The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure.

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

Functional, structural, and molecular remodelling of the goldfish (Carassius auratus) heart under moderate hypoxia.

The goldfish (Carassius auratus) is known for its physiologic ability to survive even long periods of oxygen limitation (hypoxia), adapting the cardiac performance to the requirements of peripheral tissue perfusion. We here investigated the effects of short-term moderate hypoxia on the heart, focusing on ventricular adaptation, in terms of hemodynamics and structural traits. Functional evaluations revealed that animals exposed to 4 days of environmental hypoxia increased the hemodynamic performance evaluated on ex vivo cardiac preparations. This was associated with a thicker and more vascularized ventricular compact layer and a reduced luminal lacunary space. Compared to normoxic animals, ventricular cardiomyocytes of goldfish exposed to hypoxia showed an extended mitochondrial compartment and a modulation of proteins involved in mitochondria dynamics. The enhanced expression of the pro-fission markers DRP1 and OMA1, and the modulation of the short and long forms of OPA1, suggested a hypoxia-related mitochondria fission. Our data propose that under hypoxia, the goldfish heart undergoes a structural remodelling associated with a potentiated cardiac activity. The energy demand for the highly performant myocardium is supported by an increased number of mitochondria, likely occurring through fission events.

Discovery of a novel 1,3,4-oxadiazol-2-one-based NLRP3 inhibitor as a pharmacological agent to mitigate cardiac and metabolic complications in an experimental model of diet-induced metaflammation.

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-1ß 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.

Palmitate-Induced Cardiac Lipotoxicity Is Relieved by the Redox-Active Motif of SELENOT through Improving Mitochondrial Function and Regulating Metabolic State.

Cardiac lipotoxicity is an important contributor to cardiovascular complications during obesity. Given the fundamental role of the endoplasmic reticulum (ER)-resident Selenoprotein T (SELENOT) for cardiomyocyte differentiation and protection and for the regulation of glucose metabolism, we took advantage of a small peptide (PSELT), derived from the SELENOT redox-active motif, to uncover the mechanisms through which PSELT could protect cardiomyocytes against lipotoxicity. To this aim, we modeled cardiac lipotoxicity by exposing H9c2 cardiomyocytes to palmitate (PA). The results showed that PSELT counteracted PA-induced cell death, lactate dehydrogenase release, and the accumulation of intracellular lipid droplets, while an inert form of the peptide (I-PSELT) lacking selenocysteine was not active against PA-induced cardiomyocyte death. Mechanistically, PSELT counteracted PA-induced cytosolic and mitochondrial oxidative stress and rescued SELENOT expression that was downregulated by PA through FAT/CD36 (cluster of differentiation 36/fatty acid translocase), the main transporter of fatty acids in the heart. Immunofluorescence analysis indicated that PSELT also relieved the PA-dependent increase in CD36 expression, while in SELENOT-deficient cardiomyocytes, PA exacerbated cell death, which was not mitigated by exogenous PSELT. On the other hand, PSELT improved mitochondrial respiration during PA treatment and regulated mitochondrial biogenesis and dynamics, preventing the PA-provoked decrease in PGC1-α and increase in DRP-1 and OPA-1. These findings were corroborated by transmission electron microscopy (TEM), revealing that PSELT improved the cardiomyocyte and mitochondrial ultrastructures and restored the ER network. Spectroscopic characterization indicated that PSELT significantly attenuated infrared spectral-related macromolecular changes (i.e., content of lipids, proteins, nucleic acids, and carbohydrates) and also prevented the decrease in membrane fluidity induced by PA. Our findings further delineate the biological significance of SELENOT in cardiomyocytes and indicate the potential of its mimetic PSELT as a protective agent for counteracting cardiac lipotoxicity.

Cardiac Hypoxia Tolerance in Fish: From Functional Responses to Cell Signals.

Aquatic animals are increasingly challenged by O2 fluctuations as a result of global warming, as well as eutrophication processes. Teleost fish show important species-specific adaptability to O2 deprivation, moving from intolerance to a full tolerance of hypoxia and even anoxia. An example is provided by members of Cyprinidae which includes species that are amongst the most tolerant hypoxia/anoxia teleosts. Living at low water O2 requires the mandatory preservation of the cardiac function to support the metabolic and hemodynamic requirements of organ and tissues which sustain whole organism performance. A number of orchestrated events, from metabolism to behavior, converge to shape the heart response to the restricted availability of the gas, also limiting the potential damages for cells and tissues. In cyprinids, the heart is extraordinarily able to activate peculiar strategies of functional preservation. Accordingly, by using these teleosts as models of tolerance to low O2, we will synthesize and discuss literature data to describe the functional changes, and the major molecular events that allow the heart of these fish to sustain adaptability to O2 deprivation. By crossing the boundaries of basic research and environmental physiology, this information may be of interest also in a translational perspective, and in the context of conservative physiology, in which the output of the research is applicable to environmental management and decision making.

An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System.

Alamandine is a peptide of the Renin Angiotensin System (RAS), either generated from Angiotensin A via the Angiotensin Converting Enzyme 2 (ACE2), or directly from Ang-(1-7). In mammals, it elicits cardioprotection via Mas-related G-protein-coupled receptor D (MrgD), and the NOS/NO system. In teleost fish, RAS is known to modulate heart performance. However, no information is available on the presence of a cardioactive ACE2/Alamandine axis. To fill this gap, we used the cyprinid teleost Carassius auratus (goldfish) for in silico and in vitro analyses. Via the NCBI Blast P suite we found that in cyprinids ace2 is phylogenetically detectable in a subcluster of proteins including ace2-like isoforms, and is correlated with a hypoxia-dependent pathway. By real-time PCR, Western Blotting, and HPLC, ACE2 and Alamandine were identified in goldfish heart and plasma, respectively. Both increased after chronic exposure to low O2 (2.6 mg O2 L-1). By using an ex-vivo working goldfish-heart preparation, we observed that in vitro administration of exogenous Alamandine dose-dependently stimulates myocardial contractility starting from 10-11 M. The effect that involved Mas-related receptors and PKA occurred via the NOS/NO system. This was shown by exposing the perfused heart to the NOS inhibitor L-NMMA (10-5 M) that abolished the cardiac effect of Alamandine and was supported by the increased expression of the phosphorylated NOS enzyme in the extract from goldfish heart exposed to 10-10 M Alamandine. Our data are the first to show that an ACE2/Alamandine axis is present in the goldfish C. auratus and, to elicit cardiac modulation, requires the obligatory involvement of the NOS/NO system.

The Antioxidant Selenoprotein T Mimetic, PSELT, Induces Preconditioning-like Myocardial Protection by Relieving Endoplasmic-Reticulum Stress.

Oxidative stress and endoplasmic reticulum stress (ERS) are strictly involved in myocardial ischemia/reperfusion (MI/R). Selenoprotein T (SELENOT), a vital thioredoxin-like selenoprotein, is crucial for ER homeostasis and cardiomyocyte differentiation and protection, likely acting as a redox-sensing protein during MI/R. Here, we designed a small peptide (PSELT), encompassing the redox site of SELENOT, and investigated whether its pre-conditioning cardioprotective effect resulted from modulating ERS during I/R. The Langendorff rat heart model was employed for hemodynamic analysis, while mechanistic studies were performed in perfused hearts and H9c2 cardiomyoblasts. PSELT improved the post-ischemic contractile recovery, reducing infarct size and LDH release with and without the ERS inducer tunicamycin (TM). Mechanistically, I/R and TM upregulated SELENOT expression, which was further enhanced by PSELT. PSELT also prevented the expression of the ERS markers CHOP and ATF6, reduced cardiac lipid peroxidation and protein oxidation, and increased SOD and catalase activities. An inert PSELT (I-PSELT) lacking selenocysteine was ineffective. In H9c2 cells, H2O2 decreased cell viability and SELENOT expression, while PSELT rescued protein levels protecting against cell death. In SELENOT-deficient H9c2 cells, H2O2 exacerbated cell death, that was partially mitigated by PSELT. Microscopy analysis revealed that a fluorescent form of PSELT was internalized into cardiomyocytes with a perinuclear distribution. Conclusions: The cell-permeable PSELT is able to induce pharmacological preconditioning cardioprotection by mitigating ERS and oxidative stress, and by regulating endogenous SELENOT.

The heart of the adult goldfish Carassius auratus as a target of Bisphenol A: a multifaceted analysis.

Bisphenol A (BPA) is a contaminant whose presence in aquatic environments is increasing. In fish embryos and larvae, it severely affects cardiac development; however, its influence on the heart function of adult fish has been scarcely analyzed. This study investigated the effects of the in vivo exposure to BPA on heart physiology, morphology, and oxidative balance in the goldfish Carassius auratus. Adult fish were exposed for 4 and 10 days to two BPA concentrations (10 µM and 25 µM). Ex vivo working heart preparations showed that high concentrations of BPA negatively affected cardiac hemodynamics, as revealed by an impaired Frank-Starling response. This was paralleled by increased cardio-somatic indices and by myocardial structural changes. An altered oxidative status and a modulation of stress (HSPs) and pro-apoptotic (Bax and Cytochrome C) proteins expression were also observed in the heart of animals exposed to BPA, with detrimental effects at the highest concentration and the longest exposure time. Results suggest that, in the adult goldfish, BPA may induce stressful conditions to the heart with time- and concentration-dependent deleterious morpho-functional alterations.

The Hypoxia Tolerance of the Goldfish (Carassius auratus) Heart: The NOS/NO System and Beyond.

The extraordinary capacity of the goldfish (Carassius auratus) to increase its cardiac performance under acute hypoxia is crucial in ensuring adequate oxygen supply to tissues and organs. However, the underlying physiological mechanisms are not yet completely elucidated. By employing an ex vivo working heart preparation, we observed that the time-dependent enhancement of contractility, distinctive of the hypoxic goldfish heart, is abolished by the Nitric Oxide Synthase (NOS) antagonist L-NMMA, the Nitric Oxide (NO) scavenger PTIO, as well as by the PI3-kinase (PI3-K) and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) pumps' inhibition by Wortmannin and Thapsigargin, respectively. In goldfish hearts exposed to hypoxia, an ELISA test revealed no changes in cGMP levels, while Western Blotting analysis showed an enhanced expression of the phosphorylated protein kinase B (pAkt) and of the NADPH oxidase catalytic subunit Nox2 (gp91phox). A significant decrease of protein S-nitrosylation was observed by Biotin Switch assay in hypoxic hearts. Results suggest a role for a PI3-K/Akt-mediated activation of the NOS-dependent NO production, and SERCA2a pumps in the mechanisms conferring benefits to the goldfish heart under hypoxia. They also propose protein denitrosylation, and the possibility of nitration, as parallel intracellular events.

MS-based proteomic analysis of cardiac response to hypoxia in the goldfish (Carassius auratus).

The exceptional hypoxia tolerance of the goldfish heart may be achieved through the activation of an alternative mechanism recruiting the first product of the anaerobic glycolysis (i.e. piruvate). This hypothesis led to design a classical mass spectrometry based proteomic study to identify in the goldfish cardiac proteins that may be associated with maintaining heart function under normoxia and hypoxia. A selective protein solubilization, SDS PAGE, trypsin digestion and MALDI MS/MS analysis allowed the identification of the 12 most stable hypoxia-regulated proteins. Among these proteins, five are enzymes catalyzing reversible steps of the glycolysis/gluconeogenesis network. Protein composition reveals the presence of fructose-1,6-bisphosphate aldolase B as a specific hypoxia-regulated protein. This work indicated that the key enzyme of reversible steps of the glycolysis/gluconeogenesis network is fructose-1,6-bisphosphate, aldolase B, suggesting a role of gluconeogenesis in the mechanisms involved in the goldfish heart response to hypoxia.

Cardiac influence of the ß3-adrenoceptor in the goldfish (Carassius auratus): a protective role under hypoxia?

The goldfish (Carassius auratus) exhibits a remarkable capacity to survive and remain active under prolonged and severe hypoxia, making it a good model for studying cardiac function when oxygen availability is a limiting factor. Under hypoxia, the goldfish heart increases its performance, representing a putative component of hypoxia tolerance; however, the underlying mechanisms have not yet been elucidated. Here, we aimed to investigate the role of ß3-adrenoreceptors (ARs) in the mechanisms that modulate goldfish heart performance along with the impact of oxygen levels. By western blotting analysis, we found that the goldfish heart expresses ß3-ARs, and this expression increases under hypoxia. The effects of ß3-AR stimulation were analysed by using an ex vivo working heart preparation. Under normoxia, the ß3-AR-selective agonist BRL37344 (10-12 to 10-7 mol l-1) elicited a concentration-dependent increase of contractility that was abolished by a specific ß3-AR antagonist (SR59230A; 10-8 mol l-1), but not by α/ß1/ß2-AR inhibitors (phentolamine, nadolol and ICI118,551; 10-7 mol l-1). Under acute hypoxia, BRL37344 did not affect goldfish heart performance. However, SR59230A, but not phentolamine, nadolol or ICI118,551, abolished the time-dependent enhancement of contractility that characterizes the hypoxic goldfish heart. Under both normoxia and hypoxia, adenylate cyclase and cAMP were found to be involved in the ß3-AR-dependent downstream transduction pathway. In summary, we show the presence of functional ß3-ARs in the goldfish heart, whose activation modulates basal performance and contributes to a hypoxia-dependent increase of contractility.

Selenoprotein T as a new positive inotrope in the goldfish, Carassius auratus.

Selenoprotein T (SELENOT) is a thioredoxin-like protein, which mediates oxidoreductase functions via its redox active motif Cys-X-X-Sec. In mammals, SELENOT is expressed during ontogenesis and progressively decreases in adult tissues. In the heart, it is re-expressed after ischemia and induces cardioprotection against ischemia-reperfusion (IR) injury. SELENOT is present in teleost fish, including the goldfish Carassius auratus This study aimed to evaluate the cardiac expression of SELENOT, and the effects of exogenous PSELT (a 43-52 SELENOT-derived peptide) on the heart function of C. auratus, a hypoxia tolerance fish model. We found that SELENOT was expressed in cardiac extracts of juvenile and adult fish, located in the sarcoplasmic reticulum (SR) together with calsequestrin-2. Expression increased under acute hypoxia. On ex vivo isolated and perfused goldfish heart preparations, under normoxia, PSELT dose dependently increased stroke volume (VS), cardiac output [Formula: see text] and stroke work (SW), involving cAMP, PKA, L-type calcium channels, SERCA2a pumps and pAkt. Under hypoxia, PSELT did not affect myocardial contractility. Only at higher concentrations (10-8 to 10-7 mol l-1) was an increase of VS and [Formula: see text] observed. It also reduced the cardiac expression of 3-NT, a tissue marker of nitrosative stress, which increases under low oxygen availability. These data are the first to propose SELENOT 43-52 (PSELT) as a cardiac modulator in fish, with a potential protective role under hypoxia.

Quercetin and its derivative Q2 modulate chromatin dynamics in adipogenesis and Q2 prevents obesity and metabolic disorders in rats.

Recently the attention of the scientific community has focused on the ability of polyphenols to counteract adverse epigenetic regulation involved in the development of complex conditions such as obesity. The aim of this study was to investigate the epigenetic mechanisms underlying the anti-adiposity effect of Quercetin (3,3',4',5,7-pentahydroxyflavone) and of one of its derivatives, Q2 in which the OH groups have been replaced by acetyl groups. In 3 T3-L1 preadipocytes, Quercetin and Q2 treatment induce chromatin remodeling and histone modifications at the 5' regulatory region of the two main adipogenic genes, c/EBPα and PPARγ. Chromatin immunoprecipitation assays revealed a concomitant increase of histone H3 di-methylation at Lys9, a typical mark of repressed gene promoters, and a decrease of histone H3 di-methylation at Lys 4, a mark of active transcription. At the same time, both compounds inhibited histone demethylase LSD1 recruitment to the 5' region of c/EBPα and PPARγ genes, a necessary step for adipogenesis. The final effect is a significant reduction in c/EBPα and PPARγ gene expression and attenuated adipogenesis. Q2 supplementation in rats reduced the gain in body weight and in white adipose tissue, as well as the increase in adipocyte size determined by high fat diet. Moreover, Q2 improved dyslipidemia, glucose tolerance and decreased the hepatic lipid accumulation by activating the expression of beta-oxidation related genes. Our data suggest that Q2, as well as Quercetin, has the potential to revert the unfavorable epigenomic profiles associated with obesity onset. This opens the possibility to use these compounds in targeted prevention strategies against obesity.

Effect of Different Formulations of Magnesium Chloride Used As Anesthetic Agents on the Performance of the Isolated Heart of Octopus vulgaris.

Magnesium chloride (MgCl2) is commonly used as a general anesthetic in cephalopods, but its physiological effects including those at cardiac level are not well-characterized. We used an in vitro isolated perfused systemic heart preparation from the common octopus, Octopus vulgaris, to investigate: (a) if in vivo exposure to MgCl2 formulations had an effect on cardiac function in vitro and, if so, could this impact recovery and (b) direct effects of MgCl2 formulations on cardiac function. In vitro hearts removed from animals exposed in vivo to 3.5% MgCl2 in sea water (20 min) or to a mixture of MgCl2+ ethanol (1.12/1%; 20 min) showed cardiac function (heart rate, stroke volume, cardiac output) comparable to hearts removed from animals killed under hypothermia. However, 3.5% MgCl2 (1:1, sea water: distilled water, 20 min) produced a significant impairment of the Frank-Starling response as did 45 min exposure to the MgCl2+ ethanol mixture. Perfusion of the isolated heart with MgCl2± ethanol formulations produced a concentration-related bradycardia (and arrest), a decreased stroke volume and cardiac output indicating a direct effect on the heart. The cardiac effects of MgCl2 are discussed in relation to the involvement of magnesium, sodium, chloride, and calcium ions, exposure time and osmolality of the formulations and the implications for the use of various formulations of MgCl2 as anesthetics in octopus. Overall, provided that the in vivo exposure to 3.5% MgCl2 in sea water or to a mixture of MgCl2+ ethanol is limited to ~20 min, residual effects on cardiac function are unlikely to impact post-anesthetic recovery.

Cardiac and hepatic role of r-AtHSP70: basal effects and protection against ischemic and sepsis conditions.

Heat shock proteins (HSPs), highly conserved in all organisms, act as molecular chaperones activated by several stresses. The HSP70 class of stress-induced proteins is the most studied subtype in cardiovascular and inflammatory disease. Because of the high similarity between plant and mammalian HSP70, the aim of this work was to evaluate whether recombinant HSP70 of plant origin (r-AtHSP70) was able to protect rat cardiac and hepatic function under ischemic and sepsis conditions. We demonstrated for the first time that, in ex vivo isolated and perfused rat heart, exogenous r-AtHSP70 exerted direct negative inotropic and lusitropic effects via Akt/endothelial nitric oxide synthase pathway, induced post-conditioning cardioprotection via Reperfusion Injury Salvage Kinase and Survivor Activating Factor Enhancement pathways, and did not cause hepatic damage. In vivo administration of r-AtHSP70 protected both heart and liver against lipopolysaccharide-dependent sepsis, as revealed by the reduced plasma levels of interleukin-1ß, tumour necrosis factor alpha, aspartate aminotransferase and alanine aminotransferase. These results suggest exogenous r-AtHSP70 as a molecular modulator able to protect myocardial function and to prevent cardiac and liver dysfunctions during inflammatory conditions.

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.

Cardiac heterometric response: the interplay between Catestatin and nitric oxide deciphered by the frog heart.

The length-active tension relation or heterometric regulation (Frank-Starling mechanism) is modulated by nitric oxide (NO) which, released in pulsatile fashion from the beating heart, improves myocardial relaxation and diastolic distensibility. The NO signaling is also implicated in the homeometric regulation exerted by extrinsic factors such as autonomic nervous system, endocrine and humoral agents. In the in vitro working frog heart, the Chromogranin A (CGA)-derived peptide, Catestatin (CTS; bovine CGA344-364), exerts a direct cardio-suppressive action through a NOS-NO-cGMP-mediated mechanism which requires the functional integrity of the endocardial endothelium (EE) and its endothelin-1 B type (ETB) receptor. However, functional interplay between NO and CTS and their role in the Frank-Starling response of the frog heart are lacking. Here we show that CTS improves the sensitivity to preload increases similar to that exerted by NO. This effect is abolished by inhibition of NO synthase (L-NAME), guanylate cyclase (ODQ), protein kinase G (KT5823), PI3K (Wortmannin), as well as by the functional damage of EE (Triton X-100) suggesting that CTS operates through an EE-dependent NO release. On the whole, the use of the avascular frog heart revealed the EE as major sensor-transducer interface between the physical (volume load) and chemical (CTS) stimuli, NO functioning as a connector between heterometric and homeometric regulation.

Decidual endovascular trophoblast invasion in women with polycystic ovary syndrome: an experimental case-control study.

CONTEXT: Previous experimental and clinical data suggest impaired decidual trophoblast invasion in patients with polycystic ovarian syndrome (PCOS). OBJECTIVE: The objective of the study was to test the hypothesis that decidual endovascular trophoblast invasion in pregnant patients with PCOS is impaired and to clarify the potential mechanisms involved. DESIGN: This was an experimental case-control study. SETTING: The study was conducted at the academic Departments of Obstetrics and Gynecology and the Unit of Pathology (Italy). PATIENTS: Forty-five pregnant subjects screened from a wide population of women waiting for legal pregnancy termination were included in the final analysis. Specifically, 15 pregnant patients with PCOS were enrolled as cases and another 30 age- and body mass index (BMI)-matched healthy pregnant women without any feature of PCOS were enrolled as the controls. INTERVENTION: Interventions included the collection of trophoblastic and decidual tissue at the 12th week of gestation. MAIN OUTCOME MEASURES: Clinical, ultrasonographic, and biochemical data as well as the histological analysis of decidual endovascular trophoblast invasion. RESULTS: The rate of implantation site vessels with endovascular trophoblast invasion (ratio between total number of implantation site vessels and total number of vessels with endovascular trophoblast invasion) and the extent of endovascular trophoblast invasion (proportion between immunoreactive areas to cytokeratin 7 and to CD34) were significantly lower in patients with PCOS compared with healthy non-PCOS controls. Endovascular trophoblast invasion data were significantly and indirectly related to the markers of insulin resistance and testosterone concentrations in PCOS patients. CONCLUSIONS: Pregnant patients with PCOS patients have impaired decidual trophoblast invasion. Further studies are needed to evaluate the exact mechanisms through which insulin resistance and hyperandrogenemia exert this effect.

Nitrite is a positive modulator of the Frank-Starling response in the vertebrate heart.

Evidence from both mammalian and nonmammalian vertebrates indicates that intracardiac nitric oxide (NO) facilitates myocardial relaxation, ventricular diastolic distensibility, and, consequently, the Frank-Starling response, i.e., the preload-induced increase of cardiac output. Since nitrite ion (NO(2)(-)), the major storage pool of bioactive NO, recently emerged as a cardioprotective endogenous modulator, we explored its influence on the Frank-Starling response in eel, frog, and rat hearts, used as paradigms of fish, amphibians, and mammals, respectively. We demonstrated that, like NO, exogenous nitrite improves the Frank-Starling response in all species, as indicated by an increase of stroke volume and stroke work (eel and frog) and of left ventricular (LV) pressure and LVdP/dt max (rat), used as indexes of inotropism. Unlike in frog and rat, in eel, the positive influence of nitrite appeared to be dependent on NO synthase inhibition. In all species, the effect was sensitive to NO scavengers, independent on nitroxyl anion, and mediated by a cGMP/PKG-dependent pathway. Moreover, the nitrite treatment increased S-nitrosylation of lower-molecular-weight proteins in cytosolic and membrane fractions. These results suggest that nitrite acts as a physiological source of NO, modulating through different species-specific mechanisms, the stretch-induced intrinsic regulation of the vertebrate heart.

Physiological evidence for ß3-adrenoceptor in frog (Rana esculenta) heart.

ß3-Adrenergic receptors (ARs) have been recently identified in mammalian hearts where, unlike ß1- and ß2-ARs, induce cardio-suppressive effects. The aim of this study was to describe ß3-AR role in the frog (Rana esculenta) heart and to examine its signal transduction pathway. The presence of ß3-AR, by using Western blotting analysis, has been also identified. BRL(37344), a selective ß3-AR agonist, induced a dose-dependent negative inotropic effect at concentrations from 10(-12) to 10(-6)M. This effect was not modified by nadolol (ß1/ß2-AR antagonist) and by phentolamine (α-AR antagonist), but it was suppressed by the ß3-AR-specific antagonist SR(59230) and by exposure to the Gi/o proteins inhibitor Pertussis Toxin. In addition, the involvement of EE-NOS-cGMP-PKG/PDE2 pathway in the negative inotropism of BRL(37344) has been assessed. BRL(37344) treatment induced eNOS and Akt phosphorylation as well as an increase of cGMP levels. ß3-ARs activation induce a non-competitive antagonism against ISO stimulation which disappeared in presence of PKG and PDE2 inhibition. Taken together our findings provide, for the first time in the frog, a role for ß3-ARs in the cardiac performance modulation which involves Gi/o protein and occurs via an EE-NO-cGMP-PKG/PDE2 cascade.