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.

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.

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.

Shaping the cardiac response to hypoxia: NO and its partners in teleost fish.

The reduced availability of dissolved oxygen is a common stressor in aquatic habitats that affects the ability of the heart to ensure tissue oxygen supply. Among key signalling molecules activated during cardiac hypoxic stress, nitric oxide (NO) has emerged as a central player involved in the related adaptive responses. Here, we outline the role of the nitrergic control in modulating tolerance and adaptation of teleost heart to hypoxia, as well as major molecular players that participate in the complex NO network. The purpose is to provide a framework in which to depict how the heart deals with limitations in oxygen supply. In this perspective, defining the relational interplay between the multiple (sets of) proteins that, due to the gene duplication events that occurred during the teleost fish evolutive radiation, do operate in parallel with similar functions in the (different) heart (districts) and other body districts under low levels of oxygen supply, represents a next goal of the comparative research in teleost fish cardiac physiology.

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.

Hypoxic and Thermal Stress: Many Ways Leading to the NOS/NO System in the Fish Heart.

Teleost fish are often regarded with interest for the remarkable ability of several species to tolerate even dramatic stresses, either internal or external, as in the case of fluctuations in O2 availability and temperature regimes. These events are naturally experienced by many fish species under different time scales, but they are now exacerbated by growing environmental changes. This further challenges the intrinsic ability of animals to cope with stress. The heart is crucial for the stress response, since a proper modulation of the cardiac function allows blood perfusion to the whole organism, particularly to respiratory organs and the brain. In cardiac cells, key signalling pathways are activated for maintaining molecular equilibrium, thus improving stress tolerance. In fish, the nitric oxide synthase (NOS)/nitric oxide (NO) system is fundamental for modulating the basal cardiac performance and is involved in the control of many adaptive responses to stress, including those related to variations in O2 and thermal regimes. In this review, we aim to illustrate, by integrating the classic and novel literature, the current knowledge on the NOS/NO system as a crucial component of the cardiac molecular mechanisms that sustain stress tolerance and adaptation, thus providing some species, such as tolerant cyprinids, with a high resistance to stress.

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.

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.

Hypoxia Tolerance in Teleosts: Implications of Cardiac Nitrosative Signals.

Changes in environmental oxygen (O2) are naturally occurring phenomena which ectotherms have to face on. Many species exhibit a striking capacity to survive and remain active for long periods under hypoxia, even tolerating anoxia. Some fundamental adaptations contribute to this capacity: metabolic suppression, tolerance of pH and ionic unbalance, avoidance and/or repair of free-radical-induced cell injury during reoxygenation. A remarkable feature of these species is their ability to preserve a normal cardiovascular performance during hypoxia/anoxia to match peripheral (tissue pO2) requirements. In this review, we will refer to paradigms of hypoxia- and anoxia-tolerant teleost fish to illustrate cardiac physiological strategies that, by involving nitric oxide and its metabolites, play a critical role in the adaptive responses to O2 limitation. The information here reported may contribute to clarify the molecular and cellular mechanisms underlying heart vulnerability vs. resistance in relation to O2 availability.

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 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.

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.

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.

Glycyrrhizin and glycyrrhetinic acid directly modulate rat cardiac performance.

Root extract of liquorice is traditionally used to treat several diseases. Liquorice-derived constituents present several biological actions. In particular, glycyrrhizin and its aglycone, glycyrrhetinic acid, exhibit well-known cardiovascular properties. The aim of this research was to explore the direct cardiac activity of glycyrrhizin and glycyrrhetinic acid. The effects of synthetic glycyrrhizin and glycyrrhetinic acid were evaluated on the isolated and Langendorff perfused rat heart. The intracellular signaling involved in the effects of the two substances was analyzed on isolated and perfused heart and by Western blotting on cardiac extracts. Under basal conditions, both glycyrrhizin and glycyrrhetinic acid influenced cardiac contractility and relaxation. Glycyrrhizin induced significant positive inotropic and lusitropic effects starting from very low concentrations, while both inotropism and lusitropism were negatively affected by glycyrrhetinic acid. Both substances significantly increased heart rate. Analysis of the signal transduction mechanisms suggested that glycyrrhizin acts through the endothelin receptor type A/phospholipase C axis while glycyrrhetinic acid acts through endothelin receptor type B/Akt/nitric oxide synthase/nitric oxide axis. To our knowledge, these data reveal, for the first time, that both glycyrrhizin and glycyrrhetinic acid directly affect cardiac performance. Additional information on the physiological significance of these substances and their cardiac molecular targets may provide indication on their biomedical application.

The evolutionary functions of cardiac NOS/NO in vertebrates tracked by fish and amphibian paradigms.

During early ectotherm vertebrate evolution the heart was redesigned as a high pressure pump adapted to perfuse larger body sizes. To compensate the consequent higher organ complexity and heterogeneity (ventricular myoarchitecture and blood supply), conceivably the three principal cardiac cell components, the endocardium, the contractile myocardium and the epicardium recruited and diversified the cardiac NOS system for functioning not only as a major modulator, but also as a spatio-temporal integrator of heart function. In the context of NOS isoform evolution, we will use fish and amphibian paradigms to illustrate major aspects of cardiac spatial and temporal integration achieved by the NOS/NO systems. This may reveal a primordial cardiac NOS/NO function, allocating it in a wider biological framework than so far envisioned.