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.

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.

Morpho-functional remodelling of the adult zebrafish (Danio rerio) heart in response to waterborne angiotensin II exposure.

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin System, is a pluripotent humoral agent whose biological actions include short-term modulations and long-term adaptations. In fish, short-term cardio-tropic effects of AngII are documented, but information on the role of AngII in long-term cardiac remodelling is not fully understood. Here, we describe a direct approach to disclose long-term morpho-functional effects of AngII on the zebrafish heart. Adult fish exposed to waterborne teleost analogue AngII for 8 weeks showed enhanced heart weight and cardio-somatic index, coupled to myocardial structural changes (i.e. augmented compacta thickness and fibrosis), and increased heart rate. These findings were paralleled by an up-regulation of type-1 and type-2 AngII receptors expression, and by changes in the expression of GATA binding protein 4, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 and superoxide dismutase 1 soluble mRNAs, as well as of cytochrome b-245 beta polypeptide protein, indicative of cardiac remodelling. Our results suggest that waterborne AngII can sustain and robustly affect the cardiac morpho-functional remodelling of adult zebrafish.

Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

The clinical use of doxorubicin (Doxo), a widely used anticancer chemotherapeutic drug, is limited by dose-dependent cardiotoxicity. We have investigated whether chromogranin A (CgA), a cardioregulatory protein released in the blood by the neuroendocrine system and by the heart itself, may contribute to regulation of the cardiotoxic and antitumor activities of Doxo. The effects of a physiologic dose of full-length recombinant CgA on Doxo-induced cardiotoxicity and antitumor activity were investigated in rats using in vivo and ex vivo models and in murine models of melanoma, fibrosarcoma, lymphoma, and lung cancer, respectively. The effect of Doxo on circulating levels of CgA was also investigated. In vivo and ex vivo mechanistic studies showed that CgA can prevent Doxo-induced heart inflammation, oxidative stress, apoptosis, fibrosis, and ischemic injury. On the other hand, CgA did not impair the anticancer activity of Doxo in all the murine models investigated. Furthermore, we observed that Doxo can reduce the intracardiac expression and release of CgA in the blood (i.e., an important cardioprotective agent). These findings suggest that administration of low-dose CgA to patients with low levels of endogenous CgA might represent a novel approach to prevent Doxo-induced adverse events without impairing antitumor effects.-Rocca, C., Scavello, F., Colombo, B., Gasparri, A. M., Dallatomasina, A., Granieri, M. C., Amelio, D., Pasqua, T., Cerra, M. C., Tota, B., Corti, A., Angelone, T. Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

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.

Exploring cardiac plasticity in teleost: the role of humoral modulation.

The fish heart represents an established natural model for evaluating basic mechanisms of the coordinated physiological reactions which maintain cardiac steady-state. This is due to its relatively simple design, but also to its multilevel morpho-functional flexibility which allows adequate responses to a variety of intrinsic (body size and shape, swimming performance, etc.), and extrinsic (temperature, salinity, oxygen level, water chemistry, etc.) factors related to the animal life style. Nowadays, although many gaps are still present, a huge literature is available about the mechanisms that fine-tune fish cardiac performance, particularly in relation to the influence exerted by substances possessing cardio-modulatory properties. Based on these premises, this review will provide an overview of the existing current knowledge regarding the humoral control of cardiac performance in fish. The role of both classic (i.e. catecholamines, angiotensin II and natriuretic peptides), and emerging cardioactive substances (i.e. the chromogranin-A-derived peptides vasostatins, catestatin and serpinin) will be illustrated and discussed. Moreover, an example of cardiomodulation elicited by peptides (e.g., nesfatin-1) associated to the regulation of feeding and metabolism will be provided. The picture will hopefully emphasize the complex circuits that sustain fish cardiac performance, also highliting the power of the teleost heart as an experimental model to deciphering mechanisms that could be difficult to explore in more elaborated cardiac morpho-functional designs.

Phoenixin-14: detection and novel physiological implications in cardiac modulation and cardioprotection.

Phoenixin-14 (PNX) is a newly identified peptide co-expressed in the hypothalamus with the anorexic and cardioactive Nesfatin-1. Like Nesfatin-1, PNX is able to cross the blood-brain barrier and this suggests a role in peripheral modulation. Preliminary mass spectrography data indicate that, in addition to the hypothalamus, PNX is present in the mammalian heart. This study aimed to quantify PNX expression in the rat heart, and to evaluate whether the peptide influences the myocardial function under basal condition and in the presence of ischemia/reperfusion (I/R). By ELISA the presence of PNX was detected in both hypothalamus and heart. In plasma of normal, but not of obese rats, the peptide concentrations increased after meal. Exposure of the isolated and Langendorff perfused rat heart to exogenous PNX induces a reduction of contractility and relaxation, without effects on coronary pressure and heart rate. As revealed by immunoblotting, these effects were accompanied by an increase of Erk1/2, Akt and eNOS phosphorylation. PNX (EC50 dose), administered after ischemia, induced post-conditioning-like cardioprotection. This was revealed by a smaller infarct size and a better systolic recovery with respect to those detected on hearts exposed to I/R alone. The peptide also activates the cardioprotective RISK and SAFE cascades and inhibits apoptosis. These effects were also observed in the heart of obese rats. Our data provide a first evidence on the peripheral activity of PNX and on its direct cardiomodulatory and cardioprotective role under both normal conditions and in the presence of metabolic disorders.

Chromogranins: from discovery to current times.

The discovery in 1953 of the chromaffin granules as co-storage of catecholamines and ATP was soon followed by identification of a range of uniquely acidic proteins making up the isotonic vesicular storage complex within elements of the diffuse sympathoadrenal system. In the mid-1960s, the enzymatically inactive, major core protein, chromogranin A was shown to be exocytotically discharged from the stimulated adrenal gland in parallel with the co-stored catecholamines and ATP. A prohormone concept was introduced when one of the main storage proteins collectively named granins was identified as the insulin release inhibitory polypeptide pancreastatin. A wide range of granin-derived biologically active peptides have subsequently been identified. Both chromogranin A and chromogranin B give rise to antimicrobial peptides of relevance for combat of pathogens. While two of the chromogranin A-derived peptides, vasostatin-I and pancreastatin, are involved in modulation of calcium and glucose homeostasis, respectively, vasostatin-I and catestatin are important modulators of endothelial permeability, angiogenesis, myocardial contractility, and innate immunity. A physiological role is now evident for the full-length chromogranin A and vasostatin-I as circulating stabilizers of endothelial integrity and in protection against myocardial injury. The high circulating levels of chromogranin A and its fragments in patients suffering from various inflammatory diseases have emerged as challenges for future research and clinical applications.

Angiotensin II dependent cardiac remodeling in the eel Anguilla anguilla involves the NOS/NO system.

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin System (RAS), plays an important role in controlling mammalian cardiac morpho-functional remodelling. In the eel Anguilla anguilla, one month administration of AngII improves cardiac performance and influences the expression and localization of molecules which regulate cell growth. To deeper investigate the morpho-functional chronic influences of AngII on the eel heart and the molecular mechanisms involved, freshwater eels (A. anguilla) were intraperitoneally injected for 2 months with AngII (1 nmol g BW-1). Then the isolated hearts were subjected to morphological and western blotting analyses, and nitrite measurements. If compared to control animals, the ventricle of AngII-treated hearts showed an increase in compacta thickness, vascularization, muscle mass and fibrosis. Structural changes were paralleled by a higher expression of AT2 receptor and a negative modulation of the ERK1-2 pathway, together with a decrease in nitrite concentration, indicative of a reduced Nitric Oxide Synthase (NOS)-dependent NO production. Moreover, immunolocalization revealed, particularly on the endocardial endothelium (EE) of AngII-treated hearts, a significant reduction of phosphorylated NOS detected by peNOS antibody accompanied by an increased expression of the eNOS disabling protein NOSTRIN, and a decreased expression of the positive regulators of NOS activity, pAkt and Hsp90. On the whole, results suggest that, in the eel, AngII modulates cardiac morpho-functional plasticity by influencing the molecular mechanisms that control NOS activity and the ERK1-2 pathway.

Cardiac contractility in Antarctic teleost is modulated by nitrite through xanthine oxidase and cytochrome p-450 nitrite reductase.

In mammalian and non-mammalian vertebrates, nitrite anion, the largest pool of intravascular and tissue nitric oxide storage, represents a key player of many biological processes, including cardiac modulation. As shown by our studies on Antarctic teleosts, nitrite-dependent cardiac regulation is of great relevance also in cold-blooded vertebrates. This study analysed the influence elicited by nitrite on the performance of the perfused beating heart of two Antarctic stenotherm teleosts, the haemoglobinless Chionodraco hamatus (icefish) and the red-blooded Trematomus bernacchii. Since haemoglobin is crucial in nitric oxide homeostasis, the icefish, a naturally occurring genetic knockout for this protein, provides exclusive opportunities to investigate nitric oxide/nitrite signaling. In vivo, nitrite conversion to nitric oxide requires the nitrite reductase activity of xanthine oxidase and cytochrome P-450, thus the involvement of these enzymes was also evaluated. We showed that, in C. hamatus and T. bernacchii, nitrite influenced cardiac performance by inducing a concentration-dependent positive inotropic effect which was unaffected by nitric oxide scavenging by PTIO in C. hamatus, while it was abolished in T. bernacchii. Specific inhibition of xanthine oxidase and cytochrome P-450 revealed, in the two teleosts, that the nitrite-dependent inotropism required the nitrite reductase activity of both enzymes. We also found that xanthine oxidase is more expressed in C. hamatus than in T. bernacchii, while the opposite was observed concerning cytochrome P-450. Results suggested that in the heart of C. hamatus and T. bernacchii, nitrite is an integral physiological source of nitric oxide with important signaling properties, which require the nitrite reductase activity of xanthine oxidase and cytochrome P-450.

Effects of environmental hypoxia on the goldfish skeletal muscle: Focus on oxidative status and mitochondrial dynamics.

The skeletal muscle is a highly plastic tissue. Its ability to respond to external stimuli and challenges allows it to face the functional needs of the organism. In the goldfish Carassius auratus, a model of hypoxia resistance, exposure to reduced oxygen is accompanied by an improvement of the swimming performance, relying on a sustained contractile behavior of the skeletal muscle. At the moment, limited information is available on the mechanisms underlying these responses. We here evaluated the effects of short- (4 days) and long- (20 days) term exposure to moderate water hypoxia on the goldfish white skeletal muscle, focusing on oxidative status and mitochondrial dynamics. No differences in lipid peroxidation, measured as 2-thiobarbituric acid-reacting substances (TBARS), and oxidatively modified proteins (OMP) were detected in animals exposed to hypoxia with respect to their normoxic counterparts. Exposure to short-term hypoxia was characterized by an enhanced SOD activity and expression, paralleled by increased levels of Nrf2, a regulator of the antioxidant cell response, and HSP70, a chaperone also acting as a redox sensor. The expression of markers of mitochondrial biogenesis (TFAM) and abundance (VDAC) and of the mtDNA/nDNA ratio was similar under normoxia and under both short- and long-term hypoxia, thus excluding a rearrangement of the mitochondrial apparatus. Only an increase of PGC1α (a transcription factor involved in mitochondrial dynamics) was detected after 20 days of hypoxia. Our results revealed novel aspects of the molecular mechanisms that in the goldfish skeletal muscle may sustain the response to hypoxia, thus contributing to adequate tissue function to organism requirements.

The effects of ACE inhibitor Enalapril on Mytilus galloprovincialis: Insights into morphological and functional responses.

In the last decades, pharmaceuticals have emerged as a new class of environmental contaminants. Antihypertensives, including angiotensin-converting enzyme (ACE) inhibitors, are of special concern due to their increased consumption over the past years. However, the available data on their putative effects on the health of aquatic animals, as well as the possible interaction with biological systems are still poorly understood. This study analysed whether and to which extent the exposure to Enalapril, an ACE inhibitor commonly used for treating hypertension and heart failure, may induce morpho-functional alterations in the mussel Mytilus galloprovincialis, a sentinel organism of water pollution. By mainly focusing on the digestive gland (DG), a target tissue used for analysing the effects of xenobiotics in mussels, the effects of 10-days exposure to 0.6 ng/L (E1) and 600 ng/L (E2) of Enalapril were investigated in terms of cell viability and volume regulation, morphology, oxidative stress, and stress protein expression and localization. Results indicated that exposure to Enalapril compromised the capacity of DG cells from the E2 group to regulate volume by limiting the ability to return to the original volume after hypoosmotic stress. This occurred without significant effects on DG cell viability. Enalapril unaffected also haemocytes viability, although an increased infiltration of haemocytes was histologically observed in DG from both groups, suggestive of an immune response. No changes were observed in the two experimental groups on expression and tissue localization of heat shock proteins 70 (HSPs70) and HSP90, and on the levels of oxidative biomarkers. Our results showed that, in M. galloprovincialis the exposure to Enalapril did not influence the oxidative status, as well as the expression and localization of stress-related proteins, while it activated an immune response and compromised the cell ability to face osmotic changes, with potential consequences on animal performance.

From Personal Care to Coastal Concerns: Investigating Polyethylene Glycol Impact on Mussel's Antioxidant, Physiological, and Cellular Responses.

Pharmaceutical and personal care products (PPCPs) containing persistent and potentially hazardous substances have garnered attention for their ubiquitous presence in natural environments. This study investigated the impact of polyethylene glycol (PEG), a common PPCP component, on Mytilus galloprovincialis. Mussels were subjected to two PEG concentrations (E1: 0.1 mg/L and E2: 10 mg/L) over 14 days. Oxidative stress markers in both gills and digestive glands were evaluated; cytotoxicity assays were performed on haemolymph and digestive gland cells. Additionally, cell volume regulation (RVD assay) was investigated to assess physiological PEG-induced alterations. In the gills, PEG reduced superoxide dismutase (SOD) activity and increased lipid peroxidation (LPO) at E1. In the digestive gland, only LPO was influenced, while SOD activity and oxidatively modified proteins (OMPs) were unaltered. A significant decrease in cell viability was observed, particularly at E2. Additionally, the RVD assay revealed disruptions in the cells subjected to E2. These findings underscore the effects of PEG exposure on M. galloprovincialis. They are open to further investigations to clarify the environmental implications of PPCPs and the possibility of exploring safer alternatives.

Catestatin reduces myocardial ischaemia/reperfusion injury: involvement of PI3K/Akt, PKCs, mitochondrial KATP channels and ROS signalling.

Catestatin (CST) limits myocardial ischaemia/reperfusion (I/R) injury with unknown mechanisms. Clearly phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) isoforms, including intra-mitochondrial PKCε, mitochondrial KATP (mitoKATP) channels and subsequent reactive oxygen species (ROS)-signalling play important roles in postconditioning cardioprotection, preventing mitochondrial permeability transition pore (mPTP) opening. Therefore, we studied the role of these extra- and intra-mitochondrial factors in CST-induced protection. Isolated rat hearts and H9c2 cells underwent I/R and oxidative stress, respectively. In isolated hearts CST (75nM, CST-Post) given in early-reperfusion significantly reduced infarct size, limited post-ischaemic contracture, and improved recovery of developed left ventricular pressure. PI3K inhibitor, LY-294002 (LY), large spectrum PKC inhibitor, Chelerythrine (CHE), specific PKCε inhibitor (εV1-2), mitoKATP channel blocker, 5-Hydroxydecanoate (5HD) or ROS scavenger, 2-mercaptopropionylglycine (MPG) abolished the infarct-sparing effect of CST. Notably the CST-induced contracture limitation was maintained during co-infusion of 5HD, MPG or εV1-2, but it was lost during co-infusion of LY or CHE. In H9c2 cells challenged with H2O2, mitochondrial depolarization (an index of mPTP opening studied with JC1-probe) was drastically limited by CST (75nM). Our results suggest that the protective signalling pathway activated by CST includes mitoKATP channels, ROS signalling and prevention of mPTP opening, with a central role for upstream PI3K/Akt and PKCs. In fact, all inhibitors completely abolished CST-infarct-sparing effect. Since CST-anti-contracture effect cannot be explained by intra-mitochondrial mechanisms (PKCε activation and mitoKATP channel opening) or ROS signalling, it is proposed that these downstream signals are part of a reverberant loop which re-activates upstream PKCs, which therefore play a pivotal role in CST-induced protection.

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.

Humoral control of cardiac remodeling in fish: role of Angiotensin II.

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin-System (RAS), is a multipotent hormone whose biological actions include short-term modulation as well as long-term adjustments. In the eel heart, AngII elicits short-term inotropic and chronotropic effects. However, information regarding the influence of AngII on cardiac remodeling, expressed as morphological and hemodynamic changes, is lacking. To clarify the putative actions of AngII on eel cardiac remodeling, we used freshwater eels (Anguilla anguilla) intraperitoneally injected for 4 weeks with saline or AngII (0.4 or 1.2 nmol g BW(-1)) or AngII (1.2 nmol g BW(-1)) plus the AT2 receptor antagonist CGP42112. Using an in vitro working heart preparation, the cardiac response (stroke volume changes) to preload and afterload increases has been evaluated. Hearts of all groups showed similar Frank-Starling responses. However, in response to afterload increases, stroke volume rapidly decreased in control hearts, while it was better maintained in AngII-treated counterparts. These effects were abolished by an antagonist of the AT2 receptor, whose cardiac expression was revealed by western blotting analysis. We also found by immunolocalization and immunoblotting that AngII influences both expression and localization of molecules which regulate cell growth [such as c-kit, heat shock protein 90 (Hsp-90), endothelial Nitric Oxide Synthase "(eNOS)-like" isoform] and apoptosis [i.e. apoptosis repressor with CARD domain (ARC)], thus playing a role in cardiac long-term adjustments. These results point to a role of AngII in eel heart remodeling, providing new insights regarding the modulation of cardiac plasticity in fish.

Contamination by Trace Elements and Oxidative Stress in the Skeletal Muscle of Scyliorhinus canicula from the Central Tyrrhenian Sea.

Marine pollution, due to the regular discharge of contaminants by various anthropogenic sources, is a growing problem that imposes detrimental influences on natural species. Sharks, because of a diet based on smaller polluted animals, are exposed to the risk of water contamination and the subsequent bioaccumulation and biomagnification. Trace elements are very diffuse water pollutants and able to induce oxidative stress in a variety of marine organisms. However, to date, studies on sharks are rather scarce and often limited to mercury. In this context, the present study aimed to analyze the accumulation of trace elements and their putative correlation with the onset of an oxidative status in the muscle of the lesser spotted dogfish Scyliorhinus canicula, from the Central Mediterranean Sea. Ecotoxicological analysis detected the presence of Pb, As, Cd, Mn, Zn, Ni, Cu, and Fe; no significant differences were observed between sexes, while a negative correlation was found between Pb and animal length. Analysis of oxidative stress markers showed either positive or negative correlation with respect to the presence of trace elements. Lipid peroxidation (TBARS) positively correlated with Zn, Ni, and Fe; SOD enzyme activity negatively correlated with Cu and Ni; LDH was negatively correlated with Fe and positively correlated with Pb. Moreover, positive correlations between the leukocyte count and Mn and Zn, as well as with LDH activity, were also observed. The data suggested that, in sharks, trace elements accumulation may affect oxidant and antioxidant processes with important outcomes for their physiology and health.

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 ß(2)/ß(3)-adrenergic receptors (ß(2)/ß(3)-AR), showing a higher affinity for ß(2)-AR. Interaction with ß(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 ß-arrestin, suggesting an additional mechanism for intracellular calcium modulation and ß-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.