showing different biological functions in different animals. Recent works point out that, in humans, high circulating levels of TMAO are related to the progression of atherosclerosis and other cardiovascular diseases. However, studies on a direct role of TMAO in cardiomyocyte parameters are still limited. The purpose of this work is to study the effects of TMAO on isolated adult rat cardiomyocytes. TMAO in both 100 µM and 10 mM concentrations, from 1 to 24 h of treatment, does not affect cell viability, sarcomere length, intracellular ROS, and mitochondrial membrane potential. Furthermore, the simultaneous treatment with TMAO and known cardiac insults, such as H2O2 or doxorubicin, does not affect the treatment's effect. In conclusion, TMAO cannot be considered a direct cause or an exacerbating risk factor of cardiac damage at the cellular level in acute conditions.

Development of morphology and function of neonatal mouse ventricular myocytes cultured on a hyaluronan-based polymer scaffold.

In recent years cardiac tissue engineering has emerged as a promising field aimed at developing suitable techniques to repair the infarcted myocardium with a combination of cells, biomaterials, and regulative factors. In particular it could stand for an alternative strategy to simple in situ cellular implantation. In the present study our purpose was to analyze the interaction between a hyaluronan-based mesh (HYALONECT®) and neonatal murine ventricular myocytes (NMVMs). Specifically, we investigated morphological and functional characteristics of cardiomyocytes cultured on HYALONECT® in view of its employment in heart repair. Both living and fixed cells analysis was performed on in toto scaffolds with confocal microscopy. NMVMs adhesion on HYALONECT® was studied by tracking sarcomeric α-actinin immunofluorescence staining. The structural features of NMVMs adherent onto HYALONECT® were investigated at 24, 48, 72 h, and 7 days of culture by immunofluorescence for sarcomeric α-actinin and connexin-43. We observed a progressive morphological organization of the cells inside the biopolymer, with both clear sarcomeric arrangement along the scaffold fibers and gap junctions development between adjacent cells. Finally, in vivo intracellular calcium measurements performed using calcium fluorimetric confocal imaging revealed the presence of spontaneous calcium transients and contractile activity of NMVMs adherent onto HYALONECT® up to 48 h from seeding, indicating a progressive differentiation of the cells toward the adult phenotype. In conclusion, our results demonstrate that HYALONECT® allowed NMVMs to adhere to the fibers and to develop functional properties, displaying suitable features as a scaffold to perform heart tissue engineering.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

A mouse model for spatial and temporal expression of HGF in the heart.

In order to study the effects of Hepatocyte Growth Factor (HGF) in the heart, two transgenic mice were developed, one carrying a bidirectional HGF-TetO-GFP responder construct and the other carrying a α-MHC-tTA transactivator construct. Crosses were carried out between heterozygotes, so that litters contained bitransgenic α-MHC-tTA/HGF-TetO-GFP+, thus expressing HGF and GFP exclusively in the heart and only in the absence of Doxycycline. Our data show that the expression of HGF was indeed restricted to the heart and that the expression was limited to the timeframe of the absence of Doxycycline. Surprisingly the expression was variable even between bitransgenic littermates. In the setting of a model of ischemia-reperfusion, the expression of HGF ameliorates cardiac functionality, enhances proliferation and diminishes the scarred area, proving that this is a good model to study the beneficial influences and functional roles of HGF in the heart.

Skull osteology of Vipera walser (Squamata, Viperidae): Description, variability, ontogeny, and diagnostic characters in comparison to other Italian vipers.

Vipera walser is the most recently recognized European viper. This rare species is endemic to a small area in the Piedmont Alps of Italy, but its closest relatives are found among the Caucasian viper species. In order to provide a starting point for a phylogenetic and biogeographic investigation based on osteology, and including fossils remains, we analyzed four specimens of V. walser and compared them with specimens of the four other Italian viper species. Based on these specimens, we improved the diagnosis of V. walser and provided a first evaluation of intraspecific variability and ontogenetic variation. The skull of V. walser is subject to significant variation, most likely related to ontogeny in some cases (i.e., development of the parietal crest, development of the basioccipital process, shape of the posterior margin of the parabasisphenoid, shape of the quadrate). Based on the studied material, it is possible to distinguish V. walser from the other Italian vipers by the shape of the occipital crest of the supraoccipital, which is posteriorly directed, whereas it is laterally directed in the other species. The osteological diagnosibility provides further support for the validity of V. walser as a distinct species from Vipera berus.

Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan-dependent mechanism.

Accumulating evidences point to a significant role for the chromogranin A (CgA)-derived peptide vasostatin 1 (VS-1) in the protective modulation of the cardiovascular activity, because of its ability to counteract the adrenergic signal. We have recently shown that VS-1 induces a PI3K-dependent-nitric oxide (NO) release by endothelial cells, contributing to explain the mechanism of its cardio-suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exerted by this peptide are still unknown, as typical high-affinity receptors have not been identified. Here we hypothesize that in endothelial cells VS-1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparan sulfate proteoglycans (HSPGs) and activating eNOS phosphorylation (Ser1179) through a PI3K-dependent, endocytosis-coupled mechanism. In bovine aortic endothelial cells (BAE-1 cells) endocytotic vesicles trafficking was quantified by confocal microscopy with a water-soluble membrane dye; caveolin 1 (Cav1) shift from plasma membrane was studied by immunofluorescence staining; VS-1-dependent eNOS phosphorylation was assessed by immunofluorescence and immunoblot analysis. Our experiments demonstrate that VS-1 induces a marked increase in the caveolae-dependent endocytosis, (115 +/- 23% endocytotic spots/cell/field in VS-1-treated cells with respect to control cells), that is significantly reduced by both heparinase III (HEP, 17 +/- 15% above control) and Wortmannin (Wm, 7 +/- 22% above control). Heparinase, Wortmannin, and methyl-beta-cyclodextrin (MbetaCD) abolish the VS-1-dependent eNOS phosphorylation (P(Ser1179)eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPGs interaction and caveolae endocytosis, coupled with a PI3K-dependent eNOS phosphorylation.

Catestatin improves post-ischemic left ventricular function and decreases ischemia/reperfusion injury in heart.

The Chromogranin A (CgA)-derived anti-hypertensive peptide catestatin (CST) antagonizes catecholamine secretion, and is a negative myocardial inotrope acting via a nitric oxide-dependent mechanism. It is not known whether CST contributes to ischemia/reperfusion injury or is a component of a cardioprotective response to limit injury. Here, we tested whether CST by virtue of its negative inotropic activity improves post-ischemic cardiac function and cardiomyocyte survival. Three groups of isolated perfused hearts from adult Wistar rats underwent 30-min ischemia and 120-min reperfusion (I/R, Group 1), or were post-conditioned by brief ischemic episodes (PostC, 5-cycles of 10-s I/R at the beginning of 120-min reperfusion, Group 2), or with exogenous CST (75 nM for 20 min, CST-Post, Group-3) at the onset of reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated with nitroblue-tetrazolium staining. The CST (5 nM) effects were also tested in simulated ischemia/reperfusion experiments on cardiomyocytes isolated from young-adult rats, evaluating cell survival with propidium iodide labeling. Infarct size was 61 ± 6% of risk area in hearts subjected to I/R only. PostC reduced infarct size to 34 ± 5%. Infarct size in CST-Post was 36 ± 3% of risk area (P < 0.05 respect to I/R). CST-Post reduced post-ischemic rise of diastolic LVP, an index of contracture, and significantly improved post-ischemic recovery of developed LVP. In isolated cardiomyocytes, CST increased the cell viability rate by about 65% after simulated ischemia/reperfusion. These results suggest a novel cardioprotective role for CST, which appears mainly due to a direct reduction of post-ischemic myocardial damages and dysfunction, rather than to an involvement of adrenergic terminals and/or endothelium.

Squalene: More than a Step toward Sterols.

Squalene (SQ) is a natural triterpene widely distributed in nature. It is a metabolic intermediate of the sterol biosynthetic pathway and represents a possible target in different metabolic and oxidative stress-related disorders. Growing interest has been focused on SQ's antioxidant properties, derived from its chemical structure. Strong evidence provided by ex vivo models underline its scavenging activity towards free radicals, whereas only a few studies have highlighted its effect in cellular models of oxidative stress. Given the role of unbalanced free radicals in both the onset and progression of several cardiovascular diseases, an in depth evaluation of SQ's contribution to antioxidant defense mechanisms could represent a strategic approach in dealing with these pathological conditions. At present experimental results overall show a double-edged sword role of squalene in cardiovascular diseases and its function has to be better elucidated in order to establish intervention lines focused on its features. This review aims to summarize current knowledge about endogenous and exogenous sources of SQ and to point out the controversial role of SQ in cardiovascular physiology.

Homing of annexin-labeled stem cells to apoptotic cells.

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues.

Catestatin Induces Glucose Uptake and GLUT4 Trafficking in Adult Rat Cardiomyocytes.

Catestatin is a cationic and hydrophobic peptide derived from the enzymatic cleavage of the prohormone Chromogranin A. Initially identified as a potent endogenous nicotinic-cholinergic antagonist, Catestatin has recently been shown to act as a novel regulator of cardiac function and blood pressure and as a cardioprotective agent in both pre- and postconditioning through AKT-dependent mechanisms. The aim of this study is to investigate the potential role of Catestatin also on cardiac metabolism modulation, particularly on cardiomyocytes glucose uptake. Experiments were performed on isolated adult rat cardiomyocytes. Glucose uptake was assessed by fluorescent glucose incubation and confocal microscope analysis. Glut4 plasma membrane translocation was studied by immunofluorescence experiments and evaluation of the ratio peripheral vs internal Glut4 staining. Furthermore, we performed immunoblot experiments to investigate the involvement of the intracellular pathway AKT/AS160 in the Catestatin dependent Glut4 trafficking. Our results show that 10 nM Catestatin induces a significant increase in the fluorescent glucose uptake, comparable to that exerted by 100 nM Insulin. Moreover, Catestatin stimulates Glut4 translocation to plasma membrane and both AKT and AS160 phosphorylation. All these effects were inhibited by Wortmannin. On the whole, we show for the first time that Catestatin is able to modulate cardiac glucose metabolism, by inducing an increase in glucose uptake through Glut4 translocation to the plasma membrane and that this mechanism is mediated by the AKT/AS160 intracellular pathway.

Chamazulene Attenuates ROS Levels in Bovine Aortic Endothelial Cells Exposed to High Glucose Concentrations and Hydrogen Peroxide.

Endothelial cells surround the lumen of blood vessels and modulate many physiological processes, including vascular tone, blood fluidity, inflammation, immunity and neovascularization. Many pathological conditions, including hyperglycemia, may alter endothelial function through oxidative stress, leading to impaired nitric oxide bioavailability and to the onset of an inflammatory state. As widely shown in the last decade, dietary intervention could represent a good strategy to control endothelial dysfunction and atherosclerosis. In particular, extensive research in the field of antioxidant natural derivatives has been conducted. In this study, we evaluated the capability of Chamazulene (Cham), an azulene compound from chamomile essential oil, to attenuate ROS levels in bovine aortic endothelial cells (BAECs) stressed with either high glucose or H2O2. Cell viability at different concentrations of Cham was evaluated through the WST-1 assay, while ROS production acutely induced by High Glucose (HG, 4.5 g/L) treatment or H2O2 (0.5 mM) for 3 h, was quantified with 2'-7'-Dichlorofluorescein diacetate (DCFH-DA) probe using confocal microscopy and flow cytometry. Our results showed a reduction in ROS produced after simultaneous treatment with High Glucose or H2O2 and Cham, thus suggesting an in vitro antioxidant activity of the compound. On the whole, this study shows for the first time the potential role of Cham as a scavenging molecule, suggesting its possible use to prevent the rise of endothelial ROS levels and the consequent vascular damage.

Squaraines bearing halogenated moieties as anticancer photosensitizers: Synthesis, characterization and biological evaluation.

We report the synthesis and characterization of a series of symmetrical indolenine-based squaraine dyes along with the evaluation of their singlet oxygen generation efficiency. The photodynamic activity of these new photosensitizers has been evaluated on a human tumor fibrosarcoma (HT-1080) cell line. The cytotoxicity increased over time and is induced by the photoactivation of bromo (Br-C4) and iodio (I-C4) long carbon chain squaraine dyes and the consequent increase in reactive oxygen species (ROS) production (p < 0.001), which leads to necrosis 6 h after treatment. Induction of cytochrome c release, DNA damage and up-regulation of GPX1, NQO1 and SOD2 mRNA gene expression after PDT were investigated.

Phosphoinositide 3-kinase gamma controls autonomic regulation of the mouse heart through Gi-independent downregulation of cAMP level.

Cardiac beta-adrenergic and the muscarinic receptors control contractility and heart rate by triggering multiple signaling events involving downstream targets like the phosphoinositide 3-kinase gamma (PI3Kgamma). We thus investigated whether the lack of PI3Kgamma could play a role in the autonomic regulation of the mouse heart. Contractility and ICaL of mutant cardiac preparations appeared increased in basal conditions and after beta-adrenergic stimulation. However, basal and beta-adrenergic stimulated heart rate were normal. Conversely, muscarinic inhibition of heart rate was reduced without alteration of the Gbetagamma-dependent stimulation of IK,ACh current. In addition, muscarinic-mediated anti-adrenergic effect on papillary muscle contractility and ICaL was significantly depressed. Consistently, cAMP level of PI3Kgamma-null ventricles was always higher than wild-type controls. Thus, PI3Kgamma controls the cardiac function by reducing cAMP concentration independently of Gi-mediated signaling.

Nitric oxide, a key signaling molecule in the murine early embryonic heart.

Nitric oxide (NO) is thought to play an important role as a signaling molecule in embryonic and adult cardiomyocytes; however, its involvement in muscarinic signaling is still unclear. The aim of the present work was to analyze the muscarinic modulation of the L-type Ca2+ current (ICa) in early- and late-stage embryonic ventricular cardiomyocytes. Muscarinic stimulation depressed basal ICa by 30.1 +/- 3.2% (n=27) in early-stage cardiomyocytes. Pharmacological evidence suggested that the muscarinic modulation was mediated through generation of NO, activation of cGMP-dependent phosphodiesterase (PDE) 2, and ensuing lowering of cyclic AMP/protein kinase A (cAMP/PKA) levels. Conversely, in late-stage cardiomyocytes, muscarinic regulation of ICa occurred in a NO-independent manner via inhibition of prestimulated adenylyl cyclase (AC). To unequivocally prove the involvement of NO and to identify the nitric oxide synthase (NOS) isoform(s), we analyzed muscarinic signaling in embryonic ventricular cardiomyocytes of NOS2 (-/-) and NOS3 (-/-) mice. The early-stage NOS3 (-/-) cardiomyocytes lacked muscarinic modulation, whereas it was preserved in NOS2 (-/-) cells. Moreover, at the late embryonic stage, muscarinic modulation of ICa was intact in both strains. Thus, NO is the key regulator of muscarinic signaling in the early embryonic ventricle, whereas at later stages, signaling occurs through a NO-independent pathway.

Phosphoinositide 3-kinase gamma-deficient hearts are protected from the PAF-dependent depression of cardiac contractility.

OBJECTIVES: Following an ischemic insult, cardiac contractile recovery might be perturbed by the release of autacoids, like platelet-activating factor (PAF), that depress heart function by acting through G protein-coupled receptors (GPCRs). The signaling events downstream the PAF receptor that lead to the negative inotropic effect are still obscure. We thus investigated whether the GPCR-activated phosphoisositide 3-kinase gamma (PI3Kgamma) could play a role in the cardiac response to PAF. METHODS: The negative inotropic effect of PAF was studied ex vivo, in isolated electrically driven atria and in Langendorff-perfused whole hearts derived from wild-type and PI3Kgamma-null mice. Postischemic recovery of contractility was analyzed in normal and mutant whole hearts subjected to 30 min of ischemia and 40 min of reperfusion in the presence or absence of a PAF receptor antagonist. RESULTS: While wild-type hearts stimulated with PAF showed increased nitric oxide (NO) production and a consequent decreased cardiac contractility, PI3Kgamma-null hearts displayed reduced phosphorylation of nitric oxide synthase 3 (NOS3), blunted nitric oxide production and a complete protection from the PAF-induced negative inotropism. In addition, Langendorff-perfused PI3Kgamma-null hearts showed a better contractile recovery after ischemia/reperfusion, a condition where PAF is known to be an important player in depressing contractility. In agreement with a role of PI3Kgamma in this PAF-mediated signaling, postischemic contractile recovery in PI3Kgamma-null mice appeared overlapping with that of normal hearts treated with the PAF receptor antagonist WEB 2170. CONCLUSION: These data indicate a novel PAF-dependent signaling pathway that, involving PI3Kgamma and NOS3, contributes to postischemic contractile depression.

Catestatin exerts direct protective effects on rat cardiomyocytes undergoing ischemia/reperfusion by stimulating PI3K-Akt-GSK3ß 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, GSK3ß, 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, GSK3ßSer9, 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.

Obligatory role for endothelial heparan sulphate proteoglycans and caveolae internalization in catestatin-dependent eNOS activation.

The chromogranin-A peptide catestatin modulates a wide range of processes, such as cardiovascular functions, innate immunity, inflammation, and metabolism. We recently found that the cardiac antiadrenergic action of catestatin requires a PI3K-dependent NO release from endothelial cells, although the receptor involved is yet to be identified. In the present work, based on the cationic properties of catestatin, we tested the hypothesis of its interaction with membrane heparan sulphate proteoglycans, resulting in the activation of a caveolae-dependent endocytosis. Experiments were performed on bovine aortic endothelial cells. Endocytotic vesicles trafficking was quantified by confocal microscopy using a water-soluble membrane dye; catestatin colocalization with heparan sulphate proteoglycans and caveolin 1 internalization were studied by fluorimetric measurements in live cells. Modulation of the catestatin-dependent eNOS activation was assessed by immunofluorescence and immunoblot analysis. Our results demonstrate that catestatin (5 nM) colocalizes with heparan sulphate proteoglycans and induces a remarkable increase in the caveolae-dependent endocytosis and caveolin 1 internalization, which were significantly reduced by both heparinase and wortmannin. Moreover, catestatin was unable to induce Ser(1179) eNOS phosphorylation after pretreatments with heparinase and methyl-ß-cyclodextrin. Taken together, these results highlight the obligatory role for proteoglycans and caveolae internalization in the catestatin-dependent eNOS activation in endothelial cells.